Chapter 21: Genomes - Genomics: Scientific attempt to sequence, interpret and compare whole genomes (gives a list of genes that are in an organism)o Genome: Complete DNA Sequence of an Organism (Human genome sequence published 2/2001)- Functional Genomics: Observed when genes are expressed/how the productsof those genes interact o Humans have 3 billion nucleotides o Genome shotgun sequencing approach: Genome is broken up into overlapping fragments that are later sequenced/ordered Shotgun sequencing approach:- Sonication: Breaks genome into pieces by using high frequency sound saves (160 kilobases long)- Each piece inserted into a BAC plasmid (artificial chromosomes) Inserted into E.Coli cells so colonies are grown rapidly (BAC library)- Each DNA segment is broken down into a 1-kilobase segment - Each little segment is cloned into a plasmid then inserted into E.Coli cells so a colony of shotgun clones can be created- Computer programs then sequence/analyze cloned fragments- Sequences in order reconstruct the BACs- Ends of reconstructed BACs are analyzed (each 160 kb segment is put in the right place on the chromosome based on overlaps)o Pyrosequencing: Cheaper/faster way than traditional sequencing Works with 1 single DNA fragment instead of multiple copies Only works with fragments that are to small to be reconstructed into a complete genome Compares sequence of individual to a master genome - Bioinformatics: Manages, analyzes and interprets biological information o First genome sequenced was haemophilius influenza bacteriao Most basic task: Interpreting a genome to figure out which bases make which genes (tough to do in eukaryotes)o Computer programs scan genome sequences in both directions and then identify open reading frames (ORFs= possible genes, long stretches of sequence that have NO stop codons) Also look for sequences typical of promoters, operators and other sites ORF can be proven to be a gene by analyzing product and checking to see if it’s homologous to a known gene - Identifying genes in eukaryotic genomes:o Genes contain intronso Most of genome don’t code for a product (can’t scan for ORFs)o Uses reverse transcriptase to make cDNA version of each mRNA, then sequence a portion of resulting molecule to produce an expressed sequence tag (EST = protein coding gene)- Bacterial/Archael genomes: Sequencing of genomes of various strains of same prokaryotic species (compared to genomes of closely relating organisms that have different ways of life)o Bacteria: Correlation between size of genome and metabolic capabilities of organismso Huge genetic diversity between bacteria/archaea (15% unique)o Redundancy among genes is common, even within one prokaryotic genome o Mutation/genetic recombination within species are not only sources of genetic variation - Prokaryotic Genomes: Multiple chromosomes/plasmids are more common o Significant portion of genome was acquired from other distantly related specieso Lateral gene transfer: movement of DNA from one species to another (over 50% of archaen species have 1+ gene from lateral gene transfer) Evidence: If a gene is much more similar to genes in distantly related species than closely related species AND when proportion of G-C base pairs to A-T base pairs in gene or gene series is really different from base composition of the rest of the genome- Lateral gene transfer is often a result because genes are carried on plasmids- Also occurs through transformation (takes DNA fragments from environment) - Environmental sequencing/metagenomics: Practice of cataloging all genes present in a community of bacteria/archaeao Study of GENES (discovery of over 150 types of new bacteria) - Eukaryotic Genomes: Dominated by repeated DNA sequences that occur between genes/inside introns (don’t code for products used by organism)o Much larger than bacterial/archaeic genomes and have noncoding repetitive sequences (Protein sequencing is very small part of human genome) Repetitive sequences make up more than 50% of human genome, over 90% of prokaryotic genome consists of genes Repeated sequences in human genome often result of transposable elements - Transposable elements: Segments of DNA that can movefrom one location in a genome to anothero Selfish genes (parasitic DNA sequences) that survive/reproduce but don’t increase fitness ofhost genome BUT they are parasitic (can disrupt gene function when they insert in a new location)- Long interspersed nuclear elements (LINEs): One type of transposable elemento An active LINE contains all sequences required to make copies of itself, insert them into a new location in the genome Analyses of human genome have revealed that only a few LINEs appear to be complete/potentially active- Repeated sequences: Eukaryotic genomes have several thousand loci called short tandem repeats (STRs small sequences repeated down the length of a chromosome) o Hypervariable: Vary among individuals more than any other type of sequenceo 2 types of STRs: Microsatellites/simple sequence repeats that are repeating units of 2-6 bases Minisatellites or variable number terminal repeats (VNTRs) that are units of 6 to 100 bases. Hypothesis for why micro/mini satellites have so many different alleles: Highly repetitive stretches may misalign whenchromosomes synapse during meiosis (causes unequal crossover different number of repeats)- DNA Fingerprinting: Any individual can be identified by unique genome features (micro/mini satellite loci are markers of choice for DNA fingerprinting) o Process: A sample of DNA is acquired from individual PCR performed using primers that flank a region containing an STR Region is cloned Region can be analyzed to determine number of repeats present- Gene Family: Genes that are extremely similar to each other in structure/function (considered to be part of thesame gene family within species)o Genes that make up gene families are hypothesized to have arisen from a common ancestral sequence through gene duplication- Major Source of New Genes in Eukaryotes: Through duplication of existing genes o When this occurs, an extra copy of a gene is added to the genome o Most common type of gene duplication results from unequal crossing over during meiosis Redundancy of duplicated genes may allow one copy to mutateto create a new gene with different function or regulation (possibly leading to evolution of new traits)o Gene duplication is
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