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The Journal of Cell Biology Jul 2000
Review
Topics: Animals; Cytoskeleton; Drosophila melanogaster; Genome; Genomic Library; Humans
PubMed: 10908588
DOI: 10.1083/jcb.150.2.f63 -
Methods in Molecular Biology (Clifton,... 2009This chapter will discuss various adaptations of the yeast two-hybrid method for analyzing protein interactions that can be used to identify small ubiquitin-related... (Review)
Review
This chapter will discuss various adaptations of the yeast two-hybrid method for analyzing protein interactions that can be used to identify small ubiquitin-related modifier (SUMO) interacting proteins and to determine the nature of the SUMO-protein interactions that occur. SUMO binds to a protein in two different ways: covalently and noncovalently. In a covalent interaction an isopeptide bond forms between the glycine residue at the C terminus of the mature SUMO and a lysine side-chain on the substrate protein. Alternatively, SUMO can interact noncovalently with another protein, usually via insertion of a beta strand from a substrate SUMO-interacting motif (SIM) into a hydrophobic groove next to the SUMO beta2 strand. By mutating either the C-terminal diglycine motif or amino acids within the beta2 strand of SUMO, these respective interactions can be abolished. The expression of the two-hybrid SUMO constructs with either of these mutations can help distinguish the type of interaction that occurs between a SUMO and a given protein. Sumoylation can be verified by independent methods, such as a SUMO mobility shift assay. Finally, the chapter will compare the two-hybrid approach with mass spectrometric analysis as a means of identifying SUMO-interacting proteins.
Topics: Base Sequence; Genomic Library; Mass Spectrometry; Models, Biological; Molecular Sequence Data; Protein Binding; Saccharomyces cerevisiae Proteins; Small Ubiquitin-Related Modifier Proteins; Two-Hybrid System Techniques
PubMed: 19107413
DOI: 10.1007/978-1-59745-566-4_7 -
Methods (San Diego, Calif.) Jul 2009DNA methylation is a critical epigenetic mark that is essential for mammalian development and aberrant in many diseases including cancer. Over the past decade multiple...
DNA methylation is a critical epigenetic mark that is essential for mammalian development and aberrant in many diseases including cancer. Over the past decade multiple methods have been developed and applied to characterize its genome-wide distribution. Of these, reduced representation bisulfite sequencing (RRBS) generates nucleotide resolution DNA methylation bisulfite sequencing libraries that enrich for CpG-dense regions by methylation-insensitive restriction digestion. Here we provide an extensive, optimized protocol for generating RRBS libraries and discuss the power of this strategy for methylome profiling. We include information on sequence analysis and the relative coverage over genomic regions of interest for a representative mouse MspI generated RRBS library. Contemporary sequencing and array-based technologies are compared against sample throughput and coverage, highlighting the variety of options available to investigate methylation on the genome-scale.
Topics: Animals; Base Sequence; CpG Islands; DNA Methylation; DNA-Cytosine Methylases; Genomic Library; Genomics; Humans; Mice; Sequence Analysis, DNA; Sulfites
PubMed: 19442738
DOI: 10.1016/j.ymeth.2009.05.003 -
Genomics Oct 2015The natural history of the amoeba Dictyostelium discoideum has inspired scientific inquiry for seventy-five years. A genetically tractable haploid eukaryote, D....
The natural history of the amoeba Dictyostelium discoideum has inspired scientific inquiry for seventy-five years. A genetically tractable haploid eukaryote, D. discoideum appeals as a laboratory model as well. However, certain rote molecular genetic tasks, such as PCR and cloning, are difficult due to the AT-richness and low complexity of its genome. Here we report on the construction of a ~20 fold coverage D. discoideum genomic library in Escherichia coli, cloning 4-10 kilobase partial restriction fragments into a linear vector. End-sequencing indicates that most clones map to the six chromosomes in an unbiased distribution. Over 70% of these clones contain at least one complete open reading frame. We demonstrate that individual clones and library composition are stable over multiple replication cycles. Our library will enable numerous molecular biological applications and the completion of additional species' genome sequences, and suggests a path towards the long-elusive goal of genetic complementation.
Topics: Cloning, Molecular; Dictyostelium; Escherichia coli; Genome, Protozoan; Genomic Library; Sequence Analysis, DNA
PubMed: 26028264
DOI: 10.1016/j.ygeno.2015.05.008 -
Acta Biochimica Et Biophysica Sinica Feb 2012The discovery of RNA interference (RNAi) has revolutionized genetic analysis in mammalian cells. Loss-of-function RNAi screens enable rapid, functional annotation of the... (Review)
Review
The discovery of RNA interference (RNAi) has revolutionized genetic analysis in mammalian cells. Loss-of-function RNAi screens enable rapid, functional annotation of the genome. Of the various RNAi approaches, pooled shRNA libraries have received considerable attention because of their versatility. A number of genome-wide shRNA libraries have been constructed against the human and mouse genomes, and these libraries can be readily applied to a variety of screens to interrogate the function of human and mouse genes in an unbiased fashion. We provide an introduction to the technical aspects of using pooled shRNA libraries for genetic screens.
Topics: Animals; Genetic Vectors; Genome, Human; Genomic Library; Genomics; Humans; Mice; Oligonucleotide Array Sequence Analysis; RNA Interference; RNA, Small Interfering; Validation Studies as Topic
PubMed: 22271906
DOI: 10.1093/abbs/gmr116 -
Genetic Analysis, Techniques and... Sep 1990Yeast artificial chromosome (YAC) cloning of DNA in agarose is an alternative method to cloning from aqueous solutions. It minimizes any shearing that may result from... (Review)
Review
Yeast artificial chromosome (YAC) cloning of DNA in agarose is an alternative method to cloning from aqueous solutions. It minimizes any shearing that may result from handling of high molecular weight DNA and can be done with nanogram to microgram amounts of material, which facilitates construction of YACs from sources of DNA other than genomic DNA isolated from cells. The average size of the YACs recovered (200-1000 kb) and efficiency of transformation of ligation products (200-1000 cfu/micrograms) are similar to those reported using aqueous protocols. This method has been used to construct chromosome specific YACs, and it should be possible to apply the technique to the construction of chromosome specific libraries using flow sorted chromosomes as source material, and the cloning of restriction fragments isolated by preparative pulsed field gel electrophoresis.
Topics: Animals; Chromosomes, Fungal; Cloning, Molecular; Electrophoresis, Agar Gel; Genome, Human; Genomic Library; Humans; Transformation, Genetic
PubMed: 2091693
DOI: 10.1016/0735-0651(90)90016-9 -
Molecular Plant-microbe Interactions :... Dec 2006The genus Phytophthora belongs to the oomycetes and is composed of plant pathogens. Currently, there are no strategies to mutate specific genes for members of this...
The genus Phytophthora belongs to the oomycetes and is composed of plant pathogens. Currently, there are no strategies to mutate specific genes for members of this genus. Whole genome sequences are available or being prepared for Phytophthora sojae, P. ramorum, P. infestans, and P. capsici and the development of molecular biological techniques for functional genomics is encouraged. This article describes the adaptation of the reverse-genetic strategy of targeting induced local lesions in genomes (TILLING) to isolate gene-specific mutants in Phytophthora spp. A genomic library of 2,400 ethylnitrosourea (ENU) mutants of P. sojae was created and screened for induced point mutations in the genes encoding a necrosisinducing protein (PsojNIP) and a Phytophthora-specific phospholipase D (PsPXTM-PLD). Mutations were detected in single individuals and included silent, missense, and nonsense changes. Homozygous mutant isolates carrying a potentially deleterious missense mutation in PsojNIP and a premature stop codon in PsPXTM-PLD were identified. No phenotypic effect has yet been found for the homozygous mutant of PsojNIP. For those of PsPXTM-PLD, a reduction in growth rate and an appressed mycelial growth was observed. This demonstrates the feasibility of target-selected gene disruption for Phytophthora spp. and adds an important tool for functional genomic investigation.
Topics: Algal Proteins; Amino Acid Sequence; Genetic Engineering; Genomic Library; Genotype; Molecular Sequence Data; Mutagenesis; Mutation; Phenotype; Phytophthora; Sequence Alignment
PubMed: 17153920
DOI: 10.1094/MPMI-19-1359 -
The Plant Genome Mar 2017Common bermudagrass has been widely used as a major warm-season turf, forage, and soil stabilization grass in the southern United States. However, codominant marker...
Common bermudagrass has been widely used as a major warm-season turf, forage, and soil stabilization grass in the southern United States. However, codominant marker development, linkage, and quantitative trait loci (QTL) mapping resources are limited in the important taxon. Accordingly, the objectives of this study were to develop simple sequence repeat (SSR) markers, construct a genetic map, and identify genomic regions associated with establishment rate. Five genomic SSR libraries were constructed, sequenced, and used in the development of 1003 validated SSR primer pairs (PPs). A linkage map was constructed using a first-generation selfed population derived from a genotype A12359 (2 = 4 = 36). A total of 249 polymorphic SSR PPs were mapped to 18 linkage groups (LGs). The total length of the map is 1094.7 cM, with an average marker interval of 4.3 cM. Ninety-eight out of 252 mapped loci (39%) were found to be distorted from the Mendelian 1:2:1 segregation ratio. Among the other 154 nondistorted loci, 88 coupling vs. 66 repulsion linkage phases were observed to confirm the allopolyploid origin of the parent. Ground coverage (GCR) phenotypic data in the establishment stage were collected in two replicated field trials. Quantitative trait loci mapping identified five genomic regions significantly related to the trait. The findings of this study provide valuable genetic tools and resources for genomic research, genetic improvement, and breeding new cultivars in the species.
Topics: Chromosome Mapping; Chromosome Segregation; Chromosomes, Plant; Cynodon; Genes, Plant; Genetic Markers; Genomic Library; Microsatellite Repeats; Phenotype; Quantitative Trait Loci
PubMed: 28464062
DOI: 10.3835/plantgenome2016.07.0074 -
BMC Plant Biology Jan 2010The genus Arachis, originated in South America, is divided into nine taxonomical sections comprising of 80 species. Most of the Arachis species are diploids (2n = 2x =...
BACKGROUND
The genus Arachis, originated in South America, is divided into nine taxonomical sections comprising of 80 species. Most of the Arachis species are diploids (2n = 2x = 20) and the tetraploid species (2n = 2x = 40) are found in sections Arachis, Extranervosae and Rhizomatosae. Diploid species have great potential to be used as resistance sources for agronomic traits like pests and diseases, drought related traits and different life cycle spans. Understanding of genetic relationships among wild species and between wild and cultivated species will be useful for enhanced utilization of wild species in improving cultivated germplasm. The present study was undertaken to evaluate genetic relationships among species (96 accessions) belonging to seven sections of Arachis by using simple sequence repeat (SSR) markers developed from Arachis hypogaea genomic library and gene sequences from related genera of Arachis.
RESULTS
The average transferability rate of 101 SSR markers tested to section Arachis and six other sections was 81% and 59% respectively. Five markers (IPAHM 164, IPAHM 165, IPAHM 407a, IPAHM 409, and IPAHM 659) showed 100% transferability. Cluster analysis of allelic data from a subset of 32 SSR markers on 85 wild and 11 cultivated accessions grouped accessions according to their genome composition, sections and species to which they belong. A total of 109 species specific alleles were detected in different wild species, Arachis pusilla exhibited largest number of species specific alleles (15). Based on genetic distance analysis, the A-genome accession ICG 8200 (A. duranensis) and the B-genome accession ICG 8206 (A. ipaënsis) were found most closely related to A. hypogaea.
CONCLUSION
A set of cross species and cross section transferable SSR markers has been identified that will be useful for genetic studies of wild species of Arachis, including comparative genome mapping, germplasm analysis, population genetic structure and phylogenetic inferences among species. The present study provides strong support based on both genomic and genic markers, probably for the first time, on relationships of A. monticola and A. hypogaea as well as on the most probable donor of A and B-genomes of cultivated groundnut.
Topics: Alleles; Arachis; Cluster Analysis; DNA Primers; DNA, Plant; Genetic Variation; Genome, Plant; Genomic Library; Microsatellite Repeats; Phylogeny; Polymerase Chain Reaction; Sequence Analysis, DNA; Species Specificity
PubMed: 20089171
DOI: 10.1186/1471-2229-10-15 -
BMC Research Notes Mar 2014Japanese amberjack/yellowtail (Seriola quinqueradiata) is a commonly cultured marine fish in Japan. For cost effective fish production, a breeding program that increases...
BACKGROUND
Japanese amberjack/yellowtail (Seriola quinqueradiata) is a commonly cultured marine fish in Japan. For cost effective fish production, a breeding program that increases commercially important traits is one of the major solutions. In selective breeding, information of genetic markers is useful and sufficient to identify individuals carrying advantageous traits but if the aim is to determine the genetic basis of the trait, large insert genomic DNA libraries are essential. In this study, toward prospective understanding of genetic basis of several economically important traits, we constructed a high-coverage bacterial artificial chromosome (BAC) library, obtained sequences from the BAC-end, and constructed comprehensive female and male linkage maps of yellowtail using Simple Sequence Repeat (SSR) markers developed from the BAC-end sequences and a yellowtail genomic library.
RESULTS
The total insert length of the BAC library we constructed here was estimated to be approximately 11 Gb and hence 16-times larger than the yellowtail genome. Sequencing of the BAC-ends showed a low fraction of repetitive sequences comparable to that in Tetraodon and fugu. A total of 837 SSR markers developed here were distributed among 24 linkage groups spanning 1,026.70 and 1,057.83 cM with an average interval of 4.96 and 4.32 cM in female and male map respectively without any segregation distortion. Oxford grids suggested conserved synteny between yellowtail and stickleback.
CONCLUSIONS
In addition to characteristics of yellowtail genome such as low repetitive sequences and conserved synteny with stickleback, our genomic and genetic resources constructed and revealed here will be powerful tools for the yellowtail breeding program and also for studies regarding the genetic basis of traits.
Topics: Animals; Breeding; Chromosome Mapping; Chromosomes, Artificial, Bacterial; Female; Fishes; Genetic Linkage; Genetic Markers; Genome Size; Genomic Library; Male; Microsatellite Repeats; Quantitative Trait, Heritable; Synteny
PubMed: 24684753
DOI: 10.1186/1756-0500-7-200