The mammalian abasic endonuclease, APE1, has two distinct roles in the repair of oxidative DNA damage and in gene regulation. Here we show that both functions are essential for cell survival. Deletion of the APE1 gene causes embryonic lethality in mice, and no nullizygous embryo fibroblasts have been isolated. We have now established nullizygous embryo fibroblast lines from APE1 ؊/؊ mouse embryos that are transgenic with the ''floxed'' human APE1 (hAPE1) gene. Removal of hAPE1 by Cre expression through nuclear microinjection elicited apoptosis in these cells within 24 h, which was blocked by coinjection of the wild-type hAPE1 gene. In contrast, mutant hAPE1 alleles, lacking either the DNA repair or acetylation-mediated gene regulatory function, could not prevent apoptosis, although the combination of these two mutants complemented APE deficiency induced by Cre. These results indicate that distinct and separable functions of APE1 are both essential for mammalian cells even in vitro and provide the evidence that mammalian cells, unlike yeast or Escherichia coli, absolutely require APE for survival, presumably to protect against spontaneous oxidative DNA damage.conditional gene inactivation ͉ DNA repair ͉ endogenous DNA damage ͉ base excision repair A basic endonuclease (APE), a ubiquitous enzyme, plays a central role in repairing toxic and mutagenic abasic (AP) sites generated in genomes during the repair of oxidation and alkylation damage through the base excision repair (BER) pathway (1). Oxidative DNA lesions, including AP sites, are also spontaneously generated at an estimated rate of 1.5 ϫ 10 5 residues⅐cell Ϫ1 ⅐day Ϫ1 (2). Unlike two distinct APEs present in Escherichia coli and Saccharomyces cerevisiae, only one active APE, APE1, an ortholog of E. coli xth and yeast APN2, has been identified in mammalian cells (3). Based on sequence homology, a second APE-like gene, APE2, was cloned from mammalian cells. However, we could not detect APE activity in the recombinant human APE2 (4), and hAPE2, unlike hAPE1, could not complement yeast APE mutants (5). Although APE-negative bacteria and yeast are viable, very early death (3.5-7.5 days after fertilization) was observed in APE1 nullizygous mouse embryos (6-8). Unlike other BER proteins, e.g., DNA polymerase ␤ and X-ray cross complementation group 1, which are essential for embryonic survival but not for mouse embryonic fibroblasts (MEFs) cultured in vitro (9, 10), APE1-null MEF mutant lines have not been established. The mammalian APE1, independently identified as redox-enhancing factor 1 (Ref1), has a distinct regulatory function in reductively activating C-Jun, p53, and other transcription factors (3, 11) for which Cys-65 (Cys-64 in mouse APE1) was identified as the active site (12). The N-terminal region of the 36-kDa polypeptide, including Cys-65, is not conserved in the E. coli homolog exonuclease III. An additional regulatory function of APE1͞Ref1 was identified in Ca 2ϩ -dependent down-regulation of the parathyroid hormone and renin genes containing negativ...

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Multiple shoots of Spilanthes acmella Murr. were induced from nodal buds of in vivo and in vitro seedlings on Murashige and Skoog (MS) medium containing 1.0 mg dm -3 6-benzyladenine (BA) and 0.1 mg dm -3 α-naphthaleneacetic acid (NAA). Adventitious shoots were successfully regenerated from the leaf explants derived from the above mentioned multiple shoots. The efficiency of shoot regeneration was tested in the MS medium containing BA, kinetin, or 2-isopentenyl adenine in combination with NAA, indole-3-acetic acid (IAA), or indole-3-butyric acid (IBA) and gibberellic acid. Maximum number of shoots per explant (20 ± 0.47) was recorded with 3.0 mg dm -3 BA and 1.0 mg dm -3 IAA. An anatomical study confirmed shoot regeneration via direct organogenesis. About 95 % of the in vitro shoots developed roots after transfer to half strength MS medium containing 1.0 mg dm -3 IBA. 95 % of the plantlets were successfully acclimatized and established in soil. The transplanted plantlets showed normal flowering without any morphological variation.Additional key words: growth regulators, medicinal plant, tissue culture. ⎯⎯⎯⎯Spilanthes acmella is an acutely threatened plant species (Narayana Rao and Raja Reddy 1983). S. acmella (marati mogga) is one of the important medicinal plants belonging to the family Asteraceae. The active compound was found to be spilanthol (Ramsewak et al. 1999). It is a perennial herb grown in the tropics and subtropics. Direct regeneration from leaf, as another alternative step for clonal propagation and germplasm conservation, is a well established factor. Successful plant regeneration has been reported from leaf explants, e.g., in Indian spinach (Mitra and Mukherjee 2001), Plumbago species (Das and Rout 2002), safflower (Radhika et al. 2006) and Carthamus species (Sujatha and Dinesh Kumar 2007). In view of the medicinal properties of S. acmella, its threatened nature and the increased demand for it in the pharmaceutical industry, there is a need for a large scale multiplication. In the present study, the direct shoot regeneration and histological studies from leaf explants of S. acmella have been studied. Direct regeneration of this plant species has not been reported so far.Spilanthes acmella Murr. shoots 22 cm long with 5 nodes were collected from 15-week-old field grown plants raised from seeds. They were divided into shoot tips and nodes, each measuring about 1 -4 cm in length.These served as explants for multiple shoot production. Explants were initially washed in running tap water for 15 min, and then rinsed in 10 % Teepol for 5 min followed by 0.1 % Bavistin for 5 min. The explants were thoroughly washed with sterile distilled water four times. Surface sterilization was carried out in 70 % ethanol for 45 s. Then the explants were treated with 0.05 % HgCl 2 for 4 min and 5 -6 times rinses in sterile distilled water. The nodal and shoot tip explants were inoculated in the Murashige and Skoog (1962; MS) medium fortified with 1.0 mg dm -3 6-benzyladenine (BA) and 0.1 mg dm -3 α-naphthalene acetic acid...