alex lim - Academia.edu (original) (raw)
Papers by alex lim
Nature, 2001
is less than 100%). Models for the additional oligonucleotide, GTP molecules and Mg 2+ ions, have... more is less than 100%). Models for the additional oligonucleotide, GTP molecules and Mg 2+ ions, have been fitted into electron density maps and refinement of these oligo-Mn 2+polymerase and oligo-GTP-Mg-Mn-polymerase complexes against their data sets, imposing strict threefold NCS constraints, resulted in models with R factors of 23.7 and 21.4%, respectively, and good stereochemistry (Table 1).
Nature, 2001
examine the quaternary complex, LexA±AP3 and PI were expressed on the bait vector, and GAL4 AD-AG... more examine the quaternary complex, LexA±AP3 and PI were expressed on the bait vector, and GAL4 AD-AG and/or SEP3-MIK were expressed on the prey vector. When two genes were expressed on the same vector, they were both driven by ADH1 promoters. Amino-acid residues 1±167 and 1±171 were used for the truncated AP1-MIK and SEP3-MIK proteins, respectively. Other processes and the colony-lift b-gal assays were performed in accordance with the manufacturer's instructions (Clontech). Immunoprecipitation For immunoprecipitation experiments, radiolabelled AP1 or SEP3 were mixed with haemagglutinin (HA)-tagged proteins and precipitated with anti-HA antibody. Precipitated AP1 and SEP3 were separated by SDS±PAGE and detected by radio-imaging analyser, BAS2000 (Fuji®lm). Other procedures were done as described 7,12. Transactivation assay For yeast, MADS proteins cDNAs were fused in-frame to GAL4 DNA-binding domain on pAS2-1 (Clontech) and transformed into the yeast strain YRG-2 (UAS::lacZ, Stratagene). AP1-K2C (residues 125±256) and SEP3-K2C (128±257) were used as truncated MADS proteins. Yeast cells were grown at 22 8C overnight, and the b-gal activity was assayed at 30 8C using o-nitrophenyl-b-D-galactopyranoside. For onion epidermal cells, 35S promoter-driven MADS cDNAs that express native MADS proteins (effector) and CArG::LUC (reporter) were co-transfected into onion epidermal cells by using a particle delivery system (Bio-Rad). CArG::LUC has seven repeats of MADS protein binding consensus sequence 29 , 59-GGGGTGGCTTTCCTTTTTTGG TAAATTTTGGATCC-39 (CArG box is underlined), upstream of the 35S minimal promoter (-30). 35S::Renilla luciferase (RLUC) was used for the internal control. LUC assays were conducted using Dual-luciferase reporter system (Promega). Other procedures were done as described 30. Plant material Arabidopsis Columbia ecotype was used for Agrobacterium-mediated vacuum transformation 31. Plant crossing was carried out by manual cross-pollination. The presence of the transgenes was con®rmed by PCR. AP3::GUS plants have a 600-base-pair region of the AP3 promoter 16. Staining for GUS activity was done as described 16. Cryo-scanning electron micrograph We used a Hitachi S-3500N scanning electron microscope equipped with a cryo-stage. For observation and photography, the stage was chilled at-20 8C and the natural scanning electron microscopy (SEM) mode (70 Pa) was used with a 25-kV accelerating voltage.
Genome Research, 2001
We have constructed NheI and XhoI optical maps ofEscherichia coli O157:H7 solely from genomic DNA... more We have constructed NheI and XhoI optical maps ofEscherichia coli O157:H7 solely from genomic DNA molecules to provide a uniquely valuable scaffold for contig closure and sequence validation. E. coli O157:H7 is a common pathogen found in contaminated food and water. Our approach obviated the need for the analysis of clones, PCR products, and hybridizations, because maps were constructed from ensembles of single DNA molecules. Shotgun sequencing of bacterial genomes remains labor-intensive, despite advances in sequencing technology. This is partly due to manual intervention required during the last stages of finishing. The applicability of optical mapping to this problem was enhanced by advances in machine vision techniques that improved mapping throughput and created a path to full automation of mapping. Comparisons were made between maps and sequence data that characterized sequence gaps and guided nascent assemblies.
Applied and Environmental Microbiology, 2005
Rhodospirillum rubrum is a phototrophic purple nonsulfur bacterium known for its unique and well-... more Rhodospirillum rubrum is a phototrophic purple nonsulfur bacterium known for its unique and well-studied nitrogen fixation and carbon monoxide oxidation systems and as a source of hydrogen and biodegradable plastic production. To better understand this organism and to facilitate assembly of its sequence, three whole-genome restriction endonuclease maps (XbaI, NheI, and HindIII) of R. rubrum strain ATCC 11170 were created by optical mapping. Optical mapping is a system for creating whole-genome ordered restriction endonuclease maps from randomly sheared genomic DNA molecules extracted from cells. During the sequence finishing process, all three optical maps confirmed a putative error in sequence assembly, while the HindIII map acted as a scaffold for high-resolution alignment with sequence contigs spanning the whole genome. In addition to highlighting optical mapping's role in the assembly and confirmation of genome sequence, this work underscores the unique niche in resolution o...
Applied and Environmental Microbiology, 2002
Yersinia pestis is the causative agent of the bubonic, septicemic, and pneumonic plagues (also kn... more Yersinia pestis is the causative agent of the bubonic, septicemic, and pneumonic plagues (also known as black death) and has been responsible for recurrent devastating pandemics throughout history. To further understand this virulent bacterium and to accelerate an ongoing sequencing project, two whole-genome restriction maps ( Xho I and Pvu II) of Y. pestis strain KIM were constructed using shotgun optical mapping. This approach constructs ordered restriction maps from randomly sheared individual DNA molecules directly extracted from cells. The two maps served different purposes; the Xho I map facilitated sequence assembly by providing a scaffold for high-resolution alignment, while the Pvu II map verified genome sequence assembly. Our results show that such maps facilitated the closure of sequence gaps and, most importantly, provided a purely independent means for sequence validation. Given the recent advancements to the optical mapping system, increased resolution and throughput a...
Analytical Chemistry, 2004
Nature, 2001
is less than 100%). Models for the additional oligonucleotide, GTP molecules and Mg 2+ ions, have... more is less than 100%). Models for the additional oligonucleotide, GTP molecules and Mg 2+ ions, have been fitted into electron density maps and refinement of these oligo-Mn 2+polymerase and oligo-GTP-Mg-Mn-polymerase complexes against their data sets, imposing strict threefold NCS constraints, resulted in models with R factors of 23.7 and 21.4%, respectively, and good stereochemistry (Table 1).
Nature, 2001
examine the quaternary complex, LexA±AP3 and PI were expressed on the bait vector, and GAL4 AD-AG... more examine the quaternary complex, LexA±AP3 and PI were expressed on the bait vector, and GAL4 AD-AG and/or SEP3-MIK were expressed on the prey vector. When two genes were expressed on the same vector, they were both driven by ADH1 promoters. Amino-acid residues 1±167 and 1±171 were used for the truncated AP1-MIK and SEP3-MIK proteins, respectively. Other processes and the colony-lift b-gal assays were performed in accordance with the manufacturer's instructions (Clontech). Immunoprecipitation For immunoprecipitation experiments, radiolabelled AP1 or SEP3 were mixed with haemagglutinin (HA)-tagged proteins and precipitated with anti-HA antibody. Precipitated AP1 and SEP3 were separated by SDS±PAGE and detected by radio-imaging analyser, BAS2000 (Fuji®lm). Other procedures were done as described 7,12. Transactivation assay For yeast, MADS proteins cDNAs were fused in-frame to GAL4 DNA-binding domain on pAS2-1 (Clontech) and transformed into the yeast strain YRG-2 (UAS::lacZ, Stratagene). AP1-K2C (residues 125±256) and SEP3-K2C (128±257) were used as truncated MADS proteins. Yeast cells were grown at 22 8C overnight, and the b-gal activity was assayed at 30 8C using o-nitrophenyl-b-D-galactopyranoside. For onion epidermal cells, 35S promoter-driven MADS cDNAs that express native MADS proteins (effector) and CArG::LUC (reporter) were co-transfected into onion epidermal cells by using a particle delivery system (Bio-Rad). CArG::LUC has seven repeats of MADS protein binding consensus sequence 29 , 59-GGGGTGGCTTTCCTTTTTTGG TAAATTTTGGATCC-39 (CArG box is underlined), upstream of the 35S minimal promoter (-30). 35S::Renilla luciferase (RLUC) was used for the internal control. LUC assays were conducted using Dual-luciferase reporter system (Promega). Other procedures were done as described 30. Plant material Arabidopsis Columbia ecotype was used for Agrobacterium-mediated vacuum transformation 31. Plant crossing was carried out by manual cross-pollination. The presence of the transgenes was con®rmed by PCR. AP3::GUS plants have a 600-base-pair region of the AP3 promoter 16. Staining for GUS activity was done as described 16. Cryo-scanning electron micrograph We used a Hitachi S-3500N scanning electron microscope equipped with a cryo-stage. For observation and photography, the stage was chilled at-20 8C and the natural scanning electron microscopy (SEM) mode (70 Pa) was used with a 25-kV accelerating voltage.
Genome Research, 2001
We have constructed NheI and XhoI optical maps ofEscherichia coli O157:H7 solely from genomic DNA... more We have constructed NheI and XhoI optical maps ofEscherichia coli O157:H7 solely from genomic DNA molecules to provide a uniquely valuable scaffold for contig closure and sequence validation. E. coli O157:H7 is a common pathogen found in contaminated food and water. Our approach obviated the need for the analysis of clones, PCR products, and hybridizations, because maps were constructed from ensembles of single DNA molecules. Shotgun sequencing of bacterial genomes remains labor-intensive, despite advances in sequencing technology. This is partly due to manual intervention required during the last stages of finishing. The applicability of optical mapping to this problem was enhanced by advances in machine vision techniques that improved mapping throughput and created a path to full automation of mapping. Comparisons were made between maps and sequence data that characterized sequence gaps and guided nascent assemblies.
Applied and Environmental Microbiology, 2005
Rhodospirillum rubrum is a phototrophic purple nonsulfur bacterium known for its unique and well-... more Rhodospirillum rubrum is a phototrophic purple nonsulfur bacterium known for its unique and well-studied nitrogen fixation and carbon monoxide oxidation systems and as a source of hydrogen and biodegradable plastic production. To better understand this organism and to facilitate assembly of its sequence, three whole-genome restriction endonuclease maps (XbaI, NheI, and HindIII) of R. rubrum strain ATCC 11170 were created by optical mapping. Optical mapping is a system for creating whole-genome ordered restriction endonuclease maps from randomly sheared genomic DNA molecules extracted from cells. During the sequence finishing process, all three optical maps confirmed a putative error in sequence assembly, while the HindIII map acted as a scaffold for high-resolution alignment with sequence contigs spanning the whole genome. In addition to highlighting optical mapping's role in the assembly and confirmation of genome sequence, this work underscores the unique niche in resolution o...
Applied and Environmental Microbiology, 2002
Yersinia pestis is the causative agent of the bubonic, septicemic, and pneumonic plagues (also kn... more Yersinia pestis is the causative agent of the bubonic, septicemic, and pneumonic plagues (also known as black death) and has been responsible for recurrent devastating pandemics throughout history. To further understand this virulent bacterium and to accelerate an ongoing sequencing project, two whole-genome restriction maps ( Xho I and Pvu II) of Y. pestis strain KIM were constructed using shotgun optical mapping. This approach constructs ordered restriction maps from randomly sheared individual DNA molecules directly extracted from cells. The two maps served different purposes; the Xho I map facilitated sequence assembly by providing a scaffold for high-resolution alignment, while the Pvu II map verified genome sequence assembly. Our results show that such maps facilitated the closure of sequence gaps and, most importantly, provided a purely independent means for sequence validation. Given the recent advancements to the optical mapping system, increased resolution and throughput a...
Analytical Chemistry, 2004