Genomics, Proteomics and Genetic Engineering
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10 Genomics, Proteomics and Genetic Engineering Genomics and Proteomics • The field of genomics deals with the DNA sequence, organization, function, and evolution of genomes • Proteomics aims to identify all the proteins in a cell or organism including any posttranslationally modified forms, as well as their cellular localization, functions, and interactions • Genomics was made possible by the invention of techniques of recombinant DNA, also known as gene cloning or genetic engineering 2 Genetic Engineering • In genetic engineering, the immediate goal of an experiment is to insert a particular fragment of chromosomal DNA into a plasmid or a viral DNA molecule • This is accomplished by breaking DNA molecules at specific sites and isolating particular DNA fragments • DNA fragments are usually obtained by the treatment of DNA samples with restriction enzymes • Cloning from mRNA molecules depends on an unusual polymerase, reverse transcriptase, which can use a single-stranded RNA molecule as a template and 3 synthesize a complementary DNA (cDNA) cDNA Cloning • The resulting full-length cDNA contains an uninterrupted by introns coding sequence for the protein of interest • If DNA sequence is known at both ends of the cDNA for design of appropriate primers, amplification of the cDNA produced by reverse transcriptase is possible by reverse transcriptase PCR (RT-PCR) 4 Bioinformatics • Rapid automated DNA sequencing was instrumental in the success of the Human Genome Project, an international effort begun in 1990 to sequence the human genome and that of a number of organisms • However, a genomic sequence is like a book using an alphabet of only four letters, without spaces or punctuation. Identifying genes and their functions is a major challenge • The annotation of genomic sequences at this level is one aspect of bioinformatics, defined broadly as the use of computers in the interpretation and management of biological data 5 Functional Genomics • Genomic sequencing has made possible a new approach to genetics called functional genomics, which focuses on genome-wide patterns of gene expression and the mechanisms by which gene expression is coordinated • DNA microarray (or chip) - a flat surface about the size of a postage stamp with up to 100,000 distinct spots, each containing a different immobilized DNA sequence suitable for hybridization with DNA or RNA isolated from cells growing under different conditions • DNA microarrays are used to estimate the relative level of gene expression of each gene in the genome 6 Fig. 10.13 7 8 0.1 0.1 0.1 10 0.1 yceO solA yceP dinI pyrC yceB grxB yceL rimJ yceH mviM mviN flgN flgM flgA flgB flgC flgD flgE flgF flgG flgH flgI flgJ flgK 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840 940 1050 1230 850 950 1060 1150 1240 1250 1340 1350 1440 1550 860 960 970 1070 1160 1360 1450 1560 1570 870 980 1080 1170 1260 1370 1460 1470 1580 880 990 1090 1180 1270 1380 1480 1590 1600 890 1000 1190 1280 900 1 1010 1020 1 10 1100 1110 1 1200 1 1290 1300 1 10 1390 1400 1 10 1490 1500 1 1610 1 9 2820 kb 10 wild type lexA1 +UV 1 -UV oraA recA 0.1 0 20 40 minutes 60 0 20 40 60 minutes lexA -77 10 Reverse Genetics • Mutation has traditionally provided the raw material needed for genetic analysis. The customary procedure has been to use a mutant phenotype to recognize a mutant gene and then to identify the wildtype allele and its normal function • Recombinant DNA technology has made possible another approach, often called reverse genetics, in which wildtype genes are cloned, intentionally mutated in specific ways, and introduced back into the organism to study the phenotypic effects of the mutations 11 People that are unable to remove UV-induced DNA lesions are sensitive to sunlight E. coli that are unable to remove UV-induced DNA lesions are also sensitive to sunlight UV dose WT uvrA uvrB uvrC J/m2 0 5 10 20 40 80 120 12 Gene Targeting • The procedure for introducing mutations into specific genes is called gene targeting • Gene targeting in embryonic stem cells involves homologous recombination between target gene in vector and target gene in genome • Target gene in vector contains unrelated DNA so that recombination disrupts function of targeted gene • Cells with targeted gene mutations can be selected by including an selectable marker in the sequences that are incorporated into the genome 13 Transgenic Animals • Germ-line transformation involves the insertion of genes into the reproductive cells of an organism, which permanently alters the genetic content of the individual and all offspring = transgenic animals • Transgenic animals are used to study the functions of specific genes in development or disease processes 14 Applied Genetic Engineering • Crop plants with improved nutritional qualities can be created • Animal growth rate can be genetically engineered • Engineered microbes can help degrade toxic waste • The production of useful proteins is a primary impetus for recombinant DNA 15 Biomedical Applications • Recombinant DNA technology is used to produce large amounts of medically important proteins • Animal viruses such as retroviruses may prove useful vectors for gene therapy to treat single gene disorders • Recombinant DNA probes detect mutant genes in hereditary disease • A major breakthrough in disease prevention would come through the development of synthetic vaccines produced by recombinant DNA 16
