Contribution of CAF-I to anaphase-promoting-complex-mediated mitotic chromatin assembly in Saccharomyces cerevisiae

The anaphase-promoting complex (APC) is required for mitotic progression and genomic stability. Recently, we demonstrated that the APC is also required for mitotic chromatin assembly and longevity. Here, we investigated the role the APC plays in chromatin assembly. We show that apc5(CA) mutations genetically interact with the CAF-I genes as well as ASF1, HIR1, and HIR2. When present in multiple copies, the individual CAF-I genes, CAC1, CAC2, and MSI1, suppress apc5(CA) phenotypes in a CAF-1- and Asf1p-independent manner. CAF-I and the APC functionally overlap, as cac1delta cac2delta msi1delta (caf1delta) cells expressing apc5(CA) exhibit a phenotype more severe than that of apc5(CA) or caf1delta. The Ts- phenotypes observed in apc5(CA) and apc5(CA) caf mutants may be rooted in compromised histone metabolism, as coexpression of histones H3 and H4 suppressed the Ts- defects. Synthetic genetic interactions were also observed in apc5(CA) asf1delta cells. Furthermore, increased expression of genes encoding Asf1p, Hir1p, and Hir2p suppressed the apc5(CA) Ts- defect in a CAF-I-dependent manner. Together, these results suggest the existence of a complex molecular mechanism controlling APC-dependent chromatin assembly. Our data suggest the APC functions with the individual CAF-I subunits, Asf1p, and the Hir1p and Hir2p proteins. However, Asf1p and an intact CAF-I complex are dispensable for CAF-I subunit suppression, whereas CAF-I is necessary for ASF1, HIR1, and HIR2 suppression of apc5(CA) phenotypes. We discuss the implications of our observations.     

Novel interaction between Apc5p and Rsp5p in an intracellular signaling pathway in Saccharomyces cerevisiae

The ubiquitin-targeting pathway is evolutionarily conserved and critical for many cellular functions. Recently, we discovered a role for two ubiquitin-protein ligases (E3s), Rsp5p and the Apc5p subunit of the anaphase-promoting complex (APC), in mitotic chromatin assembly in Saccharomyces cerevisiae. In the present study, we investigated whether Rsp5p and Apc5p interact in an intracellular pathway regulating chromatin remodeling. Our genetic studies strongly suggest that Rsp5p and Apc5p do interact and that Rsp5p acts upstream of Apc5p. Since E3 enzymes typically require the action of a ubiquitin-conjugating enzyme (E2), we screened E2 mutants for chromatin assembly defects, which resulted in the identification of Cdc34p and Ubc7p. Cdc34p is the E2 component of the SCF (Skp1p/Cdc53p/F-box protein). Therefore, we analyzed additional SCF mutants for chromatin assembly defects. Defective chromatin assembly extracts generated from strains harboring a mutation in the Cdc53p SCF subunit or a nondegradable SCF target, Sic1(Deltaphos), confirmed that the SCF was involved in mitotic chromatin assembly. Furthermore, we demonstrated that Ubc7p physically and genetically interacts with Rsp5p, suggesting that Ubc7p acts as an E2 for Rsp5p. However, rsp5CA and Deltaubc7 mutations had opposite genetic effects on apc5CA and cdc34-2 phenotypes. Therefore, the antagonistic interplay between Deltaubc7 and rsp5CA, with respect to cdc34-2 and apc5CA, indicates that the outcome of Rsp5p's interaction with Cdc34p and Apc5p may depend on the E2 interacting with Rsp5p.     

The complete mitochondrial genome of Alligator mississippiensis and the separation between recent archosauria (birds and crocodiles)

The complete mitochondrial genome of the alligator, Alligator mississippiensis, was sequenced. The size of the molecule is 16,642 nucleotides. Previously reported rearrangements of tRNAs in crocodile mitochondrial genomes were confirmed and, relative to mammals, no other deviations of gene order were observed. The analysis of protein-coding genes of the alligator showed an evolutionary rate that is roughly the same as in mammals. Thus, the evolutionary rate in the alligator is faster than that in birds as well as that in cold-blooded vertebrates. This contradicts hypotheses of constant body temperatures or high metabolic rate being correlated with elevated molecular evolutionary rates. It is commonly acknowledged that birds are the closest living relatives to crocodiles. Birds and crocodiles represent the only archosaurian survivors of the mass extinction at the Cretaceous/Tertiary boundary. On the basis of mitochondrial protein- coding genes, the Haemothermia hypothesis, which defines birds and mammals as sister groups and thus challenges the traditional view, could be rejected. Maximum-likelihood branch length data of amino acid sequences suggest that the divergence between the avian and crocodilian lineages took place at approximately equal to 254 MYA.      

Cytochrome b nucleotide sequences and the identification of five primary lineages of extant cetaceans

Relationships among and within baleen and toothed whales were examined using the complete sequence of the mitochondrial cytochrome b gene. Based on parsimony analyses of conservative nucleotide substitutions, five primary evolutionary lineages of extant cetaceans were identified, one represented by baleen whales (Mysticeti) and four represented by odontocetes (toothed whales). Based on the most comprehensive representation of taxa, both cetaceans and artiodactyls, the most parsimonious relationship among the five lineages is (Mysticeti, Odontoceti (Platanistoidea (Physeteroidea (Ziphioidea (Delphinida))))). This relationship, however, is labile and sensitive to ingroup representation and the choice of outgroup. The short nodes among the five cetacean lineages suggest that the divergence among these lineages occurred over a narrow time period, a finding consistent with the limited fossil evidence that indicates a major cetacean radiation 30-34 Mya. The level of divergence among the five cetacean lineages, and that seen between cetaceans and artiodactyls, suggests that cetaceans and artiodactyls had a common ancestor approximately 60 Mya.      

X-linked cleft palate: the gene is localized between polymorphic DNA markers DXYS12 and DXS17

Summary The gene involved in an X-linked form of cleft palate has been finely mapped using 14 restriction fragment length polymorphic (RFLP) markers that cover the long arm of the X chromosome. By the combination of deletion mapping and linkage analysis, the gene has been localized between the anonymous DNA markers DXYS12 on the proximal side, and DXS17 distally.     

TFPI1 Mediates Resistance to Doxorubicin in Breast Cancer Cells by Inducing a Hypoxic-Like Response

Thrombin and hypoxia are important players in breast cancer progression. Breast cancers often develop drug resistance, but mechanisms linking thrombin and hypoxia to drug resistance remain unresolved. Our studies using Doxorubicin (DOX) resistant MCF7 breast cancer cells reveals a mechanism linking DOX exposure with hypoxic induction of DOX resistance. Global expression changes between parental and DOX resistant MCF7 cells were examined. Westerns, Northerns and immunocytochemistry were used to validate drug resistance and differentially expressed genes. A cluster of genes involved in the anticoagulation pathway, with Tissue Factor Pathway Inhibitor 1 (TFPI1) the top hit, was identified. Plasmids overexpressing TFPI1 were utilized, and 1% O₂ was used to test the effects of hypoxia on drug resistance. Lastly, microarray datasets from patients with drug resistant breast tumors were interrogated for TFPI1 expression levels. TFPI1 protein levels were found elevated in 3 additional DOX resistant cells lines, from humans and rats, indicating evolutionarily conservation of the effect. Elevated TFPI1 in DOX resistant cells was active, as thrombin protein levels were coincidentally low. We observed elevated HIF1α protein in DOX resistant cells, and in cells with forced expression of TFPI1, suggesting TFPI1 induces HIF1α. TFPI1 also induced c-MYC, c-SRC, and HDAC2 protein, as well as DOX resistance in parental cells. Growth of cells in 1% O₂ induced elevated HIF1α, BCRP and MDR-1 protein, and these cells were resistant to DOX. Our in vitro results were consistent with in vivo patient datasets, as tumors harboring increased BCRP and MDR-1 expression also had increased TFPI1 expression. Our observations are clinically relevant indicating that DOX treatment induces an anticoagulation cascade, leading to inhibition of thrombin and the expression of HIF1α. This in turn activates a pathway leading to drug resistance.     

A regression analysis of q’eqchi’ Maya medicinal plants from southern Belize

A previous study provided a general quantitative analysis of 169 collected medicinal plants used by the Q’eqchi’ Maya healers of southern Belize. This paper is focused on a statistical analysis of this ethnobotanical information using the method developed by Moerman (1991). The residual values obtained from the regression analysis of the Q’eqchi’ medicinal plant species versus the species listed in the checklist of the vascular plants of Belize (Balick, Nee, and Atha, 2001) placed the Piperaceae, the Rubiaceae, and the Asteraceae in the first three ranks, and the Poaceae, the Cyperaceae, and the Orchidaceae in the last three ranks. The results were compared with three northern temperate regions (Kashmir, Korea, and North America) and three southern neotropical regions (Chiapas, Ecuador, and Veracruz). The coefficients of correlation between the checklist of vascular plants of Belize and the other six floras showed, as expected, high values for regions with similar climatic type. Thus, high correlations were determined between the tropical vegetation of Belize and those of Chiapas, Ecuador, and Veracruz. The coefficients were lower with the three temperate floras but still quite high. The same analysis was done with the medicinal plants only and led to much lower coefficients, but once again, the higher results were obtained for Chiapas and Veracruz. In this case, the last rank for Ecuador demonstrated that the selection of plants in traditional medicine by the indigenous people is a complex phenomenon which depends not only on the composition of the flora but also on culture-specific factors.

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Analyse par Regression des Plantes Médicinales des Mayas Q’eqchi'’ du Sud du Belize

Résumé  Une précédente étude a fourni une analyse quantitative générale de 169 plantes médicinales utilisées par les guérisseurs Maya Q’eqchi’ du sud du Belize. Ce document se concentre sur l’analyse statistique des informations ethnobotaniques selon la méthode développée par Moerman (1991). Les valeurs résiduelles obtenues à partir des analyses de régression des plantes médicinales Q’eqchi’ vis-à-vis des espèces mentionnées dans la liste des plantes vasculaires du Belize (Balick, Nee, et Atha, 2001) ont placé les Piperacées, les Rubiacées et les Asteracées aux trois premières places, et les Poacées, les Cyperacées et les Orchidacées aux trois dernières places. Les résultats ont été comparés avec ceux de trois régions tempérées du nord (Cachemire, Corée, et Amérique du Nord) et de trois régions néotropicales du sud (Chiapas, équateur, et Veracruz). Les coefficients de corrélation entre les plantes listées dans le manuel des plantes vasculaires du Belize et les six autres flores ont montré comme attendu de hautes valeurs pour les régions possédant un type climatique similaire. Ainsi, une haute corrélation a été démontrée entre la végétation tropicale du Belize et celles du Chiapas, de l’équateur, et du Veracruz. Les coefficients étaient plus bas avec les trois régions tempérées mais tout de même passablement élevés. La même analyse a été effectuée avec les plantes médicinales et a mené à des coefficients beaucoup plus bas, mais encore une fois, les résultats les plus élevés ont été obtenus pour le Chiapas et le Veracruz. Dans ce cas, la dernière position de l’équateur a souligné que la sélection des plantes par les indigènes dans la médecine traditionnelle est un phénomène complexe qui dépend non seulement de la composition de la flore mais aussi de facteurs spécifiques à la culture.

     

Early penguin fossils, plus mitochondrial genomes, calibrate avian evolution

Testing models of macroevolution, and especially the sufficiency of microevolutionary processes, requires good collaboration between molecular biologists and paleontologists. We report such a test for events around the Late Cretaceous by describing the earliest penguin fossils, analyzing complete mitochondrial genomes from an albatross, a petrel, and a loon, and describe the gradual decline of pterosaurs at the same time modern birds radiate. The penguin fossils comprise four naturally associated skeletons from the New Zealand Waipara Greensand, a Paleocene (early Tertiary) formation just above a well-known Cretaceous/Tertiary boundary site. The fossils, in a new genus (Waimanu), provide a lower estimate of 61-62 Ma for the divergence between penguins and other birds and thus establish a reliable calibration point for avian evolution. Combining fossil calibration points, DNA sequences, maximum likelihood, and Bayesian analysis, the penguin calibrations imply a radiation of modern (crown group) birds in the Late Cretaceous. This includes a conservative estimate that modern sea and shorebird lineages diverged at least by the Late Cretaceous about 74 +/- 3 Ma (Campanian). It is clear that modern birds from at least the latest Cretaceous lived at the same time as archaic birds including Hesperornis, Ichthyornis, and the diverse Enantiornithiformes. Pterosaurs, which also coexisted with early crown birds, show notable changes through the Late Cretaceous. There was a decrease in taxonomic diversity, and small- to medium-sized species disappeared well before the end of the Cretaceous. A simple reading of the fossil record might suggest competitive interactions with birds, but much more needs to be understood about pterosaur life histories. Additional fossils and molecular data are still required to help understand the role of biotic interactions in the evolution of Late Cretaceous birds and thus to test that the mechanisms of microevolution are sufficient to explain macroevolution.