Characterization of RFLP probe sequences for gene discovery and SSR development in Sorghum bicolor (L.) Moench

In this study, we collected and analyzed DNA sequence data for 789 previously mapped RFLP probes from Sorghum bicolor (L.) Moench. DNA sequences, comprising 894 non-redundant contigs and end sequences, were searched against three GenBank databases, nucleotide (nt), protein (nr) and EST (dbEST), using BLAST algorithms. Matching ESTs were also searched against nt and nr. Translated DNA sequences were then searched against the conserved domain database (CDD) to determine if functional domains/motifs were congruent with the proteins identified in previous searches. More than half (500/894 or 56%) of the query sequences had significant matches in at least one of the GenBank searches. Overall, proteins identified for 148 sequences (17%) were consistent among all searches, of which 66 sequences (7%) contained congruent coding domains. The RFLP probe sequences were also evaluated for the presence of simple sequence repeats (SSRs) and 60 SSRs were developed and assayed in an array of sorghum germplasm comprising inbreds, landraces and wild relatives. Overall, these SSR loci had lower levels of polymorphism ( D = 0.46, averaged over 51 polymorphic loci) compared with sorghum SSRs that were isolated by library hybridization screens ( D = 0.69, averaged over 38 polymorphic loci). This result was probably due to the relatively small proportion of di-nucleotide repeat-containing markers (42% of the total SSR loci) obtained from the DNA sequence data. These di-nucleotide markers also contained shorter repeat motifs than those isolated from genomic libraries. Based on BLAST results, 24 SSRs (40%) were located within, or near, previously annotated or hypothetical genes. We determined the location of 19 of these SSRs relative to putative coding regions. In general, SSRs located in coding regions were less polymorphic ( D = 0.07, averaged over three loci) than those from gene flanking regions, UTRs and introns ( D = 0.49, averaged over 16 loci). The sequence information and SSR loci generated through this study will be valuable for application to sorghum genetics and improvement, including gene discovery, marker-assisted selection, diversity and pedigree analyses, comparative mapping and evolutionary genetic studies.     

Many gene and domain families have convergent fates following independent whole-genome duplication events in Arabidopsis, Oryza, Saccharomyces and Tetraodon

Genome duplication is potentially a good source of new genes, but such genes take time to evolve. We have found a group of "duplication-resistant" genes, which have undergone convergent restoration to singleton status following several independent genome duplications. Restoration of duplication-resistant genes to singleton status could be important to long-term survival of a polyploid lineage. Angiosperms show more frequent polyploidization and a higher degree of duplicate gene preservation than other paleopolyploids, making them well-suited to further study of duplication-resistant genes.     

Finding and comparing syntenic regions among Arabidopsis and the outgroups papaya, poplar, and grape: CoGe with rosids

In addition to the genomes of Arabidopsis (Arabidopsis thaliana) and poplar (Populus trichocarpa), two near-complete rosid genome sequences, grape (Vitis vinifera) and papaya (Carica papaya), have been recently released. The phylogenetic relationship among these four genomes and the placement of their three independent, fractionated tetraploidies sum to a powerful comparative genomic system. CoGe, a platform of multiple whole or near-complete genome sequences, provides an integrative Web-based system to find and align syntenic chromosomal regions and visualize the output in an intuitive and interactive manner. CoGe has been customized to specifically support comparisons among the rosids. Crucial facts and definitions are presented to clearly describe the sorts of biological questions that might be answered in part using CoGe, including patterns of DNA conservation, accuracy of annotation, transposability of individual genes, subfunctionalization and/or fractionation of syntenic gene sets, and conserved noncoding sequence content. This précis of an online tutorial, CoGe with Rosids (http://tinyurl.com/4a23pk), presents sample results graphically.     

The amino-terminal domain of the vacuolar proton-translocating ATPase a subunit controls targeting and in vivo dissociation, and the carboxyl-terminal domain affects coupling of proton transport and ATP hydrolysis

The 100-kDa "a" subunit of the vacuolar proton-translocating ATPase (V-ATPase) is encoded by two genes in yeast, VPH1 and STV1. The Vph1p-containing complex localizes to the vacuole, whereas the Stv1p-containing complex resides in some other intracellular compartment, suggesting that the a subunit contains information necessary for the correct targeting of the V-ATPase. We show that Stv1p localizes to a late Golgi compartment at steady state and cycles continuously via a prevacuolar endosome back to the Golgi. V-ATPase complexes containing Vph1p and Stv1p also differ in their assembly properties, coupling of proton transport to ATP hydrolysis, and dissociation in response to glucose depletion. To identify the regions of the a subunit that specify these different properties, chimeras were constructed containing the cytosolic amino-terminal domain of one isoform and the integral membrane, carboxyl-terminal domain from the other isoform. Like the Stv1p-containing complex, the V-ATPase complex containing the chimera with the amino-terminal domain of Stv1p localized to the Golgi and the complex did not dissociate in response to glucose depletion. Like the Vph1p-containing complex, the V-ATPase complex containing the chimera with the amino-terminal domain of Vph1p localized to the vacuole and the complex exhibited normal dissociation upon glucose withdrawal. Interestingly, the V-ATPase complex containing the chimera with the carboxyl-terminal domain of Vph1p exhibited a higher coupling of proton transport to ATP hydrolysis than the chimera containing the carboxyl-terminal domain of Stv1p. Our results suggest that whereas targeting and in vivo dissociation are controlled by sequences located in the amino-terminal domains of the subunit a isoforms, coupling efficiency is controlled by the carboxyl-terminal region.     

Modified miniprep method for the rapid recovery of episomes from transfected breast epithelial cells

Episomal vectors such as pCEP4 are useful in expression cloning because they can replicate in both prokaryotes and eukaryotic cells. We have found a rapid and efficient means of extracting them from transfected MCF-10A nonmalignant human breast epithelial cells. We show that a plasmid miniprep protocol, modified by the addition of an extraction that eliminates a DNase activity, can consistently harvest pCEP4 episomes from the transfected cells (516 +/- 112 pg/harvest, mean +/- standard deviation; n = 11). The quality of the episomal DNA obtained in this manner was verified by PCR, Southern blot and the retransformation of Escherichia coli. This simple method enables the efficient recovery of episomes and is applicable in the expression cloning of potential oncogenes using host MCF-10A cells.     

Role of antioxidants in the survival of normal and vitiliginous avian melanocytes.

Mutant feather melanocytes from Barred Plymouth Rock (BPR) and White Leghorn (WL) chickens are currently being used as avian models of vitiligo. Feather melanocytes in BPR and WL chickens die prematurely in vivo due to low (50-66%) antioxidant glutathione and superoxide dismutase levels when compared to the wild type Jungle Fowl (JF) melanocytes. Excess superoxide anions, generated by xanthine:xanthine oxidase (X:XO), caused a 15-20% increase in mortality after 1 and 2 hrs. in all three genotypes of in vitro melanocytes as compared to control values that received no X:XO. Overall, the JF wild type melanocytes had the lowest mortality rate, WL melanocytes had the highest mortality rate and the BPR melanocytes had an intermediate mortality rate. Superoxide anion and hydroxyl radical production in the WL feather were double the production in the JF wild type feather. The production of reactive oxygen species in BPR was intermediate to the other two genotypes. In an effort to mimic the low antioxidant levels of the BPR and WL feathers in the JF feather, JF in vitro feather melanocytes were treated with buthionine sulfoximine (BSO), a glutathione synthesis inhibitor. With BSO added to the medium, the JF mortality rates increased by 20-25%, reaching the mortality levels of the mutant BPR melanocytes. The addition of iron to the JF melanocyte X:XO medium increased their mortality rate by 20%, probably via the Fenton reaction. Thus, antioxidants play an extremely important role in both the viability of normal avian melanocytes and the premature death of the vitiliginous avian melanocytes. A working hypothesis, supported in part by the current results, is that the premature death of the mutant melanocytes could be precipitated in the poorly vascularized feather by low antioxidant protection due to both low turnover of tissue fluids which contain SOD and to genetically determined low levels of internal antioxidant protection in these melanocytes. This same mechanistic hypothesis could apply as "a" cause of premature melanocyte cell death in human vitiligo wherein the vitiliginous melanocytes may have a genetic defect in their antioxidant protection system and blood flow to an area may be restricted.     

Avian fissura prima: differential accumulation of extracellular matrix at a fold

Extracellular matrix components that flank the fissura prima, a primary surface infolding of the cerebellum in birds and mammals, were examined in the embryonic chick using light and transmission electron microscopy. Cerebella dissected from Day 10 embryos were perfused with a paraformaldehyde-glutaraldehyde-tannic acid primary fixative and sectioned in the sagittal plane through the mid-vermis. Ultrastructural analysis revealed a distinct, continuous basal lamina separating the organ parenchyma (epithelia) from pia mater (mesenchyme) at the fissure surface (arbitrarily labeled; fissure floor, folia wall, and folia apex). The basal lamina was significantly thicker (P < 0.001) at the fissure floor compared to that found at the folia wall, which was significantly thicker (P < 0.001) than that observed at the folia apex. Folds in the basal lamina were observed exclusively at the fissure floor. Surface-associated collagen fibrils were distributed in an aligned, relatively dense manner at the fissure floor, compared with fibrils observed in various orientations and widely separated or absent at the folia wall and folia apex. Metachromasia was more pronounced in the fissure floor than in either the folia wall or folia apex in methylene blue-stained tissue sections. Together, the thicker, folded basal lamina and densely aligned collagen fibrils at the fissure floor provide a chemical rationale for this color change. These findings suggest that the differential accumulation of extracellular matrix at the fissura prima is positioned to play a structural and/or biochemical role in the maintenance of this fold.