Ubiquitination of mammalian AP endonuclease (APE1) regulated by the p53-MDM2 signaling pathway.

APE1/Ref-1 is an essential DNA repair/gene regulatory protein in mammals of which intracellular level significantly affects cellular sensitivity to genotoxicants. The apurinic/apyrimidinic endonuclease 1 (APE1) functions are altered by phosphorylation and acetylation. We here report that APE1 is also modified by ubiquitination. APE1 ubiquitination occurred specifically at Lys residues near the N-terminus, and was markedly enhanced by mouse double minute 2 (MDM2), the major intracellular p53 inhibitor. Moreover, DNA-damaging reagents and nutlin-3, an inhibitor of MDM2-p53 interaction, increased APE1 ubiquitination in the presence of p53. Downmodulation of MDM2 increased APE1 level, suggesting that MDM2-mediated ubiquitination can be a signal for APE1 degradation. In addition, unlike the wild-type APE1, ubiquitin-APE1 fusion proteins were predominantly present in the cytoplasm. Therefore, monoubiquitination not only is a prerequisite for degradation, but may also alter the APE1 activities in cells. These results reveal a novel regulation of APE1 through ubiquitination.

A linkage map of meadow fescue ( Festuca pratensis Huds.) and comparative mapping with other Poaceae species.

A genetic linkage map has been constructed for meadow fescue ( Festuca pratensis Huds.) (2n=2 x=14) using a full-sib family of a cross between a genotype from a Norwegian population (HF2) and a genotype from a Yugoslavian cultivar (B14). The two-way pseudo-testcross procedure has been used to develop separate maps for each parent, as well as a combined map. A total number of 550 loci have been mapped using homologous and heterologous RFLPs, AFLPs, isozymes and SSRs. The combined map consists of 466 markers, has a total length of 658.8 cM with an average marker density of 1.4 cM/marker. A high degree of orthology and colinearity was observed between meadow fescue and the Triticeae genome(s) for all linkage groups, and the individual linkage groups were designated 1F-7F in accordance with the orthologous Triticeae chromosomes. As expected, the meadow fescue linkage groups were highly orthologous and co-linear with Lolium, and with oat, maize and sorghum, generally in the same manner as the Triticeae chromosomes. It was shown that the evolutionary 4AL/5AL translocation, which characterises some of the Triticeae species, is not present in the meadow fescue genome. A putative insertion of a segment orthologous to Triticeae 2 at the top of 6F, similar to the rearrangement found in the wheat B and the rye R genome, was also observed. In addition, chromosome 4F is completely orthologous to rice chromosome 3 in contrast to the Triticeae where this rice chromosome is distributed over homoeologous group 4 and 5 chromosomes. The meadow fescue genome thus has a more ancestral configuration than any of the Triticeae genomes. The extended meadow fescue map reported here provides the opportunity for beneficial cross-species transfer of genetic knowledge, particularly from the complete genome sequence of rice.

Comparative sequence analysis of colinear barley and rice bacterial artificial chromosomes.

Colinearity of a large region from barley (Hordeum vulgare) chromosome 5H and rice (Oryza sativa) chromosome 3 has been demonstrated by mapping of several common restriction fragment-length polymorphism clones on both regions. One of these clones, WG644, was hybridized to rice and barley bacterial artificial chromosome (BAC) libraries to select homologous clones. One BAC from each species with the largest overlapping segment was selected by fingerprinting and blot hybridization with three additional restriction fragment-length polymorphism clones. The complete barley BAC 635P2 and a 50-kb segment of the rice BAC 36I5 were completely sequenced. A comparison of the rice and barley DNA sequences revealed the presence of four conserved regions, containing four predicted genes. The four genes are in the same orientation in rice, but the second gene is in inverted orientation in barley. The fourth gene is duplicated in tandem in barley but not in rice. Comparison of the homeologous barley and rice sequences assisted the gene identification process and helped determine individual gene structures. General gene structure (exon number, size, and location) was largely conserved between rice and barley and to a lesser extent with homologous genes in Arabidopsis. Colinearity of these four genes is not conserved in Arabidopsis compared with the two grass species. Extensive similarity was not found between the rice and barley sequences other than within the exons of the structural genes, and short stretches of homology in the promoters and 3' untranslated regions. The larger distances between the first three genes in barley compared with rice are explained by the insertion of different transposable retroelements.

Analysis of recombination rate in female and male gametogenesis in pearl millet (Pennisetum glaucum) using RFLP markers.

Sex as a factor affecting recovered recombination in plant gametes was investigated in pearl millet, Pennisetum glaucum, by using reciprocal three-way crosses [(AxB)xCvCx(A x B)]. The two populations were mapped at 42 loci pre-selected to cover the majority of the genome. No differences in recombination distances were observed at the whole-genome level and only a few individual linkage intervals were found to differ, all in favour of increased recombination through the male. Distorted segregations found in the three-way crosses provide evidence of post-gametic selection for particular gene(s) or chromosome regions. The significance of these results for the design of pearl millet breeding programmes and inheritance experiments, as well as for other experimental strategies, is discussed.

An RFLP-based genetic map of pearl millet (Pennisetum glaucum).

Analysis of a sample of diverse pearl millet genotypes with 200 genomic DNA probes revealed this crop species to be extremely polymorphic. Among these genotypes, 85% of probes detected polymorphism using only two restriction enzymes, with an average pair-wise polymorphism between all of the probe-enzyme combinations of 56%. Two crosses were employed to construct an RFLP-based genetic map. In an intervarietal F2 population, derived from a single F1 plant, 181 loci were placed on a linkage map. The total length of this map, which comprised seven linkage groups, was 303 cM and the average map distance between loci was about 2 cM, although a few intervals in excess of 10 cM were present at the ends of a few linkage groups. Very few clones, including those which hybridized to more than one copy, detected more than one locus in the pearl millet genome. The analysis was complicated initially because 83 of the 181 loci mapped to a single linkage group. Analysis of a second cross identified a probable translocation breakpoint in the middle of this large linkage group.