A tree-based method for the rapid screening of chemical fingerprints

Background  

The fingerprint of a molecule is a bitstring based on its structure, constructed such that structurally similar molecules will have similar fingerprints. Molecular fingerprints can be used in an initial phase of drug development for identifying novel drug candidates by screening large databases for molecules with fingerprints similar to a query fingerprint.     

Recrafting the neighbor-joining method

Background  

The neighbor-joining method by Saitou and Nei is a widely used method for constructing phylogenetic trees. The formulation of the method gives rise to a canonical Θ(n ³) algorithm upon which all existing implementations are based.     

FSH-initiated differentiation of newt spermatogonia to primary spermatocytes in germ-somatic cell reaggregates cultured within a collagen matrix

We previously cultured fragments of newt testes in chemically defined media and showed that mammalian follicle-stimulating hormone (FSH) stimulates proliferation of spermatogonia as well as their differentiation into primary spermatocytes (Ji et al., 1992; Abe and Ji, 1994). Next, we indicated in cultures composed of spermatogonia and somatic cells (mainly Sertoli cells) that FSH stimulates germ cell proliferation via Sertoli cells (Maekawa et al., 1995). However, the spermatogonia did not differentiate into primary spermatocytes, but instead died. In the present study, we embedded large reaggregates of spermatogonia and somatic cells (mainly Sertoli cells) within a collagen matrix and cultured the reaggregates on a filter that floated on chemically defined media containing FSH; in this revised culture system, spermatogonia proliferated and differentiated into primary spermatocytes. The viability and percentage of germ cells differentiating into primary spermatocytes were proportional to the percentage of somatic cells in the culture, indicating that differentiation of spermatogonia into primary spermatocytes is mediated by Sertoli cells.     

Microarray-based method for combinatorial library sequence mapping and characterization

Here we describe a DNA-chip-based method for high-throughput sequence mapping. This involves competitive hybridization between short and differentially labeled fluorescent oligonucleotide probes and glass-supported PCR products. Competition between an excess of oligonucleotide probes targeting the same sequence segment improves sequence discrimination and reduces sensitivity to experimental conditions such as probe concentrations, hybridization, and washing temperatures and durations. The method was found to be particularly adapted to sequence mapping of combinatorial libraries obtained by DNA shuffling between members of a gene family. We present an application of this technique for the characterization of recombination biases in combinatorial libraries used in directed evolution.     

A relationship between seed development, Arabinogalactan-proteins (AGPs) and the AGP mediated promotion of somatic embryogenesis

Arabinogalactan-protein (AGP) epitopes are known to display developmentally regulated patterns of expression in several plant tissues. Therefore, AGPs have been suggested to play a role in plant development. Somatic embryogenesis is regulated by AGPs as well as by EP3 endochitinases. Using four different methods we have analysed the composition of AGPs in immature carrot seeds. The results obtained show that: (1) the native electrophoretic mobility of such AGPs changes during development; (2) AGP epitopes in immature seeds are developmentally regulated; (3) enzymatically released fragments of AGPs show that the composition of these molecules changes as a function of development; and (4) the biological activity of AGPs on the formation of somatic embryos changes depending on the age of the seeds. Our results suggest that degradation of maternally derived AGPs occurs after fertilization, while cellularization of the endosperm leads to synthesis of a new set of AGPs. The presence of an endochitinase cleavage site as well as the capacity to increase somatic embryogenesis only occurred in AGPs that were isolated from seeds in which the endosperm had been cellularized. Apparently, both EP3 endochitinases and somatic embryogenesis-promoting AGPs are developmentally regulated in immature carrot seeds.     

Isolation of a chromosomally engineered durum wheat line carrying the Aegilops ventricosa pchl gene for resistance to eyespot.

The chromosome 7Dv of Aegilops ventricosa (syn. Triticum ventricosum, 2n = 4x = 28, genome DvDvMvMv) carries the gene Pch1 for resistance to eyespot. This gene has previously been transferred to chromosome 7D of bread wheat, T. aestivum (2n = 6x = 42, genome AABBDD). To (1) enhance the level of resistance of bread wheat by increasing the copy number of Pch1, and (2) create eyespot-resistant triticales, meiotically stable Pch1-carrying durum lines were selected from the backcross progenies of a cross between Ae. ventricosa and T. durum cv. Creso ph1c (2n = 4x = 28, genome AABB). The Pch1 transfer, likely resulting from homoeologous recombination, was located at the distal position on the long arm of chromosome 7A. The 7A microsatellite marker Xgwm 698 was found closely linked in repulsion to the introgression in the resistant recombination lines, and the endopeptidase allele located on chromosome 7A of cv. Creso ph1c was lost.     

Attempts to induce homoeologous pairing between wheat and Agropyron cristatum genomes.

Agropyron cristatum (2n = 4x = 28, PPPP) possesses potentially valuable traits that could be used in wheat (Triticum aestivum) improvement through interspecific hybridization. Homoeologous pairing between wheat chromosomes and P chromosomes added to wheat in a set of wheat - A. cristatum addition lines was assessed. First, the Ph-suppressing effect of P chromosomes (except 7P) was analyzed. It was concluded that this system is polygenic with no major gene, and consequently, has no prospect in the transfer of alien genes from wild relatives. In a second step, the potential of the deletion ph1b of the Ph1 gene for inducing P-ABD pairing was evaluated. Allosyndetic associations between P and ABD genomes are very rare. This very low level of pairing is likely due to divergence in the repeated sequences between Agropyron and wheat genomes. Development of translocation lines using ionizing radiation seems to be a more suitable technique than homoeologous recombination to exploit the A. cristatum genome in wheat improvement.     

Estrogen-inducible and liver-specific expression of the chicken Very Low Density Apolipoprotein II gene locus in transgenic mice.

We have examined the chicken Very Low Density Apolipoprotein II (apoVLDL II) gene locus in transgenic mice. A DNA fragment composed of the transcribed region, 16 kb of 5' flanking and 400 bp of 3' flanking sequences contained all the information sufficient for estrogen-inducible, liver-specific expression of the apoVLDL II gene. The far-upstream region contains a Negative Regulating Element coinciding with a DNaseI-hypersensitive site at -11 kb. In transgenic mice, the NRE at -11 kb is used for downregulating the expression to a lower maximum level. The NRE might be used for modulating apoVLDL II gene expression, and may be involved in the rapid shut-down of the expression after hormone removal.     

The pea late nodulin gene PsNOD6 is homologous to the early nodulin genes PsENOD3/14 and is expressed after the leghaemoglobin genes

The pea late nodulin gene PsNOD6 has been cloned and sequenced. PsNOD6 is homologous to the pea early nodulin genes PsNOD3 and PsENOD14. In situ hybridization experiments showed that, like the PsENOD3 and PsENOD14 genes, the PsNOD6 gene is only expressed in the infected cell type. The PsNOD6 gene is first expressed at the transition of the pre-fixation zone II into the interzone II–III (the amyloplast-rich zone preceding the fixation zone III), whereas the early nodulin genes PsENOD3 and PsENOD14 are already induced in the pre-fixation zone II. Thus these nodulin genes encoding homologous proteins are induced at consecutive stages of nodule development.The expression of the late nodulin genes encoding leghaemoglobin precedes the expression of the late nodulin gene PsNOD6. Therefore these late nodulin genes have to be regulated by different mechanisms despite the fact they are expressed in the same cell type. This conclusion is consistent with the fact that PsNOD6 lacks one of the conserved regions occurring in the promoters of all other late nodulin genes studied.