Chapter 14 - Genomes and Genomics

• One of the most powerful means to advance the analysis and annotation of genomes is by comparing with the genomes of related species.
• In bacterial genomics, comparisons of pathogenic and nonpathogenic strains have revealed many differences in gene content that contribute to pathogenicity.
• Two key elements in functional genomics are the transcriptome, the set of all transcripts produced, and the interactome, the set of interacting gene products and other molecules that together enable a cell to be produced and to function.
• The function of individual genes and gene products for which classical mutations are not available can be tested through reverse genetics—by targeted mutation or phenocopying.
• The reporter gene is lacZ, which resides on a yeast chromosome.
• Yeast two-hybrid systems are used to study the protein–DNA interactome.
• ChIP (chromatin immunoprecipitation) is a technique for isolating the DNA and its associated proteins in a specific region of chromatin so that both can be analyzed together.
• In ChIP, proteins are cross-linked to DNA, the chromatin is broken into small pieces, an antibody to the target protein is added, and the DNA bound in the immune complex is analyzed after cross-linking is reversed.
• ChIP-seq is a variation of ChIP that identifies all the binding sites of a protein in a sequenced genome.
• Reverse genetics involves disrupting the function of a gene to understand phenotypes in native conditions.
• Advances in genomic technologies have made it possible to catalog the transcripts and proteins as well as protein–DNA and protein–protein interactions found in normal and diseased cells.
• Reverse genetics is the gold standard for establishing the function of a gene or genetic element.
• One approach to reverse genetics is to introduce random mutations into the genome, but then to hone in on the gene of interest by molecular identification of mutations in the gene.
• A second approach to reverse genetics is to conduct a targeted mutagenesis that produces mutations directly in the gene of interest.
• A third approach to reverse genetics is to create phenocopies—effects comparable to mutant phenotypes—usually by treatment with agents that interfere with the mRNA transcript of the gene.
• Functional genomics is the use of an expanding variety of methods, including reverse genetics, to understand gene and protein function in biological processes.
• In the 1980s, some scientists realized that a large team of researchers making a concerted effort could clone and sequence the entire genome of a selected organism.
• Having the entire sequence of the human genome raises questions such as: How many genes does it contain? How are they distributed, and why? What fraction of the genome is coding sequence? What fraction is regulatory sequence? How is our genome similar to or different from other animals?
• The basic techniques needed for sequencing entire genomes were already available in the 1980s, but the scale that was needed to sequence a complex genome was far beyond the capacity of the research community then.
• Genomics in the late 1980s and the 1990s evolved out of large research centers that could integrate these elemental technologies into an industrial-level production line.
• Advances in information technology aided the analysis of the resulting data.
• New technologies can now obtain more than 1 × 10^ bases of sequence in a working day on a single instrument, representing an approximately 1 million-fold increase in throughput over earlier instruments used to obtain the first human genome sequences.
• Random mutagenesis is a method that allows the recovery of mutations in a gene of interest from a genome containing random mutations.
• Targeted mutagenesis is a method that allows the recovery of mutations in a gene of interest from a genome containing targeted mutations.
• Creating phenocopies is a method that allows the recovery of mutations in a gene of interest from a genome containing phenocopies.
• In reverse genetics, the gene of interest is identified in the mutagenized genome and checked for the presence of mutations.
• Targeted mutagenesis can be labor intensive, but, after the targeted mutation has been obtained, its characterization is more straightforward.
• In reverse genetics, the gene of interest is identified in the mutagenized genome and checked for the presence of mutations.
• Targeted mutagenesis is the most precise means of obtaining mutations in a specific gene and can now be practiced in a variety of model systems, including mice and flies.
• Reverse genetics by phenocopying involves creating phenocopies, which are organisms that exhibit the same phenotype as an organism with a mutation in a specific gene.
• Phenocopying can be applied to a great many organisms regardless of how well developed the genetic technology is for a given species.
• One of the most exciting discoveries of the past decade or so has been the discovery of a widespread mechanism whose natural function seems to be to protect a cell from foreign DNA.
• The expression pattern of the reporter gene corresponds to the native pattern of expression of the mouse ISL1 gene on day 11.5 of gestation.
• The expression pattern of the reporter gene strongly suggests that the conserved element is a regulatory region for the ISL1 gene in each species.
• The success of this approach suggests that many additional human noncoding regulatory elements will likely be identified on the basis of sequence conservation and the activity of those elements in reporter assays.
• An ultraconserved element lying near the human ISL1 gene was coupled to a reporter gene and injected into fertilized mouse oocytes.
• The expression pattern of the reporter gene strongly suggests that the conserved element is a regulatory region for the ISL1 gene in each species.
• The success of this approach suggests that many additional human noncoding regulatory elements will likely be identified on the basis of sequence conservation and the activity of those elements in reporter assays.
• Predictions of mRNA and polypeptide structure from genomic DNA sequence depend on the integration of information from cDNA and EST sequence, binding-site predictions, polypeptide similarities, and codon bias.
• Only a small part of the human genome encodes polypeptides; that is, somewhat less than 3 percent of it encodes exons of mRNAs.