Meristem-Specific Suppression of Mitosis and a Global Switch in Gene Expression in the Root Cap of Pea by Endogenous Signals1

Two functionally distinct sets of meristematic cells exist within root tips of pea (Pisum sativum): the root apical meristem, which gives rise to the body of the root; and the root cap meristem, which gives rise to cells that differentiate progressively through the cap and separate ultimately from its periphery as border cells. When a specific number of border cells has accumulated on the root cap periphery, mitosis within the root cap meristem, but not the apical meristem, is suppressed. When border cells are removed by immersion of the root tip in water, a transient induction of mitosis in the root cap meristem can be detected starting within 5 min. A corresponding switch in gene expression throughout the root cap occurs in parallel with the increase in mitosis, and new border cells begin to separate from the root cap periphery within 1 h. The induction of renewed border cell production is inhibited by incubating root tips in extracellular material released from border cells. The results are consistent with the hypothesis that operation of the root cap meristem and consequent turnover of the root cap is self-regulated by a signal from border cells.

wrinkled1: A Novel, Low-Seed-Oil Mutant of Arabidopsis with a Deficiency in the Seed-Specific Regulation of Carbohydrate Metabolism1

During oil deposition in developing seeds of Arabidopsis, photosynthate is imported in the form of carbohydrates into the embryo and converted to triacylglycerols. To identify genes essential for this process and to investigate the molecular basis for the developmental regulation of oil accumulation, mutants producing wrinkled, incompletely filled seeds were isolated. A novel mutant locus, wrinkled1 (wri1), which maps to the bottom of chromosome 3 and causes an 80% reduction in seed oil content, was identified. Wild-type and homozygous wri1 mutant plantlets or mature plants were indistinguishable. However, developing homozygous wri1 seeds were impaired in the incorporation of sucrose and glucose into triacylglycerols, but incorporated pyruvate and acetate at an increased rate. Because the activities of several glycolytic enzymes, in particular hexokinase and pyrophosphate-dependent phosphofructokinase, are reduced in developing homozygous wri1 seeds, it is suggested that WRI1 is involved in the developmental regulation of carbohydrate metabolism during seed filling.

High Aluminum Resistance in Buckwheat1 : I. Al-induced Specific Secretion of Oxalic Acid from Root Tips

High Al resistance in buckwheat (Fagopyrum esculentum Moench. cv Jianxi) has been suggested to be associated with both internal and external detoxification mechanisms. In this study the characteristics of the external detoxification mechanism, Al-induced secretion of oxalic acid, were investigated. Eleven days of P depletion failed to induce secretion of oxalic acid. Exposure to 50 μm LaCl3 also did not induce the secretion of oxalic acid, suggesting that this secretion is a specific response to Al stress. Secretion of oxalic acid was maintained for 8 h by a 3-h pulse treatment with 150 μm Al. A nondestructive method was developed to determine the site of the secretion along the root. Oxalic acid was found to be secreted in the region 0 to 10 mm from the root tip. Experiments using excised roots also showed that secretion was located on the root tip. Four kinds of anion-channel inhibitors showed different effects on Al-induced secretion of oxalic acid: 10 μm anthracene-9-carboxylic acid and 4,4′-diisothiocyanatostilbene-2,2′-disulfonate had no effect, niflumic acid stimulated the secretion 4-fold, and phenylglyoxal inhibited the secretion by 50%. Root elongation in buckwheat was not inhibited by 25 μm Al or 10 μm phenylglyoxal alone but was inhibited by 40% in the presence of Al and phenylglyoxal, confirming that secretion of oxalic acid is associated with Al resistance.

Tissue Culture-Specific Expression of a Naturally Occurring Tobacco Feedback-Insensitive Anthranilate Synthase1

A cDNA and corresponding promoter region for a naturally occurring, feedback-insensitive anthranilate synthase (AS) α-subunit gene, ASA2, has been isolated from an unselected, but 5-methyl-tryptophan-resistant (5MTr), tobacco (Nicotiana tabacum) cell line (AB15–12-1). The ASA2 cDNA contains a putative transit peptide sequence, and Southern hybridization shows that more than one closely related sequence is present in the tobacco genome. The ASA2 cDNA complemented a trpE nonsense mutant Escherichia coli strain, allowing growth on 300 μm 5MT-containing minimal medium without tryptophan, and cell extracts contained feedback-insensitive AS activity. The 5MTr was lost when the E. coli strain was transformed with an ASA2 site-directed mutant (phenylalanine-107-arginine-108 → serine-107-glutamine-108). Identical nucleotide sequences encoding the phenylalanine-107-arginine-108 region have been found in polymerase chain reaction-amplified 326-bp ASA2 genomic fragments of wild-type (5-methyl-tryptophan-sensitive [5MTs]) tobacco and a progenitor species. High-level ASA2 transcriptional expression was detected only in 5MTr-cultured cells, not in 5MTs cells or in plants. Promoter studies indicate that tissue specificity of ASA2 is controlled by the promoter region between −2252 and −607. Since the ASA2 promoter sequences are not substantially different in the 5MTr and 5MTs lines, the increased levels of ASA2 mRNA in the 5MTr lines are most likely due to changes in a regulatory gene affecting ASA2 expression.

Substrate-specific regulation of the ribosome– translocon junction by N-terminal signal sequences

Amino-terminal signal sequences target nascent secretory and membrane proteins to the endoplasmic reticulum for translocation. Subsequent interactions between the signal sequence and components of the translocation machinery at the endoplasmic reticulum are thought to be important for the productive engagement of the translocon by the ribosome-nascent chain complex. However, it is not clear whether all signal sequences carry out these posttargeting steps identically, or if there are differences in the interactions directed by one signal sequence versus another. In this study, we find substantial differences in the ability of signal sequences from different substrates to mediate closure of the ribosome–translocon junction early in translocation. We also show that these differences in some cases necessitate functional coordination between the signal sequence and mature domain for faithful translocation. Accordingly, the translocation of some proteins is sensitive to replacement of their signal sequences. In a particularly dramatic example, the topology of the prion protein was found to depend highly on the choice of signal sequence used to direct its translocation. Taken together, our results reveal an unanticipated degree of substrate-specific functionality encoded in N-terminal signal sequences.

SPO21 Is Required for Meiosis-specific Modification of the Spindle Pole Body in Yeast

During meiosis II in the yeast Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body changes from a site of microtubule initiation to a site of de novo membrane formation. These membranes are required to package the haploid meiotic products into spores. This functional change in the spindle pole body involves the expansion and modification of its cytoplasmic face, termed the outer plaque. We report here that SPO21 is required for this modification. The Spo21 protein localizes to the spindle pole in meiotic cells. In the absence of SPO21 the structure of the outer plaque is abnormal, and prospore membranes do not form. Further, decreased dosage of SPO21 leaves only two of the four spindle pole bodies competent to generate membranes. Mutation of CNM67, encoding a known component of the mitotic outer plaque, also results in a meiotic outer plaque defect but does not block membrane formation, suggesting that Spo21p may play a direct role in initiating membrane formation.

Specific infection of CD4+ target cells by recombinant rabies virus pseudotypes carrying the HIV-1 envelope spike protein.

A recombinant rabies virus (RV) mutant deficient for the surface spike glycoprotein (G) gene was used to study the incorporation of envelope proteins from HIV-1 expressed from transfected plasmids. A hybrid HIV-1 protein in which the cytoplasmic domain was replaced with that of RV G was incorporated into the virus envelope and rescued the infectivity of the RV mutant. The RV(HIV-1) pseudotype viruses could infect only CD4+ cells, and their infectivity was neutralized specifically by anti-HIV-1 sera. In contrast to the chimeric protein, wild-type HIV-1 envelope protein or mutants with truncated cytoplasmic domains failed to produce pseudotyped particles. This indicates the presence of a specific signal in the RV G cytoplasmic domain, allowing correct incorporation of a spike protein into the envelope of rhabdovirus particles. The possibility of directing the cell tropism of RV by replacement of the RV G with proteins of defined receptor specificity should prove useful for future development of targetable gene delivery vectors.

The nuclear hormone receptor coactivator SRC-1 is a specific target of p300.

p300 and its family member, CREB-binding protein (CBP), function as key transcriptional coactivators by virtue of their interaction with the activated forms of certain transcription factors. In a search for additional cellular targets of p300/CBP, a protein-protein cloning strategy, surprisingly identified SRC-1, a coactivator involved in nuclear hormone receptor transcriptional activity, as a p300/CBP interactive protein. p300 and SRC-1 interact, specifically, in vitro and they also form complexes in vivo. Moreover, we show that SRC-1 encodes a new member of the basic helix-loop-helix-PAS domain family and that it physically interacts with the retinoic acid receptor in response to hormone binding. Together, these results implicate p300 as a component of the retinoic acid signaling pathway, operating, in part, through specific interaction with a nuclear hormone receptor coactivator, SRC-1.

Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice.

The tet regulatory system in which doxycycline (dox) acts as an inducer of specifically engineered RNA polymerase II promoters was transferred into transgenic mice. Tight control and a broad range of regulation spanning up to five orders of magnitude were monitored dependent on the dox concentration in the water supply of the animals. Administration of dox rapidly induces the synthesis of the indicator enzyme luciferase whose activity rises over several orders of magnitude within the first 4 h in some organs. Induction is complete after 24 h in most organs analyzed. A comparable regulatory potential was revealed with the tet regulatory system where dox prevents transcription activation. Directing the synthesis of the tetracycline-controlled transactivator (tTA) to the liver led to highly specific regulation in hepatocytes where, in presence of dox, less than one molecule of luciferase was detected per cell. By contrast, a more than 10(5)-fold activation of the luciferase gene was observed in the absence of the antibiotic. This regulation was homogeneous throughout but stringently restricted to hepatocytes. These results demonstrate that both tetracycline-controlled transcriptional activation systems provide genetic switches that permit the quantitative control of gene activities in transgenic mice in a tissue-specific manner and, thus, suggest possibilities for the generation of a novel type of conditional mutants.

Covalent HLA-B27/peptide complex induced by specific recognition of an aziridine mimic of arginine.

The class I major histocompatibility complex (MHC) glycoprotein HLA-B27 binds short peptides containing arginine at peptide position 2 (P2). The HLA-B27/peptide complex is recognized by T cells both as part of the development of the repertoire of T cells in the cellular immune system and during activation of cytotoxic T cells. Based on the three-dimensional structure of HLA-B27, we have synthesized a ligand with an aziridine-containing side chain designed to mimic arginine and to bind covalently in the arginine-specific P2 pocket of HLA-B27. Using tryptic digestion followed by mass spectrometry and amino acid sequencing, the aziridine-containing ligand is shown to alkylate specifically cysteine 67 of HLA-B27. Neither free cysteine in solution nor an exposed cysteine on a class II MHC molecule can be alkylated, showing that specific recognition between the anchor side-chain pocket of an MHC class I protein and the designed ligand (propinquity) is necessary to induce the selective covalent reaction with the MHC class I molecule.

Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme.

Certain matrix metalloproteinases (MMP) are expressed within the fibrous areas surrounding acellular lipid cores of atherosclerotic plaques, suggesting that these proteinases degrade matrix proteins within these areas and weaken the structural integrity of the lesion. We report that matrilysin and macrophage metalloelastase, two broad-acting MMPs, were expressed in human atherosclerotic lesions in carotid endarterectomy samples (n = 18) but were not expressed in normal arteries (n = 7). In situ hybridization and immunohistochemistry revealed prominent expression of matrilysin in cells confined to the border between acellular lipid cores and overlying fibrous areas, a distribution distinct from other MMPs found in similar lesions. Metalloelastase was expressed in these same border areas. Matrilysin was present in lipid-laden macrophages, identified by staining with anti-CD-68 antibody. Furthermore, endarterectomy tissue in organ culture released matrilysin. Staining for versican demonstrated that this vascular proteoglycan was present at sites of matrilysin expression. Biochemical studies showed that matrilysin degraded versican much more efficiently than other MMPs present in atherosclerotic lesions. Our findings suggest that matrilysin, specifically expressed in atherosclerotic lesions, could cleave structural proteoglycans and other matrix components, potentially leading to separation of caps and shoulders from lipid cores.

Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-kappa B.

Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives, is known to have antimitogenic, anticarcinogenic, antiinflammatory, and immunomodulatory properties. The molecular basis for these diverse properties is not known. Since the role of the nuclear factor NF-kappa B in these responses has been documented, we examined the effect of CAPE on this transcription factor. Our results show that the activation of NF-kappa B by tumor necrosis factor (TNF) is completely blocked by CAPE in a dose- and time-dependent manner. Besides TNF, CAPE also inhibited NF-kappa B activation induced by other inflammatory agents including phorbol ester, ceramide, hydrogen peroxide, and okadaic acid. Since the reducing agents reversed the inhibitory effect of CAPE, it suggests the role of critical sulfhydryl groups in NF-kappa B activation. CAPE prevented the translocation of the p65 subunit of NF-kappa B to the nucleus and had no significant effect on TNF-induced I kappa B alpha degradation, but did delay I kappa B alpha resynthesis. The effect of CAPE on inhibition of NF-kappa B binding to the DNA was specific, in as much as binding of other transcription factors including AP-1, Oct-1, and TFIID to their DNA were not affected. When various synthetic structural analogues of CAPE were examined, it was found that a bicyclic, rotationally constrained, 5,6-dihydroxy form was superactive, whereas 6,7-dihydroxy variant was least active. Thus, overall our results demonstrate that CAPE is a potent and a specific inhibitor of NF-kappa B activation and this may provide the molecular basis for its multiple immunomodulatory and antiinflammatory activities.

Specific accumulation of tumor-derived adhesion factor in tumor blood vessels and in capillary tube-like structures of cultured vascular endothelial cells.

Tumor-derived adhesion factor (TAF) was previously identified as a cell adhesion molecule secreted by human bladder carcinoma cell line EJ-1. To elucidate the physiological function of TAF, we examined its distribution in human normal and tumor tissues. Immunochemical staining with an anti-TAF monoclonal antibody showed that TAF was specifically accumulated in small blood vessels and capillaries within and adjacent to tumor nests, but not in those in normal tissues. Tumor blood vessel-specific staining of TAF was observed in various human cancers, such as esophagus, brain, lung, and stomach cancers. Double immunofluorescent staining showed apparent colocalization of TAF and type IV collagen in the vascular basement membrane. In vitro experiments demonstrated that TAF preferentially bound to type IV collagen among various extracellular matrix components tested. In cell culture experiments, TAF promoted adhesion of human umbilical vein endothelial cells to type IV collagen substrate and induced their morphological change. Furthermore, when the endothelial cells were induced to form capillary tube-like structures by type I collagen, TAF and type IV collagen were exclusively detected on the tubular structures. The capillary tube formation in vitro was prevented by heparin, which inhibited the binding of TAF to the endothelial cells. These results strongly suggest that TAF contributes to the organization of new capillary vessels in tumor tissues by modulating the interaction of endothelial cells with type IV collagen.

Sequence-specific recognition of cytosine C5 and adenine N6 DNA methyltransferases requires different deformations of DNA.

DNA methyltransferases modify specific cytosines and adenines within 2-6 bp recognition sequences. We used scanning force microscopy and gel shift analysis to show that M.HhaI, a cytosine C-5 DNA methyltransferase, causes only a 2 degree bend upon binding its recognition site. Our results are consistent with prior crystallographic analysis showing that the enzyme stabilizes an extrahelical base while leaving the DNA duplex otherwise unperturbed. In contrast, similar analysis of M.EcoRI, an adenine N6 DNA methyltransferase, shows an average bend angle of approximately 52 degrees. This distortion of DNA conformation by M.EcoRI is shown to be important for sequence-specific binding.