Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases

Xeroderma pigmentosum is a monogenic disease characterized by hypersensitivity to ultraviolet light. The cells of xeroderma pigmentosum patients are defective in nucleotide excision repair, limiting their capacity to eliminate ultraviolet-induced DNA damage, and resulting in a strong predisposition to develop skin cancers. The use of rare cutting DNA endonucleases-such as homing endonucleases, also known as meganucleases-constitutes one possible strategy for repairing DNA lesions. Homing endonucleases have emerged as highly specific molecular scalpels that recognize and cleave DNA sites, promoting efficient homologous gene targeting through double-strand-break-induced homologous recombination. Here we describe two engineered heterodimeric derivatives of the homing endonuclease I-CreI, produced by a semi-rational approach. These two molecules-Amel3-Amel4 and Ini3-Ini4-cleave DNA from the human XPC gene (xeroderma pigmentosum group C), in vitro and in vivo. Crystal structures of the I-CreI variants complexed with intact and cleaved XPC target DNA suggest that the mechanism of DNA recognition and cleavage by the engineered homing endonucleases is similar to that of the wild-type I-CreI. Furthermore, these derivatives induced high levels of specific gene targeting in mammalian cells while displaying no obvious genotoxicity. Thus, homing endonucleases can be designed to recognize and cleave the DNA sequences of specific genes, opening up new possibilities for genome engineering and gene therapy in xeroderma pigmentosum patients whose illness can be treated ex vivo.     

Delayed ageing through damage protection by the Arf/p53 pathway

The tumour-suppressor pathway formed by the alternative reading frame protein of the Cdkn2a locus (Arf) and by p53 (also called Trp53) plays a central part in the detection and elimination of cellular damage, and this constitutes the basis of its potent cancer protection activity. Similar to cancer, ageing also results from the accumulation of damage and, therefore, we have reasoned that Arf/p53 could have anti-ageing activity by alleviating the load of age-associated damage. Here we show that genetically manipulated mice with increased, but otherwise normally regulated, levels of Arf and p53 present strong cancer resistance and have decreased levels of ageing-associated damage. These observations extend the protective role of Arf/p53 to ageing, revealing a previously unknown anti-ageing mechanism and providing a rationale for the co-evolution of cancer resistance and longevity.     

The common biology of cancer and ageing

At first glance, cancer and ageing would seem to be unlikely bedfellows. Yet the origins for this improbable union can actually be traced back to a sequence of tragic--and some say unethical--events that unfolded more than half a century ago. Here we review the series of key observations that has led to a complex but growing convergence between our understanding of the biology of ageing and the mechanisms that underlie cancer.     

Discrimination between alternate membrane protein topologies in living cells using GFP/YFP tagging and pH exchange

Membrane protein function is determined by the relative organization of the protein domains with respect to the membrane. We have experimentally verified the topology of a protein with diverse orientations arising from a single primary sequence (the cellular prion protein, PrP^C), a novel somatostatin truncated receptor, and the Golgi-associated protein GPBP₉₁. Tagging with fluorescent proteins (FP) allows location of their expression at the plasma membrane or at endomembranes, but does not inform about their orientation. Exploiting the pH dependency of some FPs, we developed a pH exchange assay in which extracellularly exposed FPs are quenched by application of low pH buffer. We constructed standards to demonstrate and calibrate the assay, and the method was adapted for acidic organelle membrane proteins. This method can serve as a proof of concept, experimentally confirming and/or discriminating in living cells among theoretical topology predictions, providing the proportion of inside/outside orientation for proteins with multiple forms.     

XY1Y2, a new sex-chromosome system among caraboid beetles

Summary Calathus ascendens (Caraboidea, Pterostichiae, Sphodrini) has 2n=20, XY₁Y₂: XX. This multiple sexchromosome system seems to be derived from a neo-XY one through an X-autosome fusion.Thanks go to A. Machado for collecting some specimens from Last Lagunets. This work has been supported by a postdoctoral fellowship of the C.S.I.C.     

Untersuchungen über die Nikotinbildung des Tabaks

Summary During germination the tobacco seedling produces nicotine from proteins accumulated in the seed. Later on the nicotine formation of the growing tobacco plant is dependent on the protein synthesis in its root. The comparison of theprotein production by the tobacco root with thenicotine content of the whole plant shows an interesting parallelism.     

Yeast plasma membrane ATPase is essential for growth and has homology with (Na+ + K+), K+- and Ca2+-ATPases

The plasma membrane ATPase of plants and fungi is a hydrogen ion pump. The proton gradient generated by the enzyme drives the active transport of nutrients by H+-symport. In addition, the external acidification in plants and the internal alkalinization in fungi, both resulting from activation of the H+ pump, have been proposed to mediate growth responses. This ATPase has a relative molecular mass (Mr) similar to those of the Na+-, K+- and Ca2+-ATPases of animal cells and, like these proteins, forms an aspartylphosphate intermediate. We have cloned, mapped and sequenced the gene encoding the yeast plasma membrane ATPase (PMA1) and report here that it maps to chromosome VII adjacent to LEU1. The strong homology between the amino-acid sequence encoded by PMA1 and those of (Na+ + K+), Na+-, K+- and Ca2+- ATPases is consistent with the notion that the family of cation pumps which form a phosphorylated intermediate evolved from a common ancestral ATPase. The function of the PMA1 gene is essential because a null mutation is lethal in haploid cells.