The Experts below are selected from a list of 15 Experts worldwide ranked by ideXlab platform
Daniel M. Vernon - One of the best experts on this subject based on the ideXlab platform.
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T-DNA-Associated Duplication/Translocations in Arabidopsis. Implications for Mutant Analysis and Functional Genomics
Plant physiology, 2001Co-Authors: Frans E. Tax, Daniel M. VernonAbstract:T-DNA insertion mutants have become a valuable resource for studies of gene function in Arabidopsis. In the course of both forward and reverse genetic projects, we have identified novel interchromosomal rearrangements in two Arabidopsis T-DNA insertion lines. Both rearrangements were unilateral translocations associated with the left borders of T-DNA inserts that exhibited normal Mendelian segregation. In one study, we characterized the embryo-defective 88 mutation. Although emb 88 had been mapped to chromosome I, molecular analysis of DNA adjacent to the T-DNA left border revealed sequence from chromosome V. Simple sequence length polymorphism mapping of the T-DNA insertion demonstrated that a >40-kbp Region of chromosome V had inserted with the T-DNA into the emb 88 locus on chromosome I. A similar scenario was observed with a prospective T-DNA knockout allele of the LIGHT-REGULATED RECEPTOR PROTEIN KINASE ( LRRPK ) gene. Whereas wild-type LRRPK is on lower chromosome IV, mapping of the T-DNA localized the disrupted LRRPK allele to chromosome V. In both these cases, the sequence of a single T-DNA-Flanking Region did not provide an accurate picture of DNA disruption because Flanking sequences had duplicated and inserted, with the T-DNA, into other chromosomal locations. Our results indicate that T-DNA insertion lines—even those that exhibit straightforward genetic behavior—may contain an unexpectedly high frequency of rearrangements. Such duplication/translocations can interfere with reverse genetic analyses and provide misleading information about the molecular basis of mutant phenotypes. Simple mapping and polymerase chain reaction methods for detecting such rearrangements should be included as a standard step in T-DNA mutant analysis.
Gabriel Waksman - One of the best experts on this subject based on the ideXlab platform.
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DNA helicases, motors that move along nucleic acids: Lessons from the SF1 helicase superfamily
The Enzymes, 2008Co-Authors: Timothy M. Lohman, Sergey Korolev, John Hsieh, Nasib K. Maluf, Wei Cheng, Aaron L. Lucius, Christopher J. Fischer, Katherine M. Brendza, Gabriel WaksmanAbstract:Publisher Summary This chapter focuses on the mechanistic aspects of superfamily 1 (SFl) DNA helicases. Helicases are allosteric enzymes, many of which are known to function as oligomeric assemblies. Such oligomerization is exemplified by the class of hexameric DNA helicases, including the Escherichia coli DNAB helicase, the phage T7 gene 4 helicase, and the SV40 large T antigen. The initial characterizations of a DNA helicase generally include studies of the features of DNA substrates that are required for efficient unwinding by the helicase. With few exceptions, DNA helicases show a preference for unwinding duplex DNA possessing an single-stranded (ss)-DNA Flanking Region or tail in vitro . In fact, the unwinding reaction generally displays a defined polarity of unwinding with respect to the backbone polarity of the ss-DNA tail that flanks the duplex DNA. Two operational classes of helicases are helicases that initiate unwinding more efficiently on DNA substrates with a 3'-ss-DNA tail and are referred to as “3' to 5' helicases,” whereas those that prefer DNA substrates possessing a 5'-ss-DNA tail are referred to as “5' to 3' helicases.”
Harcharan Singh Dhaliwal - One of the best experts on this subject based on the ideXlab platform.
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A candidate gene OsAPC6 of anaphase-promoting complex of rice identified through T-DNA insertion
Functional & Integrative Genomics, 2010Co-Authors: Mankesh Kumar, P. Osman Basha, Anju Puri, Deepak Rajpurohit, Gursharn Singh Randhawa, Tilak Raj Sharma, Harcharan Singh DhaliwalAbstract:A dwarf mutant ( Oryza sativa anaphase-promoting complex 6 ( OsAPC6 )) of rice cultivar Basmati 370 with 50% reduced plant height as compared to the wild type was isolated by Agrobacterium tumefaciens -mediated transformation using Hm^R Ds cassette. This mutant was found to be insensitive to exogenous gibberellic acid (GA_3) application. Homozygous mutant plants showed incomplete penetrance and variable expressivity for plant height and pleiotropic effects including gibberellic acid insensitivity, reduced seed size, panicle length, and female fertility. Single copy insertion of T-DNA and its association with OsAPC6 was confirmed by Southern hybridization, germination on hygromycin, and 3:1 segregation of HPT gene in F_2 from OsAPC6 × Basmati 370 cross. The T-DNA Flanking Region sequenced through thermal asymmetric interlaced polymerase chain reaction showed a single hit on chromosome 3 of japonica rice cultivar Nipponbare in the second exonic Region of a gene which encodes for sixth subunit of anaphase-promoting complex/cyclosome. The candidate gene of 8.6-kb length encodes a 728-amino acid protein containing a conserved tetratricopeptide repeat (TPR) domain and has only a paralog, isopenicillin N -synthase family protein on the same chromosome without the TPR domain. There was no expression of the gene in the mutant while in Basmati 370, it was equal in both roots and shoots. The knockout mutant OsAPC6 interferes with the gibberellic acid signaling pathway leading to reduced height and cell size probably through ubiquitin-mediated proteolysis. Further functional validation of the gene through RNAi is in progress.
Frans E. Tax - One of the best experts on this subject based on the ideXlab platform.
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T-DNA-Associated Duplication/Translocations in Arabidopsis. Implications for Mutant Analysis and Functional Genomics
Plant physiology, 2001Co-Authors: Frans E. Tax, Daniel M. VernonAbstract:T-DNA insertion mutants have become a valuable resource for studies of gene function in Arabidopsis. In the course of both forward and reverse genetic projects, we have identified novel interchromosomal rearrangements in two Arabidopsis T-DNA insertion lines. Both rearrangements were unilateral translocations associated with the left borders of T-DNA inserts that exhibited normal Mendelian segregation. In one study, we characterized the embryo-defective 88 mutation. Although emb 88 had been mapped to chromosome I, molecular analysis of DNA adjacent to the T-DNA left border revealed sequence from chromosome V. Simple sequence length polymorphism mapping of the T-DNA insertion demonstrated that a >40-kbp Region of chromosome V had inserted with the T-DNA into the emb 88 locus on chromosome I. A similar scenario was observed with a prospective T-DNA knockout allele of the LIGHT-REGULATED RECEPTOR PROTEIN KINASE ( LRRPK ) gene. Whereas wild-type LRRPK is on lower chromosome IV, mapping of the T-DNA localized the disrupted LRRPK allele to chromosome V. In both these cases, the sequence of a single T-DNA-Flanking Region did not provide an accurate picture of DNA disruption because Flanking sequences had duplicated and inserted, with the T-DNA, into other chromosomal locations. Our results indicate that T-DNA insertion lines—even those that exhibit straightforward genetic behavior—may contain an unexpectedly high frequency of rearrangements. Such duplication/translocations can interfere with reverse genetic analyses and provide misleading information about the molecular basis of mutant phenotypes. Simple mapping and polymerase chain reaction methods for detecting such rearrangements should be included as a standard step in T-DNA mutant analysis.
Lane Chapman - One of the best experts on this subject based on the ideXlab platform.
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Determining An Unknown Genomic Sequence of Arhodomonas sp. Seminole Through Polymerase Chain Reaction
2014Co-Authors: Shanell Shoop, Bethany Andrews, Robert Pokoo, Amber Anderson, Kelli Perkins, Kelly Stoup, Tiffany Ging, Lane ChapmanAbstract:Our central focus is to see if halo.Contig697rc with an alignment at the head joins with halo.Contig478 with an alignment at the tail. From our initial analysis using BLASTX, we showed possibility of the gap of the amino acid to be filled. We used BLASTX in order to determine the alignment of the given fused contigs and the related protein. Primer Quest allowed us to use the fused contig in order to develop forward and reverse primers to be used in PCR. The gel showed that further testing needed to be done due to no definite sequence being found. We used BLASTX of the genomic DNA Flanking Region by using the fused contig sequence in order to determine a reference protein. No domains were detected. Dr. Canaan ran our PCR a second time, and we were able to utilize ClustalW2 for multiple sequencing alignments to find our final alignment results. Results were limited to the forward primer, so further testing is required.