Understanding bamboo flowering based on large-scale analysis of expressed sequence tags.

Unlike other plants, bamboo (Bambusoideae) flowering is an elusive physiological phenomena, because it is unpredictable, long-periodic, gregarious, and uncontrollable; also, bamboo plants usually die after flowering. The flowering mechanism in Arabidopsis thaliana, a eudicot model species, is well established, but it remains unknown in bamboo species. We found 4470 and 3878 expressed sequence tags in the flower bud and vegetative shoot cDNA libraries, respectively, of the bamboo species, Bambusa oldhamii. Different genes were found expressed in bamboo flower buds compared to vegetative shoots, based on the Munich Information Center for Protein Sequences functional categorization; flowering-related genes were also identified in this species. We also identified Arabidopsis flowering-specific homologs that are involved in its photoperiod in this bamboo species, along with autonomous, vernalization and gibberellin-dependent pathways, indicating that bamboos may have a similar mechanism to control floral transition. Some bamboo expressed sequence tags shared high similarity with those of rice, but others did not match any known sequences. Our data lead us to conclude that bamboo may have its own unique flowering genes. This information can help us understand bamboo flowering and provides useful experimental methods to study the mechanisms involved.

Inhibition of poly(ADP-ribose)polymerase stimulates extrachromosomal homologous recombination in mouse Ltk-fibroblasts.

Poly(ADP-ribose)polymerase (PARP) is an abundant nuclear enzyme activated by DNA breaks. PARP is generally believed to play a role in maintaining the integrity of the genome in eukaryote cells via anti-recombinogenic activity by preventing inappropriate homologous recombination reactions at DNA double-strand breaks. While inhibition of PARP reduces non-homologous recombination, at the same time it stimulates sister chromatid exchange and intrachromosomal homologous recombination. Here we report that the inhibition of PARP with 100 microg/ml (0.622 mM) 1,5-isoquinolinediol results in an average 4.6-fold increase in the frequency of extrachromosomal homologous recombination between two linearized plasmids carrying herpes simplex virus thymidine kinase genes inactivated by non-overlapping mutations, in mouse Ltk-fibroblasts. These results are in disagreement with the previously reported observation that PARP inhibition had no effect on extrachromosomal homologous recombination in Ltk-cells.

Lesions in post-ribosomal supernatant fractions associated with loss of viability in pea (Pisum arvense) seed.

RNA synthesis and protein synthesis in viable pea embryonic axis tissue commences during the first hour of water imbibition whilst DNA synthesis commences after 8 h of imbibition. Neither DNA synthesis nor protein synthesis could be detected in non-viable axis tissue during the first 24 h of imbibition but some RNA synthesis is detectable during this period. Both post-ribosomal supernatant and ribosomal fractions from imbibed non-viable embryonic axis tissue were impaired in their ability to support polyphenylalanine synthesis in a cell-free protein-synthesising system, yet the same fractions isolated from unimbibed non-viable axis tissue were as efficient as equivalent fractions from unimbibed viable axis tissue in the support of polyphenylalanine synthesis in the cell-free system. A major lesion in elongation factor 1 activity and additional lesions in elongation factor 2 and phenylalanyl-tRNA synthetase activities were detected in the post-ribosomal supernatants isolated from non-viable embryonic axis tissue.

Lesions in the ribosomes of non-viable pea (Pisum arvense) embryonic axis tissue.

Ribosomes isolated from either dry viable or non-viable pea embryonic axis tissue were equally effective in the support of polyphenylalanine synthesis in a poly(U)-directed cell-free protein-synthesising system. Ribosomes isolated from imbibed non-viable axis tissue were impaired in their ability to support polyphenylalanine synthesis in the cell-free system. RNA isolated from ribosomes and 40-S ribosomal subunits of dry or imbibed viable axis tissue was found not to be degraded, whereas the equivalent RNA species isolated from non-viable axis tissue showed an increased degree of breakdown as imbibition proceeded. Even though rRNA of imbibed non-viable axis tissue was degraded, the ribosomes and ribosomal subunits of these embryos appeared intact. In viable embryonic axis tissue the percentage of ribosomes present in the cell in the form of polysomes increased during imbibition whereas no polysomes could be detected in ribosomal preparations from dry or imbibed non-viable axis tissue. The breakdown of rRNA in ribosomal particles from non-viable axis tissue may be a contributory factor to senescence and loss of viability in Pisum arvense.

Nucleotide sequence and expression of the isoamylase gene from an isoamylase-hyperproducing mutant, Pseudomonas amyloderamosa JD210.

The isoamylase gene (ISO) of Pseudomonas amyloderamosa JD210, an isoamylase-hyperproducing mutant, was cloned in an isoamylase-deficient and transformable mutant strain K31. By deletion analysis, the ISO gene was found to be located within a 3.3 kilobases BamHI fragment. Its nucleotide sequence contained an open reading frame of 2328 nucleotides (776 amino acids) encoding a secreted isoamylase precursor. The ISO gene fragment was inserted into plasmids pKT230 and pBR 322 in opposite orientations. The expression of the ISO gene in the constructed plasmids was compared in P. amyloderamosa K31, Pseudomonas aeruginosa PAO1-161, Pseudomonas putida mt-2 and Escherichia coli HB101. In all transformed cells, the majority of the isoamylase produced was secreted and higher isoamylase activities were obtained in transformats with the transcriptional direction of the ISO gene similar to the nearby drug-determinant gene of the vector.

Identification of the aromatase in the breast carcinoma cell lines T47D and MCF-7.

The presence of estrogens in tumour cells is considered to be a critical factor for the development of the hormone-dependent forms of breast cancer. The last, rate-limiting step of estrogen biosynthesis is controlled by cytochrome P-450 type enzyme complex named aromatase. In the present study we determined and characterized the expression of aromatase mRNA in the breast carcinoma cell lines T47D and MCF-7. The expression was characterized by slot blot hybridization, reverse transcriptase-polymerase chain reaction technique and Northern hybridization analysis. Northern blotting revealed the presence of 4.4 kb and 2.4 kb messengers in both cell lines.

Induction of PBP74/mortalin/Grp75, a member of the hsp70 family, by low doses of ionizing radiation: a possible role in induced radioresistance.

The identification of genes whose expression is altered following exposure to a low dose of ionizing radiation (IR) is an important step in understanding the phenomenon of the adaptive response. Using the differential mRNA display method we have identified a gene whose expression is up-regulated following exposure to 0.25 Gy IR. Partial DNA sequence and restriction endonuclease analysis of this gene showed that it is identical to the gene encoding for the human peptide-binding protein 74 (PBP74/mortalin/Grp75), a member of the heat shock 70 protein family. Time-course measurement of the PBP74/mortalin/Grp75 mRNA showed that its level was elevated after a lag of at least 15 min. The maximum induction appears to be at 30 min following gamma-irradiation and there is then a steady decline to control levels within 5 h in the HT29 cell line. On the other hand, the level of the PBP74/mortalin/Grp75 mRNA in the human breast adenocarcinoma cell line MCF-7 is consistently elevated after gamma-irradiation for up to 6 h post-irradiation. Furthermore, a cell line that does not demonstrate the induced radioresistance phenomenon (SW48) shows no induction of the PBP74/mortalin/Grp75 mRNA in contrast with HT29 or MCF-7. Treatment of the HT29 cells with antisense oligonucleotide directed towards the initiation codon of PBP74 sensitized cells to ionizing radiation.

Implication of PBP74/mortalin/GRP75 in the radio-adaptive response.

PURPOSE: To investigate the relationship between expression of the human peptide-binding protein PBP74 and the occurrence of an adaptive response to ionizing radiation. MATERIALS AND METHODS: Human tumour cell lines HT29 and MCF-7 were transfected with a PBP74 or PBP74 antisense construct. For demonstration of an adaptive response, cells lines were irradiated with a conditioning dose of 0.25 Gy cobalt-60 gamma-rays followed by a second dose of 4.0 Gy after an interval of 4.5 h. Response was measured in terms of clonogenic survival. RESULTS: Transfection of a PBP74 plasmid caused transient overexpression of PBP74 mRNA in both cell lines. The optimal dose for the induction of PBP74 in the cell lines investigated was 0.1-0.25Gy and PBP74 induction occurred within 30 min of irradiation. For both cell lines, the adaptive response was repressed when cells were transfected with the anti-PBP plasmid. However, the converse, an enhancement of the adaptive response in cell lines transfected with the PBP74 construct, was seen only for HT29 cells under certain experimental conditions. CONCLUSIONS: The results support the view that while PBP74 is necessary to the adaptive response, it may not by itself be sufficient for the adaptive response to occur.

The effect of the Saccharomyces cerevisiae endo-exonuclease NUD1 gene expression on the resistance of HeLa cells to DNA-damaging agents.

HeLa cells transiently transfected with a mammalian expression DNA vector expressing the Saccharomyces cerevisiae endo-exonuclease (EE) NUD1 gene have exhibited changes in cell survival frequencies after treatment with different DNA-damaging agents as compared to HeLa cells transfected with a control plasmid. The NUD1-transfected cells showed a dose-dependent increase in sensitivity to UV irradiation resulting in up to 58% decrease in cell survival. In response to gamma-irradiation NUD1 transfected cells featured an increased survival at doses equal to and greater than 2.0 Gy, reaching a maximum enhancement in survival frequency of 17%. At the same time, the NUD1-transfectants featured an increase in resistance to 0.25 microM-0.5 microM cis-platin (up to 58% increase in cell survival) and 1.0 mM EMS (11% increase). At higher concentrations of EMS NUD1 expression resulted in a decreased cell survival of the transfected cells (17% decrease for 2.5 mM EMS). No difference in cell survival frequencies between the NUD1-transfectants and the controls was observed after treatment with different concentrations of chlorambucil and mechlorethamine. These results suggest possible roles played by EEs in different DNA repair pathways--being stimulatory for the repair of certain types of DNA lesions, such as double strand breaks (DSBs), and interfering with the endogenous DNA repair systems for the repair of other types of lesions. Furthermore, these results also provide additional indirect evidence for the role of EEs in homologous recombination.

Transient expression of Saccharomyces cerevisiae endo-exonuclease NUD1 gene increases the frequency of extrachromosomal homologous recombination in mouse Ltk- fibroblasts.

Endo-exonucleases (EEs) are nucleolytic enzymes which have been shown to participate in the processes of DNA repair and recombination in eukaryotes. Recently, we have demonstrated that transient expression of Saccharomyces cerevisiae EE NUD1 gene in HeLa cells increased the resistance of the latter to ionizing radiation and cisplatin, suggesting the involvement of the NUD1 gene product in the recombination repair of double-strand breaks (DSB). Here, we report that transient expression of NUD1 results in up to 62% increase in the frequency of homologous recombination between two co-transfected linear plasmids in mouse Ltk- cells.

The status of serum hepatitis B virus DNA in HBSAG-positive hepatocellular carcinoma.

To elucidate the status of serum hepatitis B virus (HBV) DNA in HBsAg-positive hepatocellular carcinoma (HCC), 100 type B chronic liver disease (CLD) patients and 19 HCC patients were studied. The positive rate of serum HBV DNA in HBeAg-positive CLD patients was significantly higher than that in HBeAg-negative CLD patients, and the correlation between the presence of serum HBV DNA and patients' age showed a negative trend. In contrast, the positive rates of serum HBV DNA in HCC patients were not related to the status of HBeAg and age, and the positive rate of serum HBV DNA in HBeAg-negative HCC patients was significantly higher than that in HBeAg-negative CLD patients. Nevertheless, the serum concentrations of HBV DNA in HCC patients were significantly lower than those in CLD patients. These results suggest that replication of HBV in HCC patients might differ from that in CLD patients, and that persistent low-level HBV replication might be related to the presence of HCC.

Evidence that an endo-exonuclease controlled by the NUC2 gene functions in the induction of 'petite' mutations in Saccharomyces cerevisiae.

Defects in the RAD52 gene of the yeast Saccharomyces cerevisiae reduce the levels of the NUC2 endo-exonuclease by approximately 90% compared to the levels in wild-type strains. To examine the potential role of this nuclease in the induction of mitochondrial 'petite' mutations, congenic RAD52 and rad52-1 haploids were subjected to treatment with ethidium bromide, a well-known inducer of these mutations. The rad52 strain showed a much higher resistance to ethidium bromide-induced petite formation than the corresponding wild-type strain. Two approaches were taken to confirm that this finding reflected the nuclease deficiency, and not some other effect attributable to the rad52-1 mutation. First, a multicopy plasmid (YEp213-10) carrying NUC2 was transformed into a RAD52 strain. This resulted in an increased fraction of spontaneous petite mutations relative to that seen for the same strain without the plasmid and sensitized the strain carrying the plasmid to petite induction by ethidium bromide treatment. Second, a strain having a nuc2 allele that encodes a temperature-sensitive nuclease was treated with ethidium bromide at the restrictive and permissive temperatures. Petite induction was reduced under restrictive conditions. Enzyme assays revealed that the RAD52 (YEp213-10) strain had the highest level of antibody-precipitable NUC2 endo-exonuclease whereas the nuc2 and rad52 mutants had the lowest levels. Furthermore, addition of ethidium bromide to the reaction mixture stimulated the activity of the nuclease on double-stranded DNA. Petite induction by antifolate-mediated thymine nucleotide depletion was also inhibited by inactivation of RAD52 indicating that the effect of reduced NUC2 endo-exonuclease was not restricted to ethidium bromide treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Low doses of ionizing radiation induce nuclear activity in human tumour cell lines which catalyzes homologous double-strand recombination.

Activity catalysing double-strand DNA recombination has been investigated in human tumour cell lines using an in vitro assay in which nuclear extracts from tumour cells are used to catalyse homologous recombination between deletion plasmids. The cell lines investigated showed comparable constitutive levels of recombination activity. In several cell lines a two- fold to fourfold increase in the frequency of double-strand recombinational events catalysed by nuclear extracts was observed if the cells were exposed to low doses of ionizing radiation. The response was greatest for cells harvested at 6 h after radiation exposure, and the dose to produce an optimal effect was 25 cGy. Cell lines showing this response included a relatively radioresistant human colon cancer line and two cis-DDP (cis-diamminedichloroplatinum II) resistant ovarian tumour cell lines which are cross-resistant to radiation. Sub-lethal doses of cis-DDP were also effective in inducing upregulation of recombinational activity in the cis-DDP resistant cell lines. No change in recombinational activity was seen for radiation/drug-sensitive ovarian cell line following exposure to low drug or radiation doses. These findings are of particular interest since they involve a radiation-induced process with potential for direct involvement in DNA repair. Further studies will be aimed at determining if the extent of resistance to cytotoxic agents is causally related to the degree of inducible recombination activity.

Purification and characterization of an endo-exonuclease from Saccharomyces cerevisiae that is influenced by the RAD52 gene.

An endo-exonuclease has been purified from logarithmically growing cells of the yeast Saccharomyces cerevisiae. Identification and purification of this nuclease was facilitated by its being precipitable with an antibody raised against a previously described Neurospora crassa endo-exonuclease (Resnick, M. A., Chow, T. Y.-K. Nitiss, J., and Game, J. C. (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 639-649 and T. Y.-K. Chow and M. A. Resnick (1988) Mol. Gen. Genet., in press). The enzyme which was purified to near homogeneity was composed of a molecular weight 72,000 monomer. The single-strand nuclease activity is endonucleolytic and nonprocessive, whereas the double-strand DNase activity is exonucleolytic and weakly processive. Both nuclease activities have a pH optimum of 7.5, require Mg2+ or Mn2+ but not Zn2+ or Ca2+, are not inhibited by ATP, and exhibit the same kinetics of heat inactivation. Although this protein is not the product of the RAD52 gene, the greatly reduced amounts in rad52 mutants implicate the enzyme in repair and recombination processes in both mitotic and meiotic cells.

Purification and characterization of a mammalian endo-exonuclease.

An endo-exonuclease has been purified from cultured monkey (CV-1) cells. The enzyme which was purified to near homogeneity to be a 65 kDa monomeric protein. The single-strand DNase activity is endonucleolytic and nonprocessive, whereas the double-strand DNase activity is exonucleolytic and processive. The enzyme was also found to have RNase activity using poly-rA as substrate. The pH optimum for ss-DNase is 8 and for ds-DNase it is 7.5. Both DNase activities require a divalent metal ion (Mg2+, Mn2+, Ca2+, Zn2+) for activity and exhibit the same kinetics of heat inactivation. The purified protein binds to and cleaves a synthetic Holliday junction substrate. The overall enzymatic characteristics of the mammalian protein are very similar to the putative recombination endo-exonucleases purified from Neurospora crassa, Aspergillus nidulans and Saccharomyces cerevisiae.

Over-expression of the NUD1-coded endo-exonuclease in Saccharomyces cerevisiae enhances DNA recombination and repair.

The NUD1(=NUC2) gene of Saccharomyces cerevisiae has been subcloned and over-expressed in multi-copy plasmids. Enhanced expression of this nuclear endo-exonuclease gene was confirmed by Northern hybridization (>10-fold increase), and increased enzymatic activity (2.4-fold increase) was demonstrated by direct immunological assay using antibody raised against the purified Neurospora crassa endo-exonuclease. We found that increased expression of NUD1 was associated with an increase in cell survival after irradiation treatment with gamma rays, and an increase in radiation-induced mitotic recombination frequencies between duplicated gene sequences. The results presented here are consistent with previous biochemical data and confirm a role for the NUD1 gene product in recombination/repair processes.

Purification and properties of single strand DNA-binding endo-exonuclease of Neurospora crassa.

Single strand DNA-binding endo-exonucleases purified from mitochondria, vacuoles, or a mixture of these organelles had the same high specific single strand DNase activity (910 mumol of nucleotides/min/mg), and each contained a polypeptide of Mr = 31,000-33,000 which was found to be active by sodium dodecyl sulfate-DNA-gel electrophoresis. The properties of the three preparations were identical in all respects tested. The enzyme showed distributive endonuclease activity with single strand DNA, but processive exonuclease activity with double strand DNA. In the former case, 5'-phosphoryl-terminated fragments were released at early times, while in the latter case, short 5'-oligonucleotides (n = 2-4) were released. Both activities were dependent on Mg2+ (or Mn2+), but to different extents. In 0.1 mM Mg2+, superhelical bacteriophage phi X174 (replicative form (RF II) DNA and, at converted to relaxed circular (RF II) DNA and, at higher enzyme concentrations, to unit length linear (RF III) DNA. In 10 mM Mg2+, these same conversions took place rapidly, and the RF III DNA which formed was degraded to pieces shorter than unit length. At very low enzyme concentrations, long single strand tails and gaps were detected in bacteriophage T7 linear double strand DNA molecules.

The major intracellular alkaline deoxyribonuclease activities expressed in wild-type and Rec-like mutants of Neurospora crassa.

Over 95% of the deoxyribonuclease (DNase) activity of log-phase mycelia of Neurospora crassa is expressed as single-strand (ss) specific endonucleolytic activity. This activity is associated with three nucleases (D1, D2, and D3) which after partial purification from extracts, express activity with double-strand (ds) DNA as well. All three enzymes also degrade RNA at approximately the same rates that they degrade ss-DNA. D3 has been identified as endoexonuclease, an enzyme previously shown to have endonuclease activity with ss-DNA and RNA and exonuclease activity with ds-DNA, both of which are inhibited by ATP. D3 is inhibited by ATP, is relatively resistant to p-hydroxymercuribenzoate (PHMB), and sediments with an apparent molecular weight of 75 000. D2 has the properties of the previously described mitochondrial nuclease. It is a relatively unstable Mg2+-dependent endonuclease with no appreciable strand specificity for DNA. In addition, it is not inhibited by ATP and is strongly inhibited by PHMB and by the ethylenediamine tetraacetic acid (EDTA). It also sediments with an apparent molecular weight of 75,000. The properties of D1 are quite variable from one preparation to another. Freshly isolated D1 sediments with an apparent molecular weight of 180 000. It often shows some inhibition by ATP, but is relatively resistant to both PHMB and EDTA. However, on 'ageing,' the properties of D1 gradually convert to those of D2 with concomitant decrease in molecular weight, loss of inhibition by ATP, and increase in sensitivities to PHMB and EDTA. The results indicate that D1 is very likely a second form of the mitochondrial enzyme. Evidence was obtained for the presence of protein inhibitor(s) in crude extracts which may account for the masking of the ds-DNase activities of these enzymes in extracts. Two Rec-like mutants of Neurospora (uvs-3, and nuh-4) are deficient mainly inexpressed levels of D3, the endo-exonuclease. However, the levels of inactive endo-exonuclease precursor in these two mutants are higher than in the wild type. There may, therefore, be some defect in the conversion of precursor to active enzyme in these two mutants. Another mutant, which is not sensitive to mutagens relative to the wild (nuh-3), has depressed levels of both endo-exonuclease and the mitochondrial enzyme. Nuh-3 has some defect in the conversion of D1 to D2. Proteinases probably play some role in vivo in these enzyme conversions.

An endo-exonuclease activity of yeast that requires a functional RAD52 gene.

Extracts of Rad+ and radiation-sensitive (rad) mutants of the yeast Saccharomyces cerevisiae were examined for total Mg2+-dependent alkaline deoxyribonuclease activity and the presence of a nuclease that crossreacts immunologically with an antiserum raised against an endo-exonuclease from Neurospora crassa, an enzyme exhibiting both deoxyribo- and ribonuclease activities. No significant differences were observed in total deoxyribonuclease activity between Rad+ and rad mutants. The antibody precipitable activity, however, was found to be 30%-40% of the total alkaline deoxyribonuclease activity in logarithmically growing Rad+ cells. Extracts of stationary phase cells were lacking in antibody precipitable activity. Using immunoblot methods, a 72 kDa crossreacting protein was identified from logarithmically growing cells that was absent from stationary phase cells. In all radiation-sensitive mutants examined, except rad52, at least 20% of total activity was precipitable. Extracts from logarithmically growing rad52 mutants, including a rad52::LEU2 insertion mutant, exhibited less than 10% of the Rad+ precipitable activity; however, some crossreacting material was detected. Although, the level of endo-exonuclease activity is influenced by the RAD52 gene, it is not the product of this gene. The total deoxyribonuclease and the antibody precipitable endo-exonuclease activities were also followed during meiosis. Unlike the Rad+ strain which had previously been shown to have increased levels of total and immunoprecipitable endo-exonuclease as cells underwent meiosis, the rad52 mutant exhibited no increases in either category of nuclease activity. Given the importance of the RAD52 gene in repair, recombination and mutagenesis, the endo-exonuclease may be a significant component of these processes.

Specific targeting of antisense oligonucleotides to neutrophils.

The ability of three different hydrophobic ligands (cholic acid, cholesterol, and the tetrapeptide fMLFY) to increase the uptake of an antisense (anti-actin) oligomer into neutrophils was analyzed. In agreement with the literature (Boutorin et al., 1989; Letsinger et al., 1989), we found that cholic acid and cholesterol conjugates greatly enhance the uptake of anti-actin oligomer. When fMLFY is the ligand, the cellular uptake is much less than that of anti-actin oligomer alone, but the biological consequences are much more significant. Our results are consistent with the hypothesis that the fMLFY conjugate of the anti-actin oligomer is internalized via a different route, and reaches its target site most efficiently.