Adult hippocampal neurogenesis is involved in anxiety-related behaviors.

Adult hippocampal neurogenesis is a unique example of structural plasticity, the functional role of which has been a matter of intense debate. New transgenic models have recently shown that neurogenesis participates in hippocampus-mediated learning. Here, we show that transgenic animals, in which adult hippocampal neurogenesis has been specifically impaired, exhibit a striking increase in anxiety-related behaviors. Our results indicate that neurogenesis plays an important role in the regulation of affective states and could be the target of new treatments for anxiety disorders.

Lifelong corticosterone level determines age-related decline in neurogenesis and memory.

Ageing is accompanied by an alteration of spatial memory, a decline in hippocampal neurogenesis and a dysregulation of the hypothalamic-pituitary axis (HPA) leading to elevated levels of circulating corticosterone. However, the role of the HPA axis in age-related decline in cognitive functions and in neurogenesis decline remains unclear. We found that suppression of glucocorticoids secretion from midlife to the rest of the animals' life increases neurogenesis in old animals and prevents the emergence of age-related memory disorders. Reciprocally, aged rats with a chronic upregulation of the HPA axis exhibit not only spatial memory impairments but also very low levels of hippocampal cell proliferation and survival. Altogether, these results indicate that the extent of lifetime exposure to glucocorticoids determines the extent of age-related decline in hippocampal neurogenesis and consequently age-related cognitive dysfunctions.

Smad-dependent alterations of PPT cholinergic neurons as a pathophysiological mechanism of age-related sleep-dependent memory impairments.

In humans, memory impairments are highly prevalent in the aged population, but their functional and structural origins are still unknown. We hypothesized that circadian rhythm alterations may predict spatial memory impairment in aged rats. We demonstrate an association between sleep/wake circadian rhythm disturbances (non-REM sleep fragmentation) and spatial memory impairments in aged rats. We show by light and electron microscopy that these age-related disruptions in circadian rhythm and spatial memory are also associated with degeneration of cholinergic neurons of the pedunculopontine nucleus (PPT), a structure known to be involved in sleep and cognitive functions and which is altered during aging. Finally, we demonstrate that a trophic deregulation of the PPT occur in aged impaired rats, involving an over activation of the TGFbeta-Smad cascade, a signalling pathway involved in neurodegeneration. In conclusion these results provide a new pathophysiological mechanism for age-related sleep-dependent memory impairments opening the ground for the development of new therapeutic approaches of these pathologies.

Directed evolution and Phenomics screening of new biocatalysts.

Biodiversity screening and directed evolution are two fruitful complementary approaches for the discovery and design of novel biocatalysts. A new technology for directed evolution, L-Shuffling, has been designed and patented by Proteus. L-Shuffling technology offers several competitive advantages over other technologies including (i) directed evolution of large genes: L-Shuffling" means "Large-Shuffling"; (ii) high fidelity recombination and (iii) Control over location and frequency of recombination. The thousands of new recombinants generated by L-Shuffling can be further screened for their biochemical characteristics using Phenomics. Phenomics is a proprietary functional HTS technology designed and patented by Proteus for the screening of natural biodiversity as well as biodiversity generated by combinatorial biology. Phenomics is a function to gene structure approach which provides an alternative to genomics and proteomics. The traditional limits of expression libraries are thereby circumvented especially those related to cytotoxic products in usual or specific surrogate hosts. The quality of the answer given by the screening is directed dependent on the quality of the question asked. Thanks to a new substrates synthesis technology named CLIPS-O, the company can design highly specific molecules simulating the chemical structure and energetic state of the industrial substrates. The whole process of novel biocatalysts discovery has been automated using commercially available high throughput robotics.

Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans.

The DNA polymerase I gene of a newly described deep-sea hydrothermal vent Archaea species, Thermococcus fumicolans, from IFREMERS's collection of hyperthermophiles has been cloned in Escherichia coli. As in Thermococcus litoralis, the gene is split by two intervening sequences (IVS) encoding inteins inserted in sites A and C of family B DNA polymerases. The entire DNA polymerase gene, containing both inteins, was expressed at 30 degrees C in E. coli strain BL21(DE3)pLysS using the pARHS2 expression vector. The native polypeptide precursor of 170kDa was obtained, and intein splicing as well as ligation of the three exteins was observed in vitro after heat exposure. The recombinant enzyme was purified and some of its activities were characterized: polymerization, thermostability, exonuclease activities, and fidelity.

Smoothing of the thermal stability of DNA duplexes by using modified nucleosides and chaotropic agents.

The effect of alkyltrimethylammonium ions on the thermostability of natural and modified DNA duplexes has been investigated. We have shown that the use of tetramethylammonium ions TMA+along with the chemical modification of duplexes allow the fine adjustment of T m and the possibility of obtaining several duplex systems with varied isostabilizedtemperatures, some of which show greater stability than those of natural DNA. This approach could be very useful for DNA sequencing by hybridization.

The stability of duplexes involving AT and/or G4EtC base pairs is not dependent on their AT/G4EtC ratio content. Implication for DNA sequencing by hybridization.

Sequencing by the recently reported hybridization technique requires the formation of DNA duplexes with similar stabilities. In this paper we describe a new strategy to obtain DNA duplexes with a thermal stability independent of their AT/GC ratio content. Melting data were acquired on 35 natural and 27 modified duplexes of a given length and of varying base compositions. Duplexes built with AT and/or G4EtC base pairs exhibit a thermal stability restrained to a lower range of temperature than that of the corresponding natural compounds (16 instead of 51 degrees C). The 16 degrees C difference in thermal stability observed between the least stable and the most stable duplex built with AT and/or G4EtC base pairs is mainly due to the sequence effect and not to their AT/G4EtC ratio content. Thus N -4-ethyl-2'-deoxycytidine (d4EtC) hybridizes specifically with natural deoxyguanosine leading to a G4EtC base pair whose stability is very close to that of the natural AT base pair. Oligonucleotide probes involving d4EtC can be easily prepared by chemical synthesis with phosphoramidite chemistry. Modified DNA targets were successfully amplified by random priming or PCR techniques using d4EtCTP, dATP, dGTP and dTTP in the presence of DNA polymerase. This new system might be very useful for DNA sequencing by hybridization.

Modification of DNA duplexes to smooth their thermal stability independently of their base content for DNA sequencing by hybridization.

The possibility of equalizing DNA duplex stability is essential for the application of sequencing by hybridization. In this paper we describe a new strategy to obtain DNA duplexes with a thermal stability independent of their base content. Modified *C bases have been developed and incorporated into oligonucleotides. The influence of these modifications on duplex stability has been studied by absorption spectroscopy, thus allowing selection of N -4-ethyl-2'-deoxycytidine (d4EtC), which hybridizes specifically with natural dG to give a G4EtC base pair whose stability is very close to that of natural AT base pairs. Duplexes built with AT and/or G4EtC base pairs exhibit thermal stabilities independent of their base content in a classical buffer solution, thus enabling control of the stability of DNA hybrids as a function of their length only.

Synthesis and binding properties of oligonucleotides covalently linked to an acridine derivative: new study of the influence of the dye attachment site.

2-Methoxy-6-chloro-9-aminoacridine has been coupled via a polymethylene linker to various positions of an oligonucleotide chain: the 3'-position, using a new universal support, the 5'-position, and both 5'- and 3'-positions via a phosphate. The intercalating agent was also linked to the oligonucleotide chain via an internucleotide phosphorothiolate. The mixture of diastereoisomers was obtained as well as each pure Rp and Sp isomer. Finally, the acridine moiety was introduced to the 5-position of the deoxyuridine. The binding properties of these oligonucleotide-acridine conjugates with their DNA counterparts have been studied by absorption spectroscopy.

Psoralen-modified oligonucleotide primers improve detection of mutations by denaturing gradient gel electrophoresis and provide an alternative to GC-clamping.

Denaturing gradient gel electrophoresis (DGGE), a mutation-scanning procedure separating DNA fragments differing by as little as a single base change, is widely used in studies of genomic nucleotide sequence variability. The efficiency of the technique is greatly enhanced by attaching, through polymerase chain reaction (PCR) incorporation, a long GC-tail to the test DNA sequence which, as a result, becomes analysable throughout. As synthesis of GC-rich specific PCR primers is costly and time-consuming, we attempted to clamp the DNA fragment using a psoralen derivative (ChemiClamp) that promotes photo-induced cross-linking at one end. We found that this procedure provides an attractive alternative to GC-clamp in DGGE (and temperature gradient gel electrophoresis) and should prove useful in both research and diagnostic laboratories.

Repetitive satellite-like sequences are present within or upstream from 3 avian protein-coding genes.

Peculiar DNA sequences made up by the tandem repetition of a 5 bp unit have been identified within or upstream from three avian protein-coding genes. One sequence is located within an intron of the chicken "ovalbumin-X" gene with 5'-TCTCC-3' as basic repeat unit (36 repeats). Another sequence made of 27 repeats of a 5'-GGAAG-3' basic unit is found 2500 base pairs upstream from the promoter of the chicken ovotransferrin (conalbumin) gene. A related but different sequence is present in the corresponding region of the ovotransferrin gene in the pheasant, with 5'-GGAAA-3' as the basic unit (55 repeats). These three satellite-like elements are thus characterized by a total assymetry in base distribution, with purines restricted to one strand, and pyrimidines to the other. Two of the basic repeat units can be derived from the third one (GGAAA) by a single base pair change. These related sequences are found repeated in three avian genomes, at degrees which vary both with the sequence type and the genome type. Evolution of tandemly repeated sequences (including satellites) is in general studied by analysing randomly picked elements. The presence of conserved protein-coding regions neighbouring satellite-like sequences allow to follow their evolution at a single locus, as exemplified by the striking comparison of the pheasant and chicken sequences upstream from the ovotransferrin gene.