Journal:

Article Title: Human replication protein Cdc6 prevents mitosis through a checkpoint mechanism that implicates Chk1

doi: 10.1093/emboj/cdg046

Figure Lengend Snippet: Fig. 2. Constitutively active MPF abolishes the G2 phase arrest caused by HuCdc6 overexpression. G2 phase HeLa cells were microinjected with (A) pEGFP-HuCdc6, pCyclin B1 and a constitutively active pCDK1AF or (B) with pEGFP-HuCdc6, pCyclin B1 and pCDK1wt. As controls we co-injected pCyclin B1 and pCDK1AF or pCyclin B1 and pCDK1wt were injected without HuCdc6 as a control (C). Approximately 100 fluorescent cells were counted for each sample. Fluorescence and DIC images were taken 7 h after injection. Arrows show mitotic cells. (C) The numbers of cells in G2 phase, mitosis and in early G1 phase were scored and calculated as a percentage of the total of injected cells. At least three independent experiments were performed and representative images are shown.

Article Snippet: HuCdc6 PCR product nt 129–1853 inserted into a Xho I– Hin dIII pEGFP C2 was a gift from Dr Yoshinori Takei ( Takei et al., 1999 ) and PCR product 210–1890 inserted into a Kpn I– Bam H1 pEGFP C2 produced identical data and therefore we refer in this study only to HuCdc6-pEGFP.

Techniques: Over Expression, Injection, Control, Fluorescence

Journal:

Article Title: Human replication protein Cdc6 prevents mitosis through a checkpoint mechanism that implicates Chk1

doi: 10.1093/emboj/cdg046

Figure Lengend Snippet: Fig. 1. In vivo analysis of HuCdc6 overexpression in G2 cells. (A) pEGFP and (B) pEGFP-HuCdc6 were microinjected into the nucleus of G2 phase HeLa cells. The behaviour of injected cells was observed by time-lapse fluorescence and DIC microscopy and images were taken every 30 min, or every 3 min after entry into mitosis over a 10 h period. Approximately 100 fluorescent cells expressing GFP or HuCdc6–GFP were classified according to their cell cycle stage: G2 phase, mitosis and after completion of mitosis (early G1 phase) during different time points starting from 1 h after microinjection. These numbers were compared with the total number of cells expressing GFP or HuCdc6–GFP (C and D). While 40–45% of cells expressing GFP went through mitosis [arrows in (A) and (B) show mitotic cells], only 3–5% of cells expressing HuCdc6–GFP did so. Representative images, mean values and standard deviations of five independent experiments are shown. (E) Comparison of expression of HuCdc6–GFP to endogenous HuCdc6 levels. A total of 1000 cells were injected with pEGFP-HuCdc6 and after 2 h were directly lysed in SDS buffer, proteins were separated on 10% SDS–PAGE and immunoblotted for HuCdc6. (F) Anti human Cdc6 antibody neutralizes HuCdc6 overexpression and abolishes the G2 phase arrest. G2 phase HeLa cells were microinjected with HuCdc6–GFP and a specific anti HuCdc6 antibody. As a control, cells were injected with the anti HuCdc6 antibody and Texas Red dextran. The percentages of cells expressing pEGFP-HuCdc6 in G2, M and G1 phases were calculated and compared with the control cells 7 h after injection. Mean values and standard deviation were calculated from at least three independent experiments.

Article Snippet: HuCdc6 PCR product nt 129–1853 inserted into a Xho I– Hin dIII pEGFP C2 was a gift from Dr Yoshinori Takei ( Takei et al., 1999 ) and PCR product 210–1890 inserted into a Kpn I– Bam H1 pEGFP C2 produced identical data and therefore we refer in this study only to HuCdc6-pEGFP.

Techniques: In Vivo, Over Expression, Injection, Fluorescence, Microscopy, Expressing, Microinjection, Comparison, SDS Page, Control, Standard Deviation

Journal:

Article Title: Human replication protein Cdc6 prevents mitosis through a checkpoint mechanism that implicates Chk1

doi: 10.1093/emboj/cdg046

Figure Lengend Snippet: Fig. 3. Overexpression of Cdc25 abolishes the G2 phase arrest caused by HuCdc6–GFP. G2 phase cells were microinjected with pCdc25B and pEGFP-HuCdc6 (A) or with pEGFP-Cdc25C and pHuCdc6 (B). Arrows in (A) and (B) show mitotic cells. Cells were followed by time-lapse fluorescence and DIC microscopy as described in Figure 1, starting from 2 h after microinjection during a 7 h time course. Sixty per cent of cells entered mitosis prematurely and arrested in mitosis whether they expressed HuCdc6–GFP and Cdc25B (C) or Cdc25B alone (E). (D) Forty five per cent of cells expressing Cdc25C and HuCdc6–GFP entered and progressed through mitosis, but often with a 4 h delay compared with the 2 h delay of the control cells expressing Cdc25C only (F).

Article Snippet: HuCdc6 PCR product nt 129–1853 inserted into a Xho I– Hin dIII pEGFP C2 was a gift from Dr Yoshinori Takei ( Takei et al., 1999 ) and PCR product 210–1890 inserted into a Kpn I– Bam H1 pEGFP C2 produced identical data and therefore we refer in this study only to HuCdc6-pEGFP.

Techniques: Over Expression, Fluorescence, Microscopy, Microinjection, Expressing, Control

Journal:

Article Title: Human replication protein Cdc6 prevents mitosis through a checkpoint mechanism that implicates Chk1

doi: 10.1093/emboj/cdg046

Figure Lengend Snippet: Fig. 4. An inhibitor of Chk1 kinase activity, UCN-01, abolishes HuCdc6-mediated G2 arrest. (A) G2 phase HeLa cells were microinjected with pEGFP-HuCdc6 or pEGFP as a control (not shown). UCN-01 (300 nM) was added to the medium immediately after the injections. Fluorescence and DIC images were taken during a 10 h period. G2 phase and mitotic cells (marked with an arrow) were counted from a total of ∼100 fluorescent cells over a 10 h period. (B) Percentage of cells progressing through mitosis expressing HuCdc6 or GFP. Twenty-one percent of cells expressing HuCdc6–GFP entered and progressed through mitosis comparable with the 24% of GFP expressing cells. Two experiments were performed and representative images are shown. (C) HuCdc6-mediated phosphorylation of Chk1. One thousand cells were injected with pEGFPHuCdc6 or pEGFP, uninjected but untreated or treated with HU (control cells). Proteins were separated on SDS–PAGE and immunoblotted for Chk1. Lane 1 shows a mobility shift due to phosphorylation after HU treatment, whereas control cells do not (lanes 2, 4 and 6). HuCdc6 shows a similar mobility shift as HU-treated cells (lane 1), whereas GFP alone does not (lane 3). (D) The HuCdc6-mediated G2 phase arrest is maintained in the presence of caffeine and/or wortmannin. Cells were microinjected with pEGFP-HuCdc6 in the presence or absence of 5 mM caffeine and/or 25 µM wortmannin. As a control, cells were γ-irradiated (100 kVp/min) for 15 min and caffeine and/or wortmannin were added. Cells were followed for 10 h counting cells entering and progressing through mitosis as one (%M + G1). Uninjected cells and irradiated cells entered into mitosis prematurely in the presence of caffeine and/or wortmannin. The cells expressing HuCdc6 remained arrested in G2 phase in the presence of caffeine and/or wortmannin.

Article Snippet: HuCdc6 PCR product nt 129–1853 inserted into a Xho I– Hin dIII pEGFP C2 was a gift from Dr Yoshinori Takei ( Takei et al., 1999 ) and PCR product 210–1890 inserted into a Kpn I– Bam H1 pEGFP C2 produced identical data and therefore we refer in this study only to HuCdc6-pEGFP.

Techniques: Activity Assay, Control, Fluorescence, Expressing, Phospho-proteomics, Injection, SDS Page, Mobility Shift, Irradiation

Journal:

Article Title: Human replication protein Cdc6 prevents mitosis through a checkpoint mechanism that implicates Chk1

doi: 10.1093/emboj/cdg046

Figure Lengend Snippet: Fig. 5. Behaviour of HuCdc6 mutants in G2 cells. (A) Schematic drawing of HuCdc6 depicting features such as the CDK phosphorylation sites serines (S) 54, 74 and 106, destruction box, KEN box, the cyclin-binding-motif, the ATPase/ORC homology domain and leucine-zipper, all previously identified. (B) The pEGFP-HuCdc6 mutants S54A, S74A, S106A and Δcy-motif were injected into G2 HeLa cells and their progression into mitosis compared with GFP or wtHuCdc6–GFP-expressing cells. Cells expressing the mutants S75A and Δcy-motif cannot arrest cells in G2, whereas cells expressing S54A and S106A do arrest cells in G2 in the same fashion to wtHuCdc6. (C) Illustration of the localization of the mutants. Two independent experiments were performed and representative images are shown.

Article Snippet: HuCdc6 PCR product nt 129–1853 inserted into a Xho I– Hin dIII pEGFP C2 was a gift from Dr Yoshinori Takei ( Takei et al., 1999 ) and PCR product 210–1890 inserted into a Kpn I– Bam H1 pEGFP C2 produced identical data and therefore we refer in this study only to HuCdc6-pEGFP.

Techniques: Phospho-proteomics, Binding Assay, Injection, Expressing

Journal:

Article Title: Human replication protein Cdc6 prevents mitosis through a checkpoint mechanism that implicates Chk1

doi: 10.1093/emboj/cdg046

Figure Lengend Snippet: Fig. 6. Model for potential checkpoint function of HuCdc6. Ongoing DNA replication is monitored either by proteins at the replication fork, including ATR, or by soluble factors, including HuCdc6. ATR and HuCdc6 might work in parallel pathways, both leading to the activation of Chk1 and consequent inactivation of Cdc25. The major role of the ATR pathway would be in response to stalled replication forks. Chk1 may also increase the activity of Wee1 that further prevents the activation of MPF.

Article Snippet: HuCdc6 PCR product nt 129–1853 inserted into a Xho I– Hin dIII pEGFP C2 was a gift from Dr Yoshinori Takei ( Takei et al., 1999 ) and PCR product 210–1890 inserted into a Kpn I– Bam H1 pEGFP C2 produced identical data and therefore we refer in this study only to HuCdc6-pEGFP.

Techniques: Activation Assay, Activity Assay

Journal:

Article Title: Sulfadoxine-Pyrimethamine Resistance in the Rodent Malaria Parasite Plasmodium chabaudi

doi: 10.1128/AAC.46.8.2482-2489.2002

Figure Lengend Snippet: Inheritance of 31 chromosomal markers among AJ, AS(50S/P), and 16 progeny clones from two AJ × AS(50S/P) crosses. Progeny clones are listed vertically. Roman numerals refer to chromosome number, marker numbers are shown along the bottom, and markers are ordered such that the number of crossovers per chromosome was minimal (5). An asterisk marks the inheritance profile for P. chabaudi dhfr-ts (marker 6). Black boxes indicate inheritance of AS(50S/P)-type alleles, open boxes indicate inheritance of AJ-type alleles, and grey boxes indicate data not available. Drug phenotypes are shown on the right, where black boxes indicate the inheritance of a resistant phenotype, white boxes indicate the inheritance of a sensitive phenotype, and hatched boxes identify progeny with an intermediate phenotype.

Article Snippet: We thank Mike Barrett and Chris Janssen for the gift of the initial Hin dIII P. chabaudi Vectorette II library, Richard Fawcett and Ronnie Mooney for technical assistance, and Margaret Mackinnon for helpful advice.

Techniques: Clone Assay, Marker

Journal: PLoS ONE

Article Title: An Integrated Physical, Genetic and Cytogenetic Map of Brachypodium distachyon , a Model System for Grass Research

doi: 10.1371/journal.pone.0013461

Figure Lengend Snippet: Characteristics of the two BAC libraries used to construct the Brachypodium physical map.

Article Snippet: The Hin dIII library (BD_ABa) consisted of 36,864 clones with an average insert size of 128 Kb , and the Eco RI library (BD_CBa) consisted of 36,864 clones with an average insert size of 124 Kb ( ).

Techniques: Construct

Journal: SpringerPlus

Article Title: Optimization of DNA extraction and PCR protocols for phylogenetic analysis in Schinopsis spp. and related Anacardiaceae

doi: 10.1186/s40064-016-2118-4

Figure Lengend Snippet: 0.8 % Agarose gel electrophoresis depicting results of DNA extraction of some accessions of Schinopsis spp. and related species. a DNA extracted with the optimized protocol. Lanes 1 , 2 , 8 , 14 and 15 molecular marker (Lambda Eco RI/ Hin dIII), 3 and 4 S. lorentzii , 5 and 6 S. marginata , 7 Lithraea molleoides , 9 – 13 S. brasiliensis . b DNA extracted with the DNAEasy Plant Mini Kit (Quiagen Inc. Valencia, CA) used as control. 1 Molecular weight marker (100 bp DNA Ladder), 2 – 11 different specimens of S. brasiliensis ( asterisk reference bands of the marker)

Article Snippet: The DNA yield was estimated by spectrophotometric analysis and by comparing the fluorescence intensity of each sample to 100 ng of Lambda ( Eco RI/ Hin dIII) marker (Promega, USA) (Fig. a) or 325 ng of 100 bp DNA Ladder (Promega) (Fig. b) as standard.

Techniques: Agarose Gel Electrophoresis, DNA Extraction, Marker, Control, Molecular Weight

Journal: SpringerPlus

Article Title: Optimization of DNA extraction and PCR protocols for phylogenetic analysis in Schinopsis spp. and related Anacardiaceae

doi: 10.1186/s40064-016-2118-4

Figure Lengend Snippet: 1.5 % Agarose gel electrophoresis of PCR products of several accessions of Schinopsis spp. and related species. a trn L-F amplified in two parts. Lanes 1 , 20 , 27 molecular marker, 2 – 5 S. boqueronensis , 6 – 7 , 9 S. cornuta , 10 S. peruviana , 11 – 14 S. heterophylla , 15 – 18 , 21 S. brasiliensis , 22 – 23 S. marginata , 24 S. lorentzii , 25 – 26 S. brasiliensis , 28 – 29 Astronium urundeuva and Apterokarpos gardneri , and 8 , 19 without sample. b rps 16. Lanes 1 , 13 and 21 molecular marker, 2 S. peruviana , 3 – 6 S. heterophylla , 5 – 9 , 14 – 15 S. brasiliensis , 10 – 11 S. marginata , 12 S. lorentzii , 16 – 17 Astronium urundeuva and Apterokarpos gardneri , 18 – 19 S. balansae , 20 S. lorentzii , 22 – 24 S. boqueronensis , 25 – 27 S. cornuta . c ndh F. Lanes 1 , 19 and 29 molecular marker, 11 , 12 , 17 – 19 Astronium urundeuva , Apterokarpos gardneri , Lithraea molleoides , Loxopterygium grisebachii and Schinus areira ; other lanes, Schinopsis spp. Arrows indicate the reference bands of the marker Lambda Eco RI/ Hin dIII (564 bp in a , 947 and 831 in b and 831 and 564 in c ) to estimate the amplicons size

Article Snippet: The DNA yield was estimated by spectrophotometric analysis and by comparing the fluorescence intensity of each sample to 100 ng of Lambda ( Eco RI/ Hin dIII) marker (Promega, USA) (Fig. a) or 325 ng of 100 bp DNA Ladder (Promega) (Fig. b) as standard.

Techniques: Agarose Gel Electrophoresis, Amplification, Marker

Journal: SpringerPlus

Article Title: Optimization of DNA extraction and PCR protocols for phylogenetic analysis in Schinopsis spp. and related Anacardiaceae

doi: 10.1186/s40064-016-2118-4

Figure Lengend Snippet: 1.5 % agarose gel electrophoresis of PCR products of some accessions of Schinopsis spp. and related species. a ETS. Lane 1 molecular marker, 2 – 5 S. boqueronensis , 6 – 8 , S. cornuta , 9 , S. peruviana , 10 – 13 , S. heterophylla , 14 – 18 , 21 , 22 , S. brasiliensis , 19 , 20 , S. marginata , 21 , 27 , 28 S. lorentzii , 23 , 24 , 29 – 31 Astronium urundeuva , Apterokarpos gardneri , Lithraea molleoides , Loxopterygium grisebachii and Schinus areira . b ITS2. Line 1 , 12 , 24 molecular marker, 2 S. cornuta , 3 – 6 S. heterophylla , 7 – 10 , 15 , 16 S. brasiliensis , 11 , 12 S. heterophylla , 13 , 18 , 19 , S. lorentzii , 20 – 22 , Lithraea molleoides , Loxopterygium grisebachii and Schinus areira . Arrows indicate the reference bands of the marker Lambda Eco RI/ Hin dIII (564 bp in a and b ) to estimate the amplicons size

Article Snippet: The DNA yield was estimated by spectrophotometric analysis and by comparing the fluorescence intensity of each sample to 100 ng of Lambda ( Eco RI/ Hin dIII) marker (Promega, USA) (Fig. a) or 325 ng of 100 bp DNA Ladder (Promega) (Fig. b) as standard.

Techniques: Agarose Gel Electrophoresis, Marker