François Caron

DNA: An Extensible Molecule

The force-displacement response of a single duplex DNA molecule was measured. The force saturates at a plateau around 70 piconewtons, which ends when the DNA has been stretched about 1.7 times its contour length. This behavior reveals a highly cooperative transition to a state here termed S-DNA. Addition of an intercalator suppresses this transition. Molecular modeling of the process also yields a force plateau and suggests a structure for the extended form. These results may shed light on biological processes involving DNA extension and open the route for mechanical studies on individual molecules in a previously unexplored range.

Integrated microfluidics based on multi-layered SU-8 for mass spectrometry analysis

We present a design for integrated lab-on-chip microsystems dedicated to mass spectrometry analysis based on the fabrication of watertight microchannels for the circulation of liquids. In this paper, we demonstrate how to fabricate complete polymer microchannels using the negative photoresist SU-8 which has the advantage of being compatible with protein analysis by mass spectrometry. Our method of fabrication requires novel technological steps involving SU-8 multi-layer processing, improved SU-8 adhesion and the use of SU-8 wafer bonding for the watertight closing of the microchannels with a Pyrex wafer. This technique also encompasses the design of various microfluidic elements such as tapered recesses for the housing of capillary tubes allowing the connection of the channels to external systems. Following this, the capillary tubes were used to test the hydrodynamic behaviour of the channels and consequently the efficiency of our technological process in achieving fully watertight structures within our flow rate and pressure specifications.

Developmentally Regulated Chromosome Fragmentation Linked to Imprecise Elimination of Repeated Sequences in Paramecia

ABSTRACT The chromosomes of ciliates are fragmented at reproducible sites during the development of the polyploid somatic macronucleus, but the mechanisms involved appear to be quite diverse in different species. In Paramecium aurelia, the process is imprecise and results in de novo telomere addition at locally heterogeneous positions. To search for possible determinants of chromosome fragmentation, we have studied an ∼21-kb fragmentation region from the germ line genome of P. primaurelia. The mapping and sequencing of alternative macronuclear versions of the region show that two distinct multicopy elements, a minisatellite and a degenerate transposon copy, are eliminated by an imprecise mechanism leading either to chromosome fragmentation and the formation of new telomeres or to the rejoining of flanking sequences. Heterogeneous internal deletions occur between short direct repeats containing TA dinucleotides. The complex rearrangement patterns produced vary slightly among genetically identical cell lines, show non-Mendelian inheritance during sexual reproduction, and can be experimentally modified by transformation of the maternal macronucleus with homologous sequences. These results suggest that chromosome fragmentation in Paramecium is the consequence of imprecise DNA elimination events that are distinct from the precise excision of single-copy internal eliminated sequences and that target multicopy germ line sequences by homology-dependent epigenetic mechanisms.

Sequence-Specific Epigenetic Effects of the Maternal Somatic Genome on Developmental Rearrangements of the Zygotic Genome in Paramecium primaurelia

In ciliates, the germ line genome is extensively rearranged during the development of the somatic macronucleus from a mitotic product of the zygotic nucleus. Germ line chromosomes are fragmented in specific regions, and a large number of internal sequence elements are eliminated. It was previously shown that transformation of the vegetative macronucleus of Paramecium primaurelia with a plasmid containing a subtelomeric surface antigen gene can affect the processing of the homologous germ line genomic region during development of a new macronucleus in sexual progeny of transformed clones. The gene and telomere-proximal flanking sequences are deleted from the new macronuclear genome, although the germ line genome remains wild type. Here we show that plasmids containing nonoverlapping segments of the same genomic region are able to induce similar terminal deletions; the locations of deletion end points depend on the particular sequence used. Transformation of the maternal macronucleus with a sequence internal to a macronuclear chromosome also causes the occurrence of internal deletions between short direct repeats composed of alternating thymines and adenines. The epigenetic influence of maternal macronuclear sequences on developmental rearrangements of the zygotic genome thus appears to be both sequence specific and general, suggesting that this trans-nucleus effect is mediated by pairing of homologous sequences.

A high degree of macronuclear chromosome polymorphism is generated by variable DNA rearrangements in Paramecium primaurelia during macronuclear differentiation

DNA rearrangements in Paramecium lead to the formation of macronuclear chromosomes, the sizes of which range from 50 and 800 kb (1 kb is 10(3) base-pairs). This process does not appear to be a simple size reduction of the micronuclear chromosomes by specific and reproducible DNA sequence elimination and chromosomal breakage followed by chromosomal amplification. On the contrary, this process generates a variety of different, but sequence-related, macronuclear chromosomes from a unique set of micronuclear chromosomes. This paper describes an attempt to understand the nature of the diversity of the macronuclear chromosomes and the mechanisms of their production. The structure of three macronuclear chromosomes, 480, 250 and 230 kb in size, have been determined utilizing chromosome-jumping and YAC-cloning techniques. The two smallest chromosomes correspond roughly to the two halves of the longest chromosome. The main contribution to the diversity arises from the chromosomal ends and is due to variable positions of the telomere addition sites and/or to variable rearrangements of DNA sequences. The 480 kb chromosome contains a region of variable length, which is likely to be due to a variable deletion, located at the position of telomerization seen in the two small chromosomes. A model of chromosomal breakage is proposed to rationalize this result where micronuclear DNA is first amplified, broken and degraded to various extent from the newly formed ends, which subsequently are either telomerized or religated. Potential implications of these processes for gene expression is discussed. Known phenotypes that have a macronuclear determinism could be explained by this type of process.

The Physics of DNA Electrophoresis

An area that has seen much fruitful collaboration between biologists and physicists in recent years is the development of the gel electrophoresis technique for separating DNA fragments. To some extent, the success achieved is due to the artificial nature of the system, which makes it more immediately amenable to theoretical treatment than most of the naturally occurring systems discussed in this Workshop, where the degree of complexity is far greater. The process involved here is straightforward and essentially physical - charged DNA molecules are forced to migrate through a gel by the application of an electric field - but it is of immense importance to biologists, as the efficient separation of different fragments is an inevitable requirement for the manipulation of DNA performed in molecular genetics, cancer research and, more recently, the human genome project. The physics turns out to be rather interesting, with a rich variety of dynamical behaviour displaying some unusual and unexpected features. More generally, gel electrophoresis is a good example of a driven diffusive system, many types of which are currently under investigation.

Fast HEVC Intra Mode Decision Based on Edge Detection and SATD Costs Classification

The recent High Efficiency Video Coding (HEVC) standard was designed to achieve significantly improved compression performance compared to the widely used H.264/AVC standard. This achievement was motivated by the ever-increasing popularity of high-definition video applications and the emergence of ultra-HD. Unfortunately, this comes at the expense of a significant increase in computational complexity for both inter and intra coding. To alleviate this problem, in this paper, we propose a fast intra mode decision method based on improved edge detection, consideration of most relevant modes from neighboring blocks, and classification of SATD costs permitting the elimination of several candidate modes prior to rate distortion optimization (RDO). Experimental results show that the proposed method provides time reduction up to 39.2% and an average 35.6% with negligible quality loss as compared to the HEVC reference implementation HM 15.0.

Blocking of in vitro translation of Paramecium messenger RNAs is due to messenger RNA primary structure.

Paramecium primaurelia mRNAs were translated in vitro in rabbit reticulocyte lysate and the products of translation were analyzed by their size. We show that the large majority of these products are of short but discrete sizes irrespective of the length of the mRNA which directs their synthesis. An illustrative example is given by the translation of mRNA of G surface antigen which directs the synthesis of a 50 kD polypeptide instead of the complete 250 kD protein. Control experiments suggest that the blocking is due to mRNA primary structure.

Molecular Characterization of the D Surface Protein Gene Subfamily in Paramecium primaurelia

ABSTRACT. When Paramecium primaurelia expresses the D serotype, a major high molecular weight mRNA species is detected in the cytoplasm. Using the cDNA derived from this mRNA as a probe, three very similar genes, Dα, Dβ and Dγ, were cloned. Of these three genes, we show that only the Dα mRNA is present in the cytoplasm of cells expressing the D serotype and corresponds to the major mRNA species. The nucleotide sequence of the entire coding region of the Dα gene, as well as the upstream and downstream sequences, has been determined. The 7632‐nucleotide open reading frame encodes a putative protein that displays the characteristic cysteine residue periodicity of Paramecium surface antigens but does not contain central tandemly repeated sequences. Partial sequences of the two nonexpressed genes Dβ and Dγ indicate a high percentage of identity (90%–95%) with the Dα gene, suggesting that Dβ and Dγ genes are either very similar surface protein genes whose transcription is repressed trough mutual exclusion, or perhaps are pseudogenes. A region of variable DNA rearrangement was identified 1 kb upstream of the Dγ gene. This macronuclear region arises from the same micronuclear locus by alternative excision of internal eliminated sequences during macronuclear development.

Video Error Correction Using Soft-Output and Hard-Output Maximum Likelihood Decoding Applied to an H.264 Baseline Profile

Error concealment has long been identified as the last line of defense against transmission errors. Since error handling is outside the scope of video coding standards, decoders may choose to simply ignore corrupted packets or attempt to decode their content. In this paper, we present a novel joint source-channel decoding approach that can be applied to received video packets containing transmission errors. Soft-output information is combined with our novel syntax-element-level maximum likelihood decoding framework to effectively extract valid macroblocks from corrupted H.264 slices. Simulation results show that our video error correction strategy provides an average peak signal-to-noise ratio (PSNR) improvement near 2 dB compared to the error concealment approach used by the H.264 reference software, as well as an average PSNR improvement of 0.8 dB compared to state-of-the-art error concealment. The proposed method is also applicable when only hard-information is available, in which case it performs better than state-of-the-art error concealment especially in high error conditions. Finally, in our simulations, the proposed method increased the decoder computational complexity by only 5% to 20%, making it applicable for real-time applications.