Publications by authors named "Christopher S Campbell"

24 Publications

  • Page 1 of 1

Phylogenetic relationships and chloroplast capture in the Amelanchier-Malacomeles-Peraphyllum clade (Maleae, Rosaceae): Evidence from chloroplast genome and nuclear ribosomal DNA data using genome skimming.

Mol Phylogenet Evol 2020 06 2;147:106784. Epub 2020 Mar 2.

Department of Botany, National Museum of Natural History, Smithsonian Institution, PO Box 37012, Washington, DC 20013-7012, USA. Electronic address:

The Amelanchier-Malacomeles-Peraphyllum (AMP) clade consists of ca. 26 species distributed in North and Central America, Europe, Asia, and northwestern Africa. While molecular and morphological data strongly support this clade, relationships of its genera are uncertain. Support for the monophyly of Amelanchier and for the phylogenetic positions of Malacomeles and Peraphyllum has varied between studies. Our goals were to reconstruct a robust phylogeny of the AMP clade in the framework of Maleae and clarify the phylogenetic placements of Malacomeles and Peraphyllum. This study employs sequences of the whole plastome and nuclear ribosomal DNA (nrDNA) repeats assembled using genome skimming with 131 samples representing 115 species in 31 genera of Rosaceae, especially Maleae. Maximum likelihood (ML) and Bayesian analysis (BI) of whole plastome datasets strongly supported Amelanchier as not monophyletic, with Peraphyllum sister to eastern North American Amelanchier and Malacomeles sister to the western North American-Eurasian Amelanchier. In contrast, nrDNA recovered the monophyly of Amelanchier, with Peraphyllum sister to Amelanchier and Malacomeles sister to the Amelanchier-Peraphyllum clade. The strong topological conflicts between plastome and nrDNA phylogenies of Peraphyllum and of Malacomeles are best explained by ancient chloroplast capture that occurred in SW North America.
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http://dx.doi.org/10.1016/j.ympev.2020.106784DOI Listing
June 2020

Genetic interactions between specific chromosome copy number alterations dictate complex aneuploidy patterns.

Genes Dev 2018 12 21;32(23-24):1485-1498. Epub 2018 Nov 21.

Department of Chromosome Biology, Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter, Vienna 1030, Austria.

Cells that contain an abnormal number of chromosomes are called aneuploid. High rates of aneuploidy in cancer are correlated with an increased frequency of chromosome missegregation, termed chromosomal instability (CIN). Both high levels of aneuploidy and CIN are associated with cancers that are resistant to treatment. Although aneuploidy and CIN are typically detrimental to cell growth, they can aid in adaptation to selective pressures. Here, we induced extremely high rates of chromosome missegregation in yeast to determine how cells adapt to CIN over time. We found that adaptation to CIN occurs initially through many different individual chromosomal aneuploidies. Interestingly, the adapted yeast strains acquire complex karyotypes with specific subsets of the beneficial aneuploid chromosomes. These complex aneuploidy patterns are governed by synthetic genetic interactions between individual chromosomal abnormalities, which we refer to as chromosome copy number interactions (CCNIs). Given enough time, distinct karyotypic patterns in separate yeast populations converge on a refined complex aneuploid state. Surprisingly, some chromosomal aneuploidies that provided an advantage early on in adaptation are eventually lost due to negative CCNIs with even more beneficial aneuploid chromosome combinations. Together, our results show how cells adapt by obtaining specific complex aneuploid karyotypes in the presence of CIN.
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http://dx.doi.org/10.1101/gad.319400.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6295164PMC
December 2018

An overview of extant conifer evolution from the perspective of the fossil record.

Am J Bot 2018 09 29;105(9):1531-1544. Epub 2018 Aug 29.

CSIRO National Research Collections Australia, Australian National Herbarium, Canberra, ACT, 2601, Australia.

Premise Of The Study: Conifers are an important living seed plant lineage with an extensive fossil record spanning more than 300 million years. The group therefore provides an excellent opportunity to explore congruence and conflict between dated molecular phylogenies and the fossil record.

Methods: We surveyed the current state of knowledge in conifer phylogenetics to present a new time-calibrated molecular tree that samples ~90% of extant species diversity. We compared phylogenetic relationships and estimated divergence ages in this new phylogeny with the paleobotanical record, focusing on clades that are species-rich and well known from fossils.

Key Results: Molecular topologies and estimated divergence ages largely agree with the fossil record in Cupressaceae, conflict with it in Araucariaceae, and are ambiguous in Pinaceae and Podocarpaceae. Molecular phylogenies provide insights into some fundamental questions in conifer evolution, such as the origin of their seed cones, but using them to reconstruct the evolutionary history of specific traits can be challenging.

Conclusions: Molecular phylogenies are useful for answering deep questions in conifer evolution if they depend on understanding relationships among extant lineages. Because of extinction, however, molecular datasets poorly sample diversity from periods much earlier than the Late Cretaceous. This fundamentally limits their utility for understanding deep patterns of character evolution and resolving the overall pattern of conifer phylogeny.
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http://dx.doi.org/10.1002/ajb2.1143DOI Listing
September 2018

Cdc73 suppresses genome instability by mediating telomere homeostasis.

PLoS Genet 2018 01 10;14(1):e1007170. Epub 2018 Jan 10.

Ludwig Institute for Cancer Research, San Diego Branch, San Diego, California, United States of America.

Defects in the genes encoding the Paf1 complex can cause increased genome instability. Loss of Paf1, Cdc73, and Ctr9, but not Rtf1 or Leo1, caused increased accumulation of gross chromosomal rearrangements (GCRs). Combining the cdc73Δ mutation with individual deletions of 43 other genes, including TEL1 and YKU80, which are involved in telomere maintenance, resulted in synergistic increases in GCR rates. Whole genome sequence analysis of GCRs indicated that there were reduced relative rates of GCRs mediated by de novo telomere additions and increased rates of translocations and inverted duplications in cdc73Δ single and double mutants. Analysis of telomere lengths and telomeric gene silencing in strains containing different combinations of cdc73Δ, tel1Δ and yku80Δ mutations suggested that combinations of these mutations caused increased defects in telomere maintenance. A deletion analysis of Cdc73 revealed that a central 105 amino acid region was necessary and sufficient for suppressing the defects observed in cdc73Δ strains; this region was required for the binding of Cdc73 to the Paf1 complex through Ctr9 and for nuclear localization of Cdc73. Taken together, these data suggest that the increased GCR rate of cdc73Δ single and double mutants is due to partial telomere dysfunction and that Ctr9 and Paf1 play a central role in the Paf1 complex potentially by scaffolding the Paf1 complex subunits or by mediating recruitment of the Paf1 complex to the different processes it functions in.
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http://dx.doi.org/10.1371/journal.pgen.1007170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5779705PMC
January 2018

An engineered minimal chromosomal passenger complex reveals a role for INCENP/Sli15 spindle association in chromosome biorientation.

J Cell Biol 2017 04 17;216(4):911-923. Epub 2017 Mar 17.

Department of Chromosome Biology, Max F. Perutz Laboratories, University of Vienna, 1030 Vienna, Austria

The four-subunit chromosomal passenger complex (CPC), whose enzymatic subunit is Aurora B kinase, promotes chromosome biorientation by detaching incorrect kinetochore-microtubule attachments. In this study, we use a combination of truncations and artificial dimerization in budding yeast to define the minimal CPC elements essential for its biorientation function. We engineered a minimal CPC comprised of the dimerized last third of the kinase-activating Sli15/INCENP scaffold and the catalytic subunit Ipl1/Aurora B. Although native Sli15 is not oligomeric, artificial dimerization suppressed the biorientation defect and lethality associated with deletion of a majority of its microtubule-binding domain. Dimerization did not act through a physical clustering-based kinase activation mechanism but instead promoted spindle association, likely via a putative helical domain in Sli15 that is essential even when dimerized and is required to target kinetochore substrates. Based on the engineering and characterization of a minimal CPC, we suggest that spindle association is important for active Ipl1/Aurora B complexes to preferentially destabilize misattached kinetochores.
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http://dx.doi.org/10.1083/jcb.201609123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5379952PMC
April 2017

Understanding diploid diversity: A first step in unraveling polyploid, apomictic complexity in Amelanchier.

Am J Bot 2015 Dec 7;102(12):2041-57. Epub 2015 Dec 7.

School of Biology and Ecology, University of Maine, Orono, Maine 04469, USA.

Premise Of The Study: Delimitation of Amelanchier species is difficult because of polyploidy and gametophytic apomixis. A first step in unraveling this species problem is understanding the diversity of the diploids that contributed genomes to polyploid apomicts. This research helps clarify challenging species-delimitation problems attending polyploid, apomictic complexity.

Methods: We sampled 431 diploid accessions from 13 species, of which 10 are North American and three are Old World. Quantitative morphological analyses tested the null hypothesis of no discrete groups. Using three to nine diploid accessions per species, we constructed phylogenies with DNA sequences from ETS, ITS, the second intron of LEAFY, and chloroplast regions rpoB-trnC, rpl16, trnD-trnT, and ycf6-psbM.

Key Results: Most Amelanchier diploid taxa are morphologically and ecogeographically distinct and genetically exclusive lineages. They rarely hybridize with one another. Nuclear and chloroplast DNA sequences almost completely resolve the Amelanchier phylogeny. The backbone is the mostly western North American clade A, eastern North American clade B, and Old World clade O. DNA sequences and morphology support clades A and O as sister taxa. Despite extensive paralogy, our LEAFY data are phylogenetically informative and identify a clade (T) of three arborescent taxa within clade B.

Conclusions: Amelanchier diploids differ strikingly from polyploid apomicts, in that hybridization among them is rare, and they form taxa that would qualify as species by most species concepts. Knowledge of diploid morphology, phylogeny, and ecogeography provides a foundation for understanding the evolutionary history of polyploid apomicts, their patterns of diversification, and their species status.
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http://dx.doi.org/10.3732/ajb.1500330DOI Listing
December 2015

Comparison of drug release from poly(lactide-co-glycolide) microspheres and novel fibre formulations.

J Biomater Appl 2016 Mar 29;30(8):1142-53. Epub 2015 Nov 29.

Department of Chemical Engineering, University of Bath, UK

Intraperitoneal cisplatin delivery has recently been shown to benefit ovarian cancer patients. Cisplatin-containing poly(lactide-co-glycolide) (PLGA) microspheres have been proposed for cisplatin delivery. The drug loading of cisplatin containing microspheres produced elsewhere is 3-10%w. Similar microspheres are reported here with a mean diameter of 38.8 µm, and a drug loading of 11.7%w, but using ethyl acetate as a safer solvent. In addition, novel formulations of cisplatin-containing solid and hollow PLGA 65:35 (lactide:glycolide) fibres were prepared and are reported here for the first time. PLGA hollow fibres were produced by phase inversion with a high drug loading of 27%w. Mechanistic mathematical models were applied to the cisplatin release profiles to allow quantitative comparison of microsphere, solid fibre and hollow fibre formulations. The diffusion coefficient of cisplatin eluting from a typical batch of PLGA microspheres was 4.8 × 10(-13) cm(2) s(-1); this low diffusivity of cisplatin in microspheres was caused by the low porosity of the polymer matrix. The diffusion coefficients of cisplatin eluting from a batch of PLGA solid fibres and hollow fibres were 6.1 × 10(-10) and 3.3 × 10(-10) cm(2) s(-1), respectively. These fibres allowed the controlled release of high doses of cisplatin over four days and may represent an improvement in slow release technology for treatment of ovarian cancer.
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http://dx.doi.org/10.1177/0885328215617327DOI Listing
March 2016

The CENP-A N-tail confers epigenetic stability to centromeres via the CENP-T branch of the CCAN in fission yeast.

Curr Biol 2015 Feb 22;25(3):348-356. Epub 2015 Jan 22.

Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

In most eukaryotes, centromeres are defined epigenetically by presence of the histone H3 variant CENP-A [1-3]. CENP-A-containing chromatin recruits the constitutive centromere-associated network (CCAN) of proteins, which in turn directs assembly of the outer kinetochore to form microtubule attachments and ensure chromosome segregation fidelity [4-6]. Whereas the mechanisms that load CENP-A at centromeres are being elucidated, the functions of its divergent N-terminal tail remain enigmatic [7-12]. Here, we employ the well-studied fission yeast centromere [13-16] to investigate the function of the CENP-A (Cnp1) N-tail. We show that alteration of the N-tail does not affect Cnp1 loading at centromeres, outer kinetochore formation, or spindle checkpoint signaling but nevertheless elevates chromosome loss. N-tail mutants exhibited synthetic lethality with an altered centromeric DNA sequence, with rare survivors harboring chromosomal fusions in which the altered centromere was epigenetically inactivated. Elevated centromere inactivation was also observed for N-tail mutants with unaltered centromeric DNA sequences. N-tail mutants specifically reduced localization of the CCAN proteins Cnp20/CENP-T and Mis6/CENP-I, but not Cnp3/CENP-C. Overexpression of Cnp20/CENP-T suppressed defects in an N-tail mutant, suggesting a link between reduced CENP-T recruitment and the observed centromere inactivation phenotype. Thus, the Cnp1 N-tail promotes epigenetic stability of centromeres in fission yeast, at least in part via recruitment of the CENP-T branch of the CCAN.
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http://dx.doi.org/10.1016/j.cub.2014.11.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4318777PMC
February 2015

Effects of apomixis and polyploidy on diversification and geographic distribution in Amelanchier (Rosaceae).

Am J Bot 2014 Aug;101(8):1375-87

School of Biology and Ecology, University of Maine, Orono, Maine 04469 USA.

Unlabelled: •

Premise Of The Study: Amelanchier polyploid apomicts differ from sexual diploids in their more complex diversification, greater species problems, and geographic distribution. To understand these differences, we investigated the occurrence of polyploidy and frequency of apomixis. This research helps clarify species delimitation in an evolutionarily complex genus.•

Methods: We used flow cytometry to estimate genome size of 1355 plants. We estimated the frequency of apomixis from flow-cytometrically determined ploidy levels of embryo and endosperm and from a progeny study using RAPD markers. We explored relationships of triploids to other ploidy levels and of ploidy levels to latitude plus elevation.•

Key Results: Diploids (32% of sample) and tetraploids (62%) were widespread. Triploids (6%) mostly occurred in small numbers with diploids from two or more species or with diploids and tetraploids. Seeds from diploids were 2% apomictic, the first report of apomixis in Amelanchier diploids. Seeds from triploids were 75% apomictic. We documented potential triploid bridge and triploid block from unbalanced endosperm and low pollen viability. Seeds from tetraploids were 97% apomictic, and tetraploids often formed microspecies. We did not find strong evidence for geographical parthenogenesis in North American Amelanchier. Most currently recognized species contained multiple ploidy levels that were morphologically semicryptic.•

Conclusions: Documentation of numerous transitions from diploidy to polyploidy helps clarify diversification, geographic distribution, and the species problem in Amelanchier. Despite the infrequent occurrence of triploids, their retention of 25% sexuality and capacity for triploid bridge may be important steps between sexual diploids and predominantly apomictic tetraploids.
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http://dx.doi.org/10.3732/ajb.1400113DOI Listing
August 2014

PCNA and Msh2-Msh6 activate an Mlh1-Pms1 endonuclease pathway required for Exo1-independent mismatch repair.

Mol Cell 2014 Jul 26;55(2):291-304. Epub 2014 Jun 26.

Ludwig Institute for Cancer Research, University of California, San Diego School of Medicine, La Jolla, CA 92093-0669, USA; Department of Medicine, University of California, San Diego School of Medicine, La Jolla, CA 92093-0669, USA; Department of Cellular and Molecular Medicine, University of California, San Diego School of Medicine, La Jolla, CA 92093-0669, USA; Moores-UCSD Cancer Center, University of California, San Diego School of Medicine, La Jolla, CA 92093-0669, USA; Institute of Genomic Medicine, University of California, San Diego School of Medicine, La Jolla, CA 92093-0669, USA. Electronic address:

Genetic evidence has implicated multiple pathways in eukaryotic DNA mismatch repair (MMR) downstream of mispair recognition and Mlh1-Pms1 recruitment, including Exonuclease 1 (Exo1)-dependent and -independent pathways. We identified 14 mutations in POL30, which encodes PCNA in Saccharomyces cerevisiae, specific to Exo1-independent MMR. The mutations identified affected amino acids at three distinct sites on the PCNA structure. Multiple mutant PCNA proteins had defects either in trimerization and Msh2-Msh6 binding or in activation of the Mlh1-Pms1 endonuclease that initiates excision during MMR. The latter class of mutations led to hyperaccumulation of repair intermediate Mlh1-Pms1 foci and were enhanced by an msh6 mutation that disrupted the Msh2-Msh6 interaction with PCNA. These results reveal a central role for PCNA in the Exo1-independent MMR pathway and suggest that Msh2-Msh6 localizes PCNA to repair sites after mispair recognition to activate the Mlh1-Pms1 endonuclease for initiating Exo1-dependent repair or for driving progressive excision in Exo1-independent repair.
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http://dx.doi.org/10.1016/j.molcel.2014.04.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4113420PMC
July 2014

Mlh2 is an accessory factor for DNA mismatch repair in Saccharomyces cerevisiae.

PLoS Genet 2014 May 8;10(5):e1004327. Epub 2014 May 8.

Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, La Jolla, California, United States of America; Department of Cellular and Molecular Medicine, University of California School of Medicine, San Diego, La Jolla, California, United States of America; Moores-UCSD Cancer Center, University of California School of Medicine, San Diego, La Jolla, California, United States of America; Department of Medicine, University of California School of Medicine, San Diego, La Jolla, California, United States of America.

In Saccharomyces cerevisiae, the essential mismatch repair (MMR) endonuclease Mlh1-Pms1 forms foci promoted by Msh2-Msh6 or Msh2-Msh3 in response to mispaired bases. Here we analyzed the Mlh1-Mlh2 complex, whose role in MMR has been unclear. Mlh1-Mlh2 formed foci that often colocalized with and had a longer lifetime than Mlh1-Pms1 foci. Mlh1-Mlh2 foci were similar to Mlh1-Pms1 foci: they required mispair recognition by Msh2-Msh6, increased in response to increased mispairs or downstream defects in MMR, and formed after induction of DNA damage by phleomycin but not double-stranded breaks by I-SceI. Mlh1-Mlh2 could be recruited to mispair-containing DNA in vitro by either Msh2-Msh6 or Msh2-Msh3. Deletion of MLH2 caused a synergistic increase in mutation rate in combination with deletion of MSH6 or reduced expression of Pms1. Phylogenetic analysis demonstrated that the S. cerevisiae Mlh2 protein and the mammalian PMS1 protein are homologs. These results support a hypothesis that Mlh1-Mlh2 is a non-essential accessory factor that acts to enhance the activity of Mlh1-Pms1.
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http://dx.doi.org/10.1371/journal.pgen.1004327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4014439PMC
May 2014

Dominant mutations in S. cerevisiae PMS1 identify the Mlh1-Pms1 endonuclease active site and an exonuclease 1-independent mismatch repair pathway.

PLoS Genet 2013 Oct 31;9(10):e1003869. Epub 2013 Oct 31.

Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, La Jolla, California, United States of America.

Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.
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http://dx.doi.org/10.1371/journal.pgen.1003869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814310PMC
October 2013

Tension sensing by Aurora B kinase is independent of survivin-based centromere localization.

Nature 2013 May 21;497(7447):118-21. Epub 2013 Apr 21.

Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California 92037, USA.

Accurate segregation of the replicated genome requires chromosome biorientation on the spindle. Biorientation is ensured by Aurora B kinase (Ipl1), a member of the four-subunit chromosomal passenger complex (CPC). Localization of the CPC to the inner centromere is central to the current model for how tension ensures chromosome biorientation: kinetochore-spindle attachments that are not under tension remain close to the inner centromere and are destabilized by Aurora B phosphorylation, whereas kinetochores under tension are pulled away from the influence of Aurora B, stabilizing their microtubule attachments. Here we show that an engineered truncation of the Sli15 (known as INCENP in humans) subunit of budding yeast CPC that eliminates association with the inner centromere nevertheless supports proper chromosome segregation during both mitosis and meiosis. Truncated Sli15 suppresses the deletion phenotypes of the inner-centromere-targeting proteins survivin (Bir1), borealin (Nbl1), Bub1 and Sgo1 (ref. 6). Unlike wild-type Sli15, truncated Sli15 localizes to pre-anaphase spindle microtubules. Premature targeting of full-length Sli15 to microtubules by preventing Cdk1 (also known as Cdc28) phosphorylation also suppresses the inviability of Bir1 deletion. These results suggest that activation of Aurora B kinase by clustering either on chromatin or on microtubules is sufficient for chromosome biorientation.
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http://dx.doi.org/10.1038/nature12057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3644022PMC
May 2013

What the hec is up with mouse oocyte meiosis?

Dev Cell 2013 Apr;25(1):3-4

Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92037, USA.

In this issue of Developmental Cell, Gui and Homer (2013) report that the proper execution of meiosis I in mouse oocytes requires the stabilization of cyclin B2 by the kinetochore protein Hec1, revealing unanticipated functions for both proteins.
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http://dx.doi.org/10.1016/j.devcel.2013.03.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3804012PMC
April 2013

Objective assessment of nanoparticle disposition in mammalian skin after topical exposure.

J Control Release 2012 Aug 23;162(1):201-7. Epub 2012 Jun 23.

University of Bath, Department of Pharmacy & Pharmacology, Claverton Down, Bath, BA2 7AY, UK.

The use of nanoparticles as formulation components of topical drug delivery systems for the skin has been widely investigated in the literature. Because of the conflicting conclusions resulting from these studies concerning the ultimate disposition of the nanoparticles employed, the research presented in this paper has been designed to evaluate objectively the fate of such structures when administered to mammalian skin. Confocal microscopy images of skin exposed to nanoparticles have therefore been assessed by quantitative statistical analysis. Sebum on the skin surface was naturally fluorescent and clearly defined the outermost part of the cutaneous barrier. Fluorescent polystyrene nanoparticles applied in aqueous suspension could infiltrate only the stratum disjunctum, i.e., skin layers in the final stages of desquamation. This minimal uptake was independent of contact time (up to 16 h) and of nanoparticle size tested (20-200 nm). When skin barrier function was modestly compromised, the nanoparticles remained incapable of penetration beyond the most superficial layers, corresponding to a depth of 2-3 μm, of the stratum corneum (the outermost, 15-20 μm skin layer). Overall, these results demonstrate objectively and semi-quantitatively that nanoparticles contacting intact, and even partially damaged, skin cannot penetrate beyond the superficial layers of the barrier, and are highly unlikely, therefore, to reach the viable cells of the epidermis or beyond. It follows that nanoparticulate-based, topical delivery systems may prove useful as skin surface reservoirs from which controlled drug release over time may be achieved.
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http://dx.doi.org/10.1016/j.jconrel.2012.06.024DOI Listing
August 2012

Visualization of eukaryotic DNA mismatch repair reveals distinct recognition and repair intermediates.

Cell 2011 Nov;147(5):1040-53

Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0669, USA.

DNA mismatch repair (MMR) increases replication fidelity by eliminating mispaired bases resulting from replication errors. In Saccharomyces cerevisiae, mispairs are primarily detected by the Msh2-Msh6 complex and corrected following recruitment of the Mlh1-Pms1 complex. Here, we visualized functional fluorescent versions of Msh2-Msh6 and Mlh1-Pms1 in living cells. We found that the Msh2-Msh6 complex is an S phase component of replication centers independent of mispaired bases; this localized pool accounted for 10%-15% of MMR in wild-type cells but was essential for MMR in the absence of Exo1. Unexpectedly, Mlh1-Pms1 formed nuclear foci that, although dependent on Msh2-Msh6 for formation, rarely colocalized with Msh2-Msh6 replication-associated foci. Mlh1-Pms1 foci increased when the number of mispaired bases was increased; in contrast, Msh2-Msh6 foci were unaffected. These findings suggest the presence of replication machinery-coupled and -independent pathways for mispair recognition by Msh2-Msh6, which direct formation of superstoichiometric Mlh1-Pms1 foci that represent sites of active MMR.
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http://dx.doi.org/10.1016/j.cell.2011.10.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3478091PMC
November 2011

In vivo visualization of type II plasmid segregation: bacterial actin filaments pushing plasmids.

J Cell Biol 2007 Dec 26;179(5):1059-66. Epub 2007 Nov 26.

School of Medicine, University of California, San Francisco, San Francisco, CA 94158, USA.

Type II par operons harness polymerization of the dynamically unstable actin-like protein ParM to segregate low-copy plasmids in rod-shaped bacteria. In this study, we use time-lapse fluorescence microscopy to follow plasmid dynamics and ParM assembly in Escherichia coli. Plasmids lacking a par operon undergo confined diffusion with a diffusion constant of 5 x 10(-5) microm(2)/s and a confinement radius of 0.28 microm. Single par-containing plasmids also move diffusively but with a larger diffusion constant (4 x 10(-4) microm(2)/s) and confinement radius (0.42 microm). ParM filaments are dynamically unstable in vivo and form spindles that link pairs of par-containing plasmids and drive them rapidly (3.1 microm/min) toward opposite poles of the cell. After reaching the poles, ParM filaments rapidly and completely depolymerize. After ParM disassembly, segregated plasmids resume diffusive motion, often encountering each other many times and undergoing multiple rounds of ParM-dependent segregation in a single cell cycle. We propose that in addition to driving segregation, the par operon enables plasmids to search space and find sister plasmids more effectively.
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http://dx.doi.org/10.1083/jcb.200708206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2099209PMC
December 2007

Reconstitution of DNA segregation driven by assembly of a prokaryotic actin homolog.

Science 2007 Mar;315(5816):1270-4

Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA.

Multiple unrelated polymer systems have evolved to partition DNA molecules between daughter cells at division. To better understand polymer-driven DNA segregation, we reconstituted the three-component segregation system of the R1 plasmid from purified components. We found that the ParR/parC complex can construct a simple bipolar spindle by binding the ends of ParM filaments, inhibiting dynamic instability, and acting as a ratchet permitting incorporation of new monomers and riding on the elongating filament ends. Under steady-state conditions, the dynamic instability of unattached ParM filaments provides the energy required to drive DNA segregation.
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http://dx.doi.org/10.1126/science.1138527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2851738PMC
March 2007

Nuclear ribosomal DNA internal transcribed spacer 1 (ITS1) in Picea (Pinaceae): sequence divergence and structure.

Mol Phylogenet Evol 2005 Apr 20;35(1):165-85. Epub 2005 Jan 20.

Department of Biological Sciences, University of Maine, Orono, ME 04469, USA.

The nrDNA ITS1 of Picea is 2747-3271 bp, the longest known of all plants. We obtained 24 cloned ITS1 sequences from six individuals of Picea glehnii, Picea mariana, Picea orientalis, and Picea rubens. Mean sequence divergence within these individuals (0.018+/-0.009) is more than half that between the species (0.031+/-0.011) and may be maintained against concerted evolution by separation of Picea 18S-26S rDNA repeats on multiple chromosomes. Picea ITS1 contains three subrepeats with a motif (5'-GGCCACCCTAGTC) that is conserved across Pinaceae. Two subrepeats are tandem, remote from the third, and more closely related and significantly more similar to one another than either is to the third subrepeat. This correlation between similarity and proximity may be the result of subrepeat duplication or concerted evolution within rDNA repeats. In inferred secondary structures, subrepeats generally form long hairpins, with a portion of the Pinaceae conserved motif in the terminal loop, and tandem subrepeats pair with one another over most of their length. Coalescence of ITS1 sequences occurs in P. orientalis but not in the other species.
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http://dx.doi.org/10.1016/j.ympev.2004.11.010DOI Listing
April 2005

Dynamic instability in a DNA-segregating prokaryotic actin homolog.

Science 2004 Nov;306(5698):1021-5

University of California, 600 16th Street, San Francisco, CA 94107, USA.

Dynamic instability-the switching of a two-state polymer between phases of steady elongation and rapid shortening-is essential to the cellular function of eukaryotic microtubules, especially during chromosome segregation. Since the discovery of dynamic instability 20 years ago, no other biological polymer has been found to exhibit this behavior. Using total internal reflection fluorescence microscopy and fluorescence resonance energy transfer, we observe that the prokaryotic actin homolog ParM, whose assembly is required for the segregation of large, low-copy number plasmids, displays both dynamic instability and symmetrical, bidirectional polymerization. The dynamic instability of ParM is regulated by adenosine triphosphate (ATP) hydrolysis, and filaments are stabilized by a cap of ATP-bound monomers. ParM is not related to tubulin, so its dynamic instability must have arisen by convergent evolution driven by a set of common constraints on polymer-based segregation of DNA.
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http://dx.doi.org/10.1126/science.1101313DOI Listing
November 2004

Angiosperm phylogeny based on matK sequence information.

Am J Bot 2003 Dec;90(12):1758-76

Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 USA;

Plastid matK gene sequences for 374 genera representing all angiosperm orders and 12 genera of gymnosperms were analyzed using parsimony (MP) and Bayesian inference (BI) approaches. Traditionally, slowly evolving genomic regions have been preferred for deep-level phylogenetic inference in angiosperms. The matK gene evolves approximately three times faster than the widely used plastid genes rbcL and atpB. The MP and BI trees are highly congruent. The robustness of the strict consensus tree supercedes all individual gene analyses and is comparable only to multigene-based phylogenies. Of the 385 nodes resolved, 79% are supported by high jackknife values, averaging 88%. Amborella is sister to the remaining angiosperms, followed by a grade of Nymphaeaceae and Austrobaileyales. Bayesian inference resolves Amborella + Nymphaeaceae as sister to the rest, but with weak (0.42) posterior probability. The MP analysis shows a trichotomy sister to the Austrobaileyales representing eumagnoliids, monocots + Chloranthales, and Ceratophyllum + eudicots. The matK gene produces the highest internal support yet for basal eudicots and, within core eudicots, resolves a crown group comprising Berberidopsidaceae/Aextoxicaceae, Santalales, and Caryophyllales + asterids. Moreover, matK sequences provide good resolution within many angiosperm orders. Combined analyses of matK and other rapidly evolving DNA regions with available multigene data sets have strong potential to enhance resolution and internal support in deep level angiosperm phylogenetics and provide additional insights into angiosperm evolution.
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http://dx.doi.org/10.3732/ajb.90.12.1758DOI Listing
December 2003

Ancient allopolyploid speciation in Geinae (Rosaceae): evidence from nuclear granule-bound starch synthase (GBSSI) gene sequences.

Syst Biol 2003 Jun;52(3):374-85

Bergius Foundation, Royal Swedish Academy of Sciences, and Department of Botany, Stockholm University, Sweden.

A nuclear low-copy gene phylogeny provides strong evidence for the hybrid origin of seven polyploid species in Geinae (Rosaceae). In a gene tree, alleles at homologous loci in an allopolyploid species are expected to be sisters to orthologues in the ancestral taxa rather than to each other. Alleles at a duplicated locus in an autopolyploid, however, are expected to be more closely related to each other than they are to any orthologous copies in closely related species. We cloned and sequenced about 1.9 kilobases from the 5' end of the GBSSI-1 gene from two diploid, one tetraploid, and six hexaploid species. Each of the three loci in the hexaploid species forms a separate group, two of which are more closely related to copies in other species than they are to each other. This finding indicates that the hexaploid lineage evolved through two consecutive allopolyploidization events. Based on the GBSSI-1 gene tree, we hypothesized that there was an initial hybridization between a diploid species from the ancestral lineage of Coluria and Waldsteinia and an unknown diploid species to form the tetraploid Geum heterocarpum lineage. Backcrossing of G. heterocarpum with a representative of the unknown diploid lineage then resulted in a hexaploid lineage that has radiated considerably since its origin, comprising at least 40 extant species with various morphologies. A penalized likelihood analysis indicated that Geinae may be about 17 million years old, implying that the hypothesized allopolyploid speciation events are relatively ancient. Six of the 22 cloned Geinae GBSSI-1 copies in this study, which all are duplicate copies in polyploid taxa, may have become pseudogenes. We compared the GBSSI-1 phylogeny with one from chloroplast data and explored implications for the evolution of some fruit characters.
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http://dx.doi.org/10.1080/10635150390197000DOI Listing
June 2003

Proteome analysis of Escherichia coli K-12 by two-dimensional native-state chromatography and MALDI-MS.

Mol Microbiol 2003 Jan;47(2):383-96

Department of Biochemistry, Texas A & M University, College Station TX 77843-2128, USA.

To identify proteins expressed in Escherichia coli K-12 MG1655 during exponential growth in defined medium, we separated soluble proteins of E. coli over two dimensions of native-state high-performance liquid chromatography, and examined the components of the protein mixtures in each of 380 fractions by peptide mass fingerprinting. To date, we have identified the products of 310 genes covering a wide range of cellular functions. Validation of protein assignments was made by comparing the assignments of proteins to specific first-dimension fractions to proteins visualized by two-dimensional gel electrophoresis. Co-fractionation of proteins suggests the possible identities of components of multiprotein complexes. This approach yields high-throughput gel-independent identification of proteins. It can also be used to assign identities to spots visualized by two-dimensional gels, and should be useful to evaluate differences in expressed proteome content and protein complexes among strains or between different physiological states.
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http://dx.doi.org/10.1046/j.1365-2958.2003.03294.xDOI Listing
January 2003

The origin of the apple subfamily (Maloideae; Rosaceae) is clarified by DNA sequence data from duplicated GBSSI genes.

Am J Bot 2002 Sep;89(9):1478-84

Biology Department, Acadia University, 24 University Avenue, Wolfville, Nova Scotia, B0P 1X0, Canada;

For 70 yr the leading hypothesis for the origin of the Maloideae has involved wide hybridization between ancestors of two other subfamilies. The basis of this hypothesis is that Maloideae have a base chromosome number of 17, whereas other Rosaceae are mostly x = 7, 8, or 9. To investigate this hypothesis we cloned and sequenced approximately 1.8 kilobases from the 5' portion of granule-bound starch synthase (GBSSI, or waxy) genes for 89 clones from 32 Rosaceae genera. Previous studies demonstrate the presence of two copies in all Rosaceae (GBSSI-1 and GBSSI-2) and four in Maloideae (GBSSI-1A, GBSSI-1B, GBSSI-2A, and GBSSI-2B). Parsimony and maximum likelihood analyses nest Gillenia, a genus of the southeastern United States with a base chromosome number of 9, within either Maloideae GBSSI-1 or GBSSI-2. Monophyly of Maloideae plus Gillenia is well supported by bootstrap values, loss of the sixth intron in all GBSSI-1 sequences, intron alignability between genera, and numerous nonmolecular characters. Our results falsify the wide-hybridization hypothesis and are consistent with a polyploid origin involving only members of a lineage that contained the ancestors of Gillenia. Under this hypothesis, the subfamily originated in North America, and the high Maloideae chromosome number arose via aneuploidy from x = 18.
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http://dx.doi.org/10.3732/ajb.89.9.1478DOI Listing
September 2002
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