Persistent nuclear ribosomal DNA sequence polymorphism in the Amelanchier agamic complex (Rosaceae)

Individual plants of several Amelanchier taxa contain many polymorphic nucleotide sites in the internal transcribed spacers (ITS) of nuclear ribosomal DNA (nrDNA). This polymorphism is unusual because it is not recent in origin and thus has resisted homogenization by concerted evolution. Amelanchier ITS sequence polymorphism is hypothesized to be the result of gene flow between two major North American clades resolved by phylogenetic analysis of ITS sequences. Western North American species plus A. humilis and A. sanguinea of eastern North America form one clade (A), and the remaining eastern North American Amelanchier make up clade B. Five eastern North American taxa are polymorphic at many of the nucleotide sites where clades A and B have diverged and are thought to be of hybrid origin, with A. humilis or A. sanguinea as one parent and various members of clade B as the other parent. Morphological evidence suggests that A. humilis is one of the parents of one of the polymorphic taxa, a microspecies that we refer to informally as A. "erecta." Sequences of 21 cloned copies of the ITS1- 5.8S gene-ITS2 region from one A. "erecta" individual are identical to A. humilis sequence or to the clade B consensus sequence, or they are apparent recombinants of A. humilis and clade B ITS repeats. Amelanchier "erecta" and another polymorphic taxon are suspected to be relatively old because both grow several hundred kilometers beyond the range of one of their parents. ITS sequence polymorphisms have apparently persisted in these two taxa perhaps because of polyploidy and/or agamospermy (asexual seed production), which are prevalent in the genus.      

The HOX-5 and surfeit gene clusters are linked in the proximal portion of mouse chromosome 2

Using an interspecies backcross, we have mapped the HOX-5 and surfeit (surf) gene clusters within the proximal portion of mouse chromosome 2. While the HOX-5 cluster of homeobox-containing genes has been localized to chromosome 2, bands C3-E1, by in situ hybridization, its more precise position relative to the genes and cloned markers of chromosome 2 was not known. Surfeit, a tight cluster of at least six highly conserved “housekeeping” genes, has not been previously mapped in mouse, but has been localized to human chromosome 9q, a region of the human genome with strong homology to proximal mouse chromosome 2. The data presented here place HOX-5 in the vicinity of the closely linked set of developmental mutations rachiterata, lethargic, and fidget and place surf close to the proto-oncogene Abl, near the centromere of chromosome 2.     

Defective gene expression,S phase progression,and maturation during hematopoiesis in E2F1/E2F2 mutant mice

E2F plays critical roles in cell cycle progression by regulating the expression of genes involved in nucleotide synthesis, DNA replication, and cell cycle control. We show that the combined loss of E2F1 and E2F2 in mice leads to profound cell-autonomous defects in the hematopoietic development of multiple cell lineages. E2F2 mutant mice show erythroid maturation defects that are comparable with those observed in patients with megaloblastic anemia. Importantly, hematopoietic defects observed in E2F1/E2F2 double-knockout (DKO) mice appear to result from impeded S phase progression in hematopoietic progenitor cells. During DKO B-cell maturation, differentiation beyond the large pre-BII-cell stage is defective, presumably due to failed cell cycle exit, and the cells undergo apoptosis. However, apoptosis appears to be the consequence of failed maturation, not the cause. Despite the accumulation of hematopoietic progenitor cells in S phase, the combined loss of E2F1 and E2F2 results in significantly decreased expression and activities of several E2F target genes including cyclin A2. Our results indicate specific roles for E2F1 and E2F2 in the induction of E2F target genes, which contribute to efficient expansion and maturation of hematopoietic progenitor cells. Thus, E2F1 and E2F2 play essential and redundant roles in the proper coordination of cell cycle progression with differentiation which is necessary for efficient hematopoiesis.     

Comparative modeling and analysis of microfluidic and conventional DNA microarrays

A theoretical analysis was developed to predict molecular hybridization rates for microarrays where samples flow through microfluidic channels and for conventional microarrays where samples remain stationary during hybridization. The theory was validated by using a multiplexed microfluidic microarray where eight samples were hybridized simultaneously against eight probes using 60-mer DNA strands. Mass transfer coefficients ranged over three orders of magnitude where either kinetic reaction rates or molecular diffusion rates controlled overall hybridization rates. Probes were printed using microfluidic channels and also conventional spotting techniques. Consistent with the theoretical model, the microfluidic microarray demonstrated the ability to print DNA probes in less than 1 min and to detect 10-pM target concentrations with hybridization times in less than 5 min.     

Monoclonal antibodies that recognize a polypeptide antigenic determinant shared by multiple Caenorhabditis elegans sperm-specific proteins

Four monoclonal antibodies that are directed against antigens present in sperm and absent from other worm tissues were characterized. Antibody TR20 is directed against the major sperm proteins, a family of small, abundant, cytoplasmic proteins that have been previously described (Klass, M. R., and D. Hirsh, 1981, Dev. Biol., 84:299-312; Burke, D. J., and S. Ward, 1983, J. Mol. Biol., 171:1-29). Three other antibodies, SP56, SP150, and TR11, are all directed against the same set of minor sperm polypeptides that range in size from 29 to 215 kD. More than eight different sperm polypeptides are antigenic by both immunotransfer and immunoprecipitation assays. The three antibodies are different immunoglobulin subclasses, yet they compete with each other for antigen binding so they are directed against the same antigenic determinant on the multiple sperm proteins. This antigenic determinant is sensitive to any of six different proteases, is insensitive to periodate oxidation or N-glycanase digestion, and is detectable on a polypeptide synthesized in vitro. Therefore, the antigenic determinant resides in the polypeptide chain. However, peptide fragments of the proteins are not antigenic, thus the determinant is likely to be dependent on polypeptide conformation. The antigenic determinant shared by these proteins could represent a common structural feature of importance to the localization or cellular specificity of these proteins.     

Centromere position in budding yeast: evidence for anaphase A.

Although general features of chromosome movement during the cell cycle are conserved among all eukaryotic cells, particular aspects vary between organisms. Understanding the basis for these variations should provide significant insight into the mechanism of chromosome movement. In this context, establishing the types of chromosome movement in the budding yeast Saccharomyces cerevisiae is important since the complexes that mediate chromosome movement (microtubule organizing centers, spindles, and kinetochores) appear much simpler in this organism than in many other eukaryotic cells. We have used fluorescence in situ hybridization to begin an analysis of chromosome movement in budding yeast. Our results demonstrate that the position of yeast centromeres changes as a function of the cell cycle in a manner similar to other eukaryotes. Centromeres are skewed to the side of the nucleus containing the spindle pole in G1; away from the poles in mid-M and clustered near the poles in anaphase and telophase. The change in position of the centromeres relative to the spindle poles supports the existence of anaphase A in budding yeast. In addition, an anaphase A-like activity independent of anaphase B was demonstrated by following the change in centromere position in telophase-arrested cells upon depolymerization and subsequent repolymerization of microtubules. The roles of anaphase A activity and G1 centromere positioning in the segregation of budding yeast chromosomes are discussed. The fluorescence in situ hybridization methodology and experimental strategies described in this study provide powerful new tools to analyze mutants defective in specific kinesin-like molecules, spindle components, and centromere factors, thereby elucidating the mechanism of chromosome movement.