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chapter 21 notes

by: Anna Perry

chapter 21 notes BIOSC 0160

Anna Perry
GPA 3.5
Foundations of Biology 2
Dr. Swiganova

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About this Document

Basically what she said in class and an outline of the book
Foundations of Biology 2
Dr. Swiganova
Class Notes
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This 8 page Class Notes was uploaded by Anna Perry on Sunday February 22, 2015. The Class Notes belongs to BIOSC 0160 at University of Pittsburgh taught by Dr. Swiganova in Winter2015. Since its upload, it has received 46 views. For similar materials see Foundations of Biology 2 in Biology at University of Pittsburgh.


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Date Created: 02/22/15
Chapter 21 Genomics and Beyond l WholeGenome Sequencing A As technology continues to become faster and less expensive the pace of genome sequencing accelerates B Vocabulary of the 0mics 1 2 3 4 5 Metabolomics study of metabolic activities in Genomics study of the genome Metagenomics study of the collection of genes from different genomes present in a community Proteomics study of the proteins made by cells under particular conditions Transcriptomics study of gene expression genomes C How Are Complete Genomes Sequenced 1 2 Even under the best conditions a single dideoxy sequencing reaction can analyze only about 1000 nucleotides Shotgun Sequencing a In shotgun sequencing a genome is broken up into a set of overlapping fragments that are small enough to be sequenced completely The regions of overlap are then used as guides for putting the sequenced fragments back into the correct order i Step 1 Break DNA at random locations into fragments of 160 kb using sonication ii Step 2 Clone using BACs bacterial arti cial chromosomes lnsert fragments into bacterial arti cial chromosomes grow in E coli cells to obtain large numbers of each fragment iii Step 3 Break into 1kb fragments Purify many copies of each 160kb fragment then randomly break each into a set of 1kb fragments using sonication if enough DNA is broken up there will be overlap between many fragments iv Step 4 Clone using plasmids lnsert1kb fragments into plasmids grow in E coli cells Obtain many copies of each fragment v Step 5 Sequence each fragment Find regions where different fragments overlap vi Step 6 Assemble the sequence from all of the 1kb fragments from each original 160kb fragment by matching overlapping ends Step 7 Assemble the sequence from all of the 160kb fragments from different BACs by matching overlapping ends vii b C In essence the shotgun strategy consists of breaking a genome into many small fragments sequencing each fragment and then putting the sequence data back in the correct order Essentially a quotliving PCR machinequot 3 The Impact of NextGeneration Sequencing a Much faster and cheaper than dideoxy sequencing b These nextgeneration methods are massively parallel meaning that millions of DNA fragments can be sequenced simultaneously in one run of a sequencing machine Downside They produce sequence reads of only about 50200 nucleotides depending on the technology i For piecing together a whole genome these read lengths are too short Especially helpful if a genome or closely related genome has been sequenced and assembly of the fragments uses alignment against the known sequence 4 Bioinformatics a Bioinformatics is a eld that fuses mathematics computer science and biology to manage and analyze sequence data Researchers have created searchable databases i NCBI National Center for Biotechnology Information ii EMBL EBI European Molecular Biology Laboratory European Bioinformatics Institute iii DDJ DNA Databank ofJapan D Which Genomes Are Being Sequenced and Why 1 Most of the organisms that have been selected for wholegenome sequencing have interesting biological properties represent a particular branch of life informative for evolutionary investigations or cause disease a b Ex Genomes of bacteria and archaea from hot environments have been sequenced in the hopes of discovering enzymes useful for hightemperature industrial applications Genomes such as those of rice and maize have been sequenced for crop improvement applications E Which Sequences Are Genes 1 The most basic task in interpreting a genome is to identify which bases constitute genes This is called genome annotation 2 Identifying Genes in Bacterial and Archaeal Genomes a To interpret these genomes biologists begin with computer programs that scan the sequence of a genome in both directions i These programs identify each reading frame possible on the two strands of DNA ii Codons consist of 3 bases so 3 reading frames are possible on each DNA strand for a total of 6 possible reading frames The computer programs draw attention to any quotgene sizedquot stretches of sequence that lack an internal stop codon and are anked by a stop codon and a start codon They also look for sequences typical of promoters ribosome binding sits or other regulatory sites DNA segments identi ed in this way are called open reading frames ORFs If an ORF shares a signi cant amount of sequence with a known gene from another species then it is very likely to be a gene Similarities between genes in different species are usually due to homology i If genes are homologous it means they are similar because they are related by descent from a common ancestor ii Homologous genes have similar base sequences and frequently the same or a similar func on 3 Identifying Genes in Eukaryotic Genomes a b Because coding regions are broken up by introns it is not possible to scan for long ORFs The most productive gene nding strategy has been to isolate mRNAs from cells use reverse transcriptase to produce a cDNA version of each mRNA and sequence a portion of the resulting molecule to produce an expressed sequence tag orEST i ESTs represent proteincoding genes ii To locate the gene researchers use the EST to nd the matching sequence in genomic DNA ll Bacterial and Archaeal Genomes A The Natural History of Prokaryotic Genomes 1 These are compact genomes no introns little inter genic regions between genes extensive use of operons few regulatory sequences 2 The larger the genome the more genes and the more metabolic activity 3 The function of many genes is still unknown 4 Most of the genes found in one species are not shared widely with others a Conserved genes include i Genes coding for ribosomes ii Genes coding for enzymes in DNA replication transcription and translation 5 The content of genomes varies widely even within species 6 Prokaryotic genomes are frequently rearranged during evolution 7 The movement of DNA from one species to another is lateral gene transfer LGT and is a major evolutionary force in evolution of bacteria and archaea a Mechanisms of LGT i Plasmids plasmid genes get integrated into the bacterial chromosome via genetic recombination ii Transformation getting raw DNA from the environment iii Transduction Viruses transfer DNA from one host to another iv Conjugation when bacteria combine they switch genes sometimes B Lateral Gene Transfer in Bacteria and Archaea 1 Biologists use two criteria to identify laterally transferred genes a A gene is much more similar to genes in distantly related species than to those in closely related species b The proportion of GC base pairs to AT base pairs in a particular gene or series of genes is markedly different from the base composition of the rest of the genome C Metagenomics 1 Cataloging all the genes present in a community of bacteria and archaea is called metagenomics or environmental sequencing The subject is genes not species 2 A common strategy is to start by studying a simple system then go on to more complex systems lll Eukaryotic Genomes A Genome size varies widely but the number of genes is much more similar B Many eukaryotic genomes are dominated by repeated DNA sequences that occur between genes or inside introns and do not code for products used by the organism 1 These repeated sequences greatly complicate the work of aligning and interpreting sequence data C Transposable Elements and Other Repeated Sequences 1 The exons and regulatory sequences associated with genes make up a relatively small percentage of the genome a Over 90 of bacterial genome consists of genes b About 50 of eukaryotic genome consists of repeated sequences that do not code for a product used by the cell 2 Transposable elements are segments of DNA that are capable of being inserted into new locations or transposing in a genome a They behave similarly to some viruses that insert into the genome b However they seldom leave their host cell instead they make copies of themselves that become inserted in new locations c They are passed from mother to daughter cell and from pa rents to offspring 3 A transposable element is an example of sel sh DNA a DNA sequence that has invaded a host and persists and reproduces using the resources of the host 4 Like any mutation a transposable element insertion into a new site in the genome can have negative neutral or positive effects on tness They play a major role in evolution 5 How Do Transposable Elements Work a A transposable element called a long interspersed nuclear element LINE exists in the genome b RNA polymerase transcribes the LINE producing LlNE mRNA c LlNE mRNA exits the nucleus and is translated d LlNE mRNA and proteins enter the nucleus e Reverse transcriptase makes LlNE cDNA from mRNA then makes cDNA double stranded f lntegrase cuts chromosomal DNA and inserts LlNE cDNA g New copy of the LlNE is integrated into a new position in the genome 6 Repeated Sequences and DNA Fingerprinting a Many eukaryotic genomes have several thousand loci of relatively short DNA sequences They are repeated in tandem one after another contiguoust along part of a chromosome b These tandem repetitive DNA sequences make up 3 of the genome and fall into two major classes i Repeating units that are just 26 bases long are microsatellites Also known as short tandem repeats STRs o The origin of microsatellites is when DNA polymerase skips or mistakenly adds extra bases during replication ii Repeating units that are long about 6100 bases are minisatellites or variable number tandem repeats VNTRs c Unequal crossover When homologous chromosomes align incorrectly during prophase of meiosis l Instead of lining up in exactly the same location the two chromosomes pair in a way that matches up bases in different DNA repeats i Resulting chromosomes have different numbers of repeats ii Repeated sequences are prone to unequal crossover because their homologs are so similar that they are likely to misalign d DNA ngerprinting refers to any technique for identifying individuals based on the unique features of their genomes i Because microsatellite and minisatellite sequences vary so much they are now the sequences of choice for DNA ngerprinting D Gene Families 1 In eukaryotes the major source of new genes is the duplication of existing genes a Within a species genes that are similar to each other in structure and function are considered to be part of the same gene family b The degree of sequence similarity among members of a gene family varies 2 How Do Gene Families Arise a Genes that make up gene families are hypothesized to have arisen from a common ancestral sequence through gene duplication When gene duplication occurs an extra copy of a gene is added to the genome b The most common type of gene duplication results from unequal crossover during meiosis c Genesized segments of chromosomes can be deleted or duplicated if homologous chromosomes misalign during prophase of meiosis l and an unequal crossover occurs 3 New Genes New Functions a If mutations in the duplicated sequence alter the protein product so that it performs a valuable new function then an important new gene has been created b A member of a gene family that resembles a working gene but does not code for a functional product because of a mutation is a pseudogene i In the human genome there are roughly as many pseudogenes as functional genes 4 Lateral Gene Transfer in Eukaryotes a LGT seems to be relatively rare in eukaryotes compared with prokaryotes E Insights from the Human Genome Project 1 Less than 2 of the human genome consists of protein coding exons and nearly half is made of transposable elements 2 lntrons make up over 14 of the genome and are 17 times more abundant than proteincoding exons 3 Of all the observations the most striking is that organisms with complex morphology biochemistry and behavior do not have particularly large numbers of genes a This can be explained by alternative splicing b The alternative splicing hypothesis claims that multicellular eukaryotes do not need enormous numbers of distinct genes Instead more extensive use of alternative splicing in these organisms creates different proteins from the same gene c About 90 of genes produce transcripts that are alternatively spliced with an average of more than 3 distinct transcripts per gene IV Functional Genomics Proteomics and Systems Biology A What Is Functional Genomics 1 Largescale analysis of gene expression is part of functional genomics research on how genes work together to produce a phenotype 2 A DNA microarray consists of as many as 1 million different singlestranded DNA segments that are permanently attached at one end to a glass slide or silicon chip a Each DNA sequence is linked to the slide or chip in a known location and serves as a probe for a speci c transcript 3 Using A DNA Microarray a Isolate mRNAs and use reverse transcriptase to prepare singlestranded cDNA b Label cDNA use uorescent tags to mark each cDNA c Hybridize a microarray labeled cDNAs will bind to complementary DNA probe sequences on the slide d Shine laser light on one spot at a time to induce uorescence 4 A microarray lets researchers study the expression of thousands of genes at a time As a result they can identify which sets of genes are expressed in concert under speci c sets of conditions 5 The approach of extensive sequencing of populations of DNA or cDNA molecules is called deep sequencing B What Is Proteomics 1 Instead of studying individual proteins or how two proteins might interact proteomics is based on studying all the proteins present at once 2 One approach to studying proteinprotein interactions is similar to the use of DNA microarrays a Large numbers of proteins are attached to a glass plate b They are treated with an assortment of proteins produced by the same organism and are labeled with a uorescent or radioactive tag c If any labeled proteins bind to the proteins in the microarray the two molecules may also interact in the cell C What Is Systems Biology 1 Systems biology aims to understand how interactions between the individual parts of a biological system create new properties a Complex properties that arise from the interaction of simpler elements are emergent properties b DNA replication metabolism cancer and the development of an organism are all emergent properties because they come about from the interactions of simpler elements genes amp proteins 2 Genomics and proteomics provide the parts list needed to start systems biology studies which opens the door to knowing how cells work in health and disease


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