Biosynthesis and analysis of 3Z-nonenal

Nonenal synthesis from 9(S)-hydroperoxy-linoleic acid by hydroperoxide-lyase-catalysed cleavage was followed by a precise headspace analysis with GC-MS. Enzyme activity was extracted from plant leaves and fruits. 3Z-nonenal was the main product and a small amount of 2E-isomer was detected since the reaction-analysis procedure used avoided supplementary volatile fraction transformation. The substrate transformation and volatile purge were carried out successively and without delay or any contact with air.     

A Whole-Genome Radiation Hybrid Map of the Dog Genome

A whole genome radiation hybrid (RH) map of the canine genome was constructed by typing 400 markers, including 218 genes and 182 microsatellites, on a panel of 126 radiation hybrid cell lines. Fifty-seven RH groups have been determined with lod scores greater than 6, and 180 framework landmarks were ordered with odds greater than 1000:1. Average spacing between adjacent markers is 23 cR₅₀₀₀, an estimated physical distance of 3.8 Mb. Fourteen groups have been assigned to 9 of the canine chromosomes, and a comparison of RH and genetic groups allowed the successful bridging of both types of data on one map composed of 31 RH and 13 syntenic RH groups. Comparison of canine, human, mouse, and pig maps underlined regions of conserved synteny. This integrated map, covering an estimated 80% of the dog genome, should prove a powerful tool for localizing and identifiying genes implicated in pathological and phenotypical traits.     

Traced orthologous amplified sequence tags (TOASTs) and mammalian comparative maps

Progress on mammalian comparative maps could be significantly accelerated by developing reagents defining orthologous landmarks in the genome of many mammalian species. Using the large databases of gene sequences, we designed 225 orthologous gene-specific primer pairs corresponding to 146 functional genes. Of these 225 primer pairs, 155 (68.9%), 182 (80.9%), 126 (56.0%), and 82 (36.4%) produced a single PCR product when tested against human, pig, dog, and hamster genomic DNA, respectively. In addition to the general rules of primer designing, particular factors must be taken into consideration when choosing gene-specific universal primers—for instance, preference for single-exon regions or highly conserved segments among species, avoidance of GC-rich regions. Sequencing all the canine PCR products traced by these primers demonstrated that of 123 traced canine fragments with readable and reliable sequences, 121 (98.4%) were found to match the GenBank orthologous sequences used for designing the primers, after a BLAST search. Comparative characterization of PCR fragments among human, pig, dog, and hamster revealed that the length of a single exon was much conserved among species, with few exceptions. As the fragments were traced with amplification by orthologous gene-specific primers, we suggest they be termed Traced Orthologous Amplified Sequence Tags (TOASTs). Received: 22 December 1997 / Accepted: 16 March 1998     

Base specificity of mismatch repair in Streptococcus pneumoniae

DNA sequence analysis was undertaken to investigate the structural basis of mutations showing different integration efficiencies in Streptococcus pneumoniae. Wild type, mutant and revertant sequences at two sites in the amiA locus were determined. It appears that markers which transform efficiently or inefficiently can result from single base pair changes. A low efficiency (LE) marker corresponds to a C:G to T:A change and a high efficiency (HE) marker to a G:C to T:A change. In the latter case, two mismatches, G/A and T/C, can exist at the heteroduplex stage in transformation; only T/C appears to be recognized by the hex system which controls transforming efficiencies in pneumococcus. Each of the recognized mismatches, T/G and C/A, which result from transitional change, and T/C appears to involve at least one pyrimidine. It is proposed that the mismatch repair system of S. pneumoniae is directed against mismatched pyrimidines. DNA sequence analysis also reveals that short deletions (33 or 34 bases long) behave as very high efficiency markers, confirming that deletions are not recognized by the hex system.     

Sequence determination of the 3'' terminal T1 oligonucleotide of 18S ribosomal RNA.

We have reexamined the primary structure of the 3' terminal oligonucleotide of 18S RNA from chicken fibroblasts and have shown, contrary to previously published results that this extremity G-A-U-C-A-U-U-AOH is identical to that of the rabbit, drosophila and bombyx. Furthermore the electrophoretic mobility and composition of the 3' terminal oligonucleotides of 18S RNA from rat and human cells are similar to that of other RNAs and show that the identity of structure for this region of 18S RNA extends to include all tested species between yeast and man. This finding reveals a marked degree of evolutionary constraint on the structure of this region.     

The carboxy-terminal extension of the collagen binding domain of fibronectin mediates interaction with a 165 kDa membrane protein involved in odontoblast differentiation

Terminal differentiation of the odontoblast is characterized by an elongation and a polarization of the cell. The change in the cell shape and the reorganization of the cytoplasm involve the microfilament system. An immunological approach has previously implicated a transmembrane interaction between fibronectin and vinculin in the control of odontoblast differentiation. A 165 kDa protein localized on the cell-surface of odontoblasts mediated this interaction. In order to define the nature of the interaction of the 165 kDa protein with fibronectin, peptides were prepared by proteolytic cleavage of fibronectin with alpha-chymotrypsin. The results indicate that the 165 kDa protein interacted with a 62 kDa peptide located towards the amino-terminal extremity of fibronectin, but not with a 47 kDa related fragment. Both these 62 kDa and 47 kDa peptides included the collagen-binding domain and were retarded on a heparin-Ultrogel column. Microsequences demonstrated that the 62 kDa and 47 kDa fragments had the same amino-terminal extremity and that the larger fragment was extended in the carboxy-terminal direction. This carboxy-terminal extension of the collagen binding domain of fibronectin is implicated in the interaction of this molecule with the 165 kDa protein. On the other hand, odontoblasts differentiated normally when tooth germs were cultured in the presence of GRGDS synthetic peptide, suggesting that RGD-dependent integrins were not involved in odontoblast differentiation. Staining of dental mesenchymal cells in primary culture and of differentiated odontoblasts in situ with antibodies directed against the beta 1-subunit of integrins confirmed previous observations and showed that although beta 1 integrins are involved in the attachment of cultured dental cells, they are not implicated in the process of odontoblast differentiation.     

The Saccharomyces cerevisiae protein YJR043C (Pol32) interacts with the catalytic subunit of DNA polymerase α and is required for cell cycle progression in G2?/?M

We have analysed the YJR043c gene of Saccharomyces cerevisiae, previously identified by systematic sequencing. The deletion mutant (yjr043cΔ) shows slow growth at low temperature (15° C), while at 30° C and 37° C the growth rate of mutant cells is only moderately affected. At permissive and nonpermissive temperatures, mutant cells were larger and showed a high proportion of large-budded cells with a single duplicated nucleus at or beyond the bud neck and a short spindle. This phenotype was even more striking at low temperature, the mutant cells becoming dumbbell shaped. All these phenotypes suggest a role for YJR043C in cell cycle progression in G2/M phase. In two-hybrid assays, the YJR043c gene product specifically interacted with Poll, the catalytic subunit of DNA polymerase α. The pol1-1 /yjr043cΔ double mutant showed a more severe growth defect than the pol1-1 single mutant at permissive temperature. Centromeric plasmid loss rate elevated in yjr043cΔ. Analysis of the sequence upstream of the YJR043c ORF revealed the presence of an MluI motif (ACGCGT), a sequence associated with many genes involved in DNA replication in budding yeast. The cell cycle phenotype of the yjr043cΔ mutant, the evidence for genetic interaction with Pol1, the presence of an MluI motif upstream and the elevated rate of CEN plasmid loss in mutants all support a function for YJR043C in DNA replication. Received: 22 July 1998 / Accepted: 22 September 1998     

Recombination every day: abundant recombination in a virus during a single multi-cellular host infection

Viral recombination can dramatically impact evolution and epidemiology. In viruses, the recombination rate depends on the frequency of genetic exchange between different viral genomes within an infected host cell and on the frequency at which such co-infections occur. While the recombination rate has been recently evaluated in experimentally co-infected cell cultures for several viruses, direct quantification at the most biologically significant level, that of a host infection, is still lacking. This study fills this gap using the cauliflower mosaic virus as a model. We distributed four neutral markers along the viral genome, and co-inoculated host plants with marker-containing and wild-type viruses. The frequency of recombinant genomes was evaluated 21 d post-inoculation. On average, over 50% of viral genomes recovered after a single host infection were recombinants, clearly indicating that recombination is very frequent in this virus. Estimates of the recombination rate show that all regions of the genome are equally affected by this process. Assuming that ten viral replication cycles occurred during our experiment—based on data on the timing of coat protein detection—the per base and replication cycle recombination rate was on the order of 2 × 10⁻⁵ to 4 × 10⁻⁵. This first determination of a virus recombination rate during a single multi-cellular host infection indicates that recombination is very frequent in the everyday life of this virus.     

High-Resolution Physical Map of the pSymb Megaplasmid and Comparison of the Three Replicons of Sinorhizobium melilotiStrain 1021

A high-resolution physical map of the larger megaplasmid (pSymb) of Sinorhizobium meliloti strain 1021 has been constructed by using BAC libraries and an original two-step PCR screening method. This method, previously used to map both the chromosome and the smaller megaplasmid (pSyma), allowed us to position over the genome a total of 842 markers with an average density of one marker every 8.3 kb. In addition, we used BLASTX and PRODOM analysis to predict a function for a number of STSs. This work led to the discovery of several interesting loci and to a comparison of the genetic information carried by each replicon. The two main results emerging from this study are (i) a biased distribution of housekeeping genes, mainly detected on chromosome, and (ii) the presence of an unexpected number of transporters, mainly belonging to the ABC superfamily. These are broadly distributed across the whole genome, but particularly found on pSymb. Received: 25 January 2000 / Accepted: 28 February 2000