microorganisms applications in molecular biology

8/12/2019 Microorganisms Applications in Molecular Biology

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Microorganisms:Applications inMolecular BiologyJill B Keeney, Juniata College, Huntingdon, Pennsylvania, USA

Microorganisms, most notably yeast and bacteria, are used in research and industry for cloning genes, replicating DNA and producing purified proteins. Yeast, in particular, iswidely used as a model organism for studying a variety of cell functions.

Growth of MicroorganismsA variety of microorganisms are used by molecularbiologists to study gene structure and function. Somescientists study microorganisms because they are patho-

genic to plants, humans or other animals, and throughlearning more about these pathogenic organisms, effectivedrugs and strategies for infection control can be developed.Microorganisms, particularly bacteria and yeasts, are alsoused by many scientists as a tool for molecular biologyresearch. Bacteria cultures grow very quickly, and moststrains used in molecular research divide in less than45 min. Thus, a single bacterial cell can, in 16 h, producesufficient numbers of cells for isolating deoxyribonucleicacid (DNA) and many proteins. A yeast cell takesapproximately 1.5 h to divide, and thus also requires fairlyshort culture times. By contrast, a mammalian cell requires18 h to complete cell division, and several days of growth

are needed to produce comparable cell numbers.Microorganisms are grown in the laboratory on solidmedia in Petri dishes or in liquid media in asks. Mediacontain essential components needed for cell growth,including a carbon source, a nitrogen source, and essentialvitamins and cofactors. Additionally, media often containantibiotics or dened components that allow for selectivegrowth of cells, especially cells containing recombinantDNA. An essential skill for culturing microorganisms isaseptic technique. Media prepared in the laboratory mustbe kept sterile, and cultures must be free of contaminationby other microorganisms present in the laboratory.

Bacteria: Prokaryotic Unicellular OrganismsOrganisms are generally classied as prokaryotic (nonucleus or other organelles) or eukaryotic (containingorganelles). Bacteria are unicellular prokaryotes: theorganism is one cell. Some bacteria have very simplegrowth requirements.This,in combination with their rapid

division time, makes themideal research organisms. By farthe most frequently used bacterial species in molecularbiology is Escherichia coli . E . coli is a Gram-negativebacteria found in the intestines of many mammals and canoften be pathogenic. However, the commonly usedlaboratory strains do not carry toxins and therefore arenot considered pathogenic. E . coli is regularly used forcloning genes and growing plasmid DNA (discussedbelow) from many different organisms. Since the geneticcode is conserved among living organisms, DNA from anysource can be replicated in E . coli .

Not all bacteria are, however, easy to culture. Forexample, thebacterium that causes tuberculosis grows veryslowly in the laboratory, and thus is difficult to culture.Other bacteria are naturally found in extreme or uniqueenvironments and thus require specialized conditions forculture in the laboratory. Some examples include bacteriathat grow in deep oceanic thermal vents, in anaerobicenvironments,or in acid lakes. Although these bacteria arenot routinely cultured in the laboratory, they containenzymes that are essential to molecular biology research.Probably the most widely used are the DNA polymerasesisolated from thermophilic (heat-loving) bacteria, used in atechnique called polymerase chain reaction (PCR). Mole-cular biologists also use restriction enzymes to clone andanalyse DNA. Restriction enzymes, isolated from manydifferent species of bacteria, act as a natural host defencemechanism to prevent bacteriophage infection. Theseenzymes cut DNA molecules at specic sequences andmany are routinely used in research and forensic DNAanalysis.

Article Contents

Introductory article

. Growth of Microorganisms

. Bacteria: Prokaryotic Unicellular Organisms

. Bacterial Genes and Genome

. Bacterial Genetics

. Plasmids as Vectors for Amplifying Foreign DNA

. BacteriophagesasVectorsfor Amplifying ForeignDN

. Yeast: EukaryoticUnicellular Organisms

. Yeast Genes and Genome

. Yeast Genetics

. Tissue Culture Cells

. Shuttle Vectors: Plasmids that can Replicate in TwoDifferent Hosts

. Summary

ENCYCLOPEDIA OF LIFE SCIENCES 2001 , John Wiley & Sons, L td. www.els.net


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Bacterial Genes and GenomeThe bacterial genome consists of one circular chromo-some, attached to the bacterial cell wall. This chromosomecontains all the genes the bacterium needs to replicateDNA and make proteins needed for cell growth. Each timea bacterium divides, the entire chromosome is replicatedonce and a copy goes with the new cell. This is regarded asasexual reproduction, and the new cell is an exact copy, orclone, of the original cell.

Bacterial genes and promotersBacterial cells regulate when genes areexpressedat thelevelof transcription and translation. This regulation assuresthat the cell does not wasteenergy expressing a protein thatis not needed.One of the best understood examples of generegulation is the control of the lac operon of E . coli , whichcontains the genes needed to break down the sugar lactose(Figure 1). These genes are regulated together so that theyare only expressed when the bacterium has lactoseavailable to use as an energy source. Expression of theoperon is regulatedby DNAsequences locatednear the siteon the DNA where transcription begins. Ribonucleic acid(RNA) polymerase is able to specically bind to a specicsequence, called the promoter, and initiate transcription.Operons also contain operator sequences bound byproteins which help RNA polymerase bind better (activa-tors) or which prevent binding of RNA polymerase(repressors). For example, the operator of the lac operonnormally has a repressor bound to it, so RNA polymerase

cannot bind the promoter and transcribe the genes. Thepresence of lactose in the cell causes the removal of therepressor protein, andthegenes are then expressed.Thestudy of transcriptional regulation in E . coli has led tomajor advances in understanding how transcription isregulated in all organisms.

Bacterial genomeMost bacterial genomes are between 1 million and 10million base pairs in size. Although the function andregulation of some bacterial genes have been studiedextensively, the function of many bacterial genes remainsunknown. Recent sequencing initiatives have producedcompleted genomic sequences of several bacterial species,which are available to scientists on the Internet. Thesequences of these different species can then be comparedto each other, yielding valuable clues about gene function,mechanisms of pathogenicity and evolutionary relation-ships.

Bacterial GeneticsAs mentioned above, bacterial cells reproduce asexually,always producing an exact clone. For evolutionarysurvival, bacteria also have several mechanisms by whichthey trade or share their DNA with other individualbacterial cells, resulting in variability in genomic content.Genes that are critical for survival in specic situations,such as genes for antibiotic resistance, are exchangedbetween individuals, including cross-species exchange.There are three main mechanisms for exchanging DNAbetween individual bacterial cells:

. Transformation is the process by which bacteria pick upDNAfromtheir immediate surroundings. In the naturalenvironment, the source canbe DNAfragments releasedfrom dead bacterial cells. In the laboratory, this is themost common method used in molecular biology toclone and replicate genes in bacteria (see below).

. Transduction is the exchange of genetic informationmediated by bacteriophages (discussed below). Bacter-iophages are also commonly used tools for cloning andmanipulating DNA.

. Conjugation is the process by which bacteria exchangeDNA through specialized protein structures called sexpili.

Each of these mechanisms of DNA transfer is employed ina variety of molecular biology techniques to manipulateDNA. Some applications of transformation and phageinfection are discussed below.

O P lac Z Y A

Repressor

RNA polymerase

O P lac Z Y A

(a)

(b)

Figure 1 The Escherichia coli lac operon. The lac operon contains threegenes ( lacZYA ) whose products are neededfor E. coli to utilize lactoseas an energy source. These genes are regulated by operator (O) andpromoter (P) sequences immediately adjacent to the genes. When nolactose is present in the bacterial cell environment (a), repressor proteinbound at the operator (O) prevents RNA polymerase from binding at thepromoter (P). When lactose is present (b), inducer (a lactose byproduct,represented as . ) is produced, which can bind to the repressor. Therepressorthenno longerbinds theoperator,and thepromoter is accessibleto RNA polymerase.

Microorganisms: Applications in Molecular Biology

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Plasmids as Vectors for AmplifyingForeign DNAIn addition to thecircular chromosome, bacteria maycarrysmaller extrachromosomal circular DNA, called plasmids,which are replicated in the cell but not necessarily carriedalong in cell division. Often these plasmids carry non-essential genes that allow bacteria to grow in specialenvironmental conditions, such as genes to degrade uniquenutrients in the environment (e.g. oil) or genes to breakdown toxins in the environment (e.g. antibiotics). Over thepast few decades, bacterial geneticists have isolated andaltered these plasmids, and now hundreds of different

specialized plasmids exist. Plasmids are used by molecularbiologists in all elds of research as vectors for cloning andamplifying DNA from many different organisms. Thestructure and composition of DNA is universal among allliving organisms. Thus, DNA from any other foreignorganism, if inserted or cloned into a bacterial vector, canbe amplied and then isolated from a bacterial cell. Thisprocess allows scientists to produce large quantities of aspecic DNA sequence for experimental study.

Most standard bacterial vectors have three essentialcomponents:1. A multiple cloning site containing several unique

restriction enzyme sites, allowing foreign DNA to beeasily inserted into the plasmid.

2. A selectable marker to select the bacteria whichreceived the vector DNA during a bacterial transfor-mation. The selectable marker is often an antibioticresistance gene such as b-lactamase, an enzyme thatdegrades ampicillins.

3. An origin of replication so that the bacterial DNApolymerase will replicate the plasmid, including theinserted foreign DNA. Origins of replication vary so

that some vectors will be present in only a few copies,while other vectors may have many copies within anindividual cell.

Circular vector DNA can be cut, or linearized, at one ortwo of the restriction sites in the multiple cloning site.Foreign DNA is then combined with the linearized vectorand the enzyme ligase joins the ends together, forming acircular plasmid containing foreign DNA ( Figure 2).

Foreign DNA

Multiplecloning

site

Originof replication

Vector

Selectablemarker

(ampicillinresistance)

LigationTransformation

into bacteria

Petri plate of mediumcontaining ampicillin

Bacterialchromosome

Productionof enzymeto degradeampicillin

Bacterialcell wall

(a)

(b)

Figure 2 Ligating, transforming and selecting plasmid DNA. (a) Linearized vector is ligated to a fragment of foreign DNA, forming a circular plasmid. When this plasmid is transformed into bacteria, the ampicillin resistance gene is expressed, producing an enzyme that degrades ampicillin. (b) Whenthebacteriaareplated on to mediacontainingampicillin, cellscontaining theplasmid (andthus ampicillin-degrading enzyme)growand formcolonies(large, dark circles).Many of thecells do not contain theplasmid andfail to divide(represented as small, faint circles, although they would not be at allvisible).



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A plasmid can be put into a bacterial cell by transforma-tion, as outlined in Figure 2. In the laboratory this is easilyaccomplished by placing freshly grown cells into calciumchloride solution, adding the plasmid DNA, and allowingthe DNA to adsorb to the bacterial membrane. Followinga brief heat shock, some of the bacterial cells will take upthe DNA. The cells are then plated and allowed to growovernight to form bacterial colonies. Electroporation isanother common transformation process, in which a quickelectrical pulse is delivered to cells. During the temporarydisruption of the cellular membrane, DNA enters the cell.

The transformation process is inefficient, so that only asmall fraction of the bacterial cells actually take up theplasmid DNA. The selectable marker on the plasmidallows for selection of these cells by plating the cells onmedia containing antibiotic. For example, after transfor-mation of vector DNA carrying the b-lactamase gene, thebacterial cells are plated on to media containing ampicillin.Bacterial cells that have obtained the plasmid beginproducing the enzyme that degrades ampicillin and areable to begin cell division, forming colonies after about16 h. The ampicillin inhibits growth of all bacterial cellslacking the plasmid. A single colony, or clone, containingthe plasmid can then be transferred into a large volume of liquid media, and allowed to grow overnight. This culturewill yield large quantities of the puried plasmid for furtherstudy and analysis.

Expressing genes in bacteriaSometimes further analysis of a gene requires expressingthe protein product. Expressing a gene from anotherspecies in E . coli requires more specialized conditions thansimply replicating the DNA. The promoters of genes fromother organisms are not likely to be recognized by thebacterial RNA polymerase. Thus, the gene to be expressedmust be cloned in such a way that the transcription startsiteis spaced properly with a bacterial promoter.Theuse of an inducible promoter, such as the promoter from the lacoperon, allows for tight control of gene expression. Thecloned gene is not expressed until the cells are cultured withinducer, a chemical substance resembling lactose. Tran-scription is then activated and the bacterial cell produceslarge quantities of the protein. If the cloned DNA is from aeukaryote, protein expression in E . coli may also requireremoval of introns from the gene prior to cloning, asbacteria lack the cellular machinery to splice RNA. This isaccomplished by isolating messenger RNA (mRNA) fromthe eukaryotic cell and reverse transcribing it into DNA,thus creating complementary DNA (cDNA). The cDNA,if cloned in proper alignment with a bacterial promoter,can then be transcribed and translated, producing largequantities of the protein for purication. This process isused to isolate large quantities of proteins for use inbiotechnology and medicine.

Bacteriophages as Vectors for Amplifying Foreign DNABacteriophages are viruses of the bacterial world. Theirentire life cycle, shown in Figure3 , exists within the connesof a bacterial cell. During this replication process, bacterial

DNA may be packaged into the phage particles with thephage DNA. This DNA is then ultimately transferred toanother bacterial cell in a process called transduction.Foreign DNA can be inserted into the phage genome sothat it is packaged into the phage particles. When bacteriaare infected with the recombinant phage, the foreign DNAis then amplied along with the phage DNA. As the phagesreplicate and lyse the bacterial cells, phage particles arereleased into theculturesupernatant.These phage particlescan then be isolated and the cloned DNA recovered inlargequantities for use in other experiments.

There are several specic situations in which a molecularbiologist would choose to use a bacteriophage, rather than

a plasmid, for working with cloned DNA. The bacter-iophage genome is much larger than plasmids, and can beused as a tool to clone fragments of DNA too large to bereplicated as a plasmid. While bacterial vectors canefficiently replicate inserts of DNA up to about 10 000 bpin length, bacteriophages are routinely used for replicatinginserts of up to 24 000 bp. Phages arealsouseful forspecicmolecular techniques that require single-stranded DNA,such as making specic mutations to cloned DNA (atechnique called site-directed mutagenesis) or DNA

Mature phageparticles

Phageproteins

Phage DNA

PhageRNA

1

Phageparticle

2

3

4

5

6

7

Bacterialribosome

Figure 3 Bacteriophage life cycle. The bacteriophage, a capsule of proteins (called a phage particle)surroundingthe phage DNA, anchors onto the surface of the bacterial cell (1) and injects its DNA into the

cell (2). Once inside the bacterial cell, the bacteriophage DNA istranscribedinto RNA(3),andalso replicated toproducemany copiesof thephageDNA(4).TheRNA istranslated bythebacterial ribosomes(5),so thatnumerous phage particles, containing phage DNA, can be assembled(6). The phages then signal lysis of the bacterial cells, releasing amultitudeof newphages (7),which can theninfect neighbouring bacteria.


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sequencing. Some phages, most notably M13, packageonly one DNA strand into the phage particle, and thus arecommonly used to isolate single-stranded DNA. Phage-mids are common vectors that contain the requiredsequences both for replicating in bacteria and for packa-ging DNA into phage particles. When transformed intobacterial cells, the bacterial replicating sequence allows forselection of clones. When a clone is infected with an M13helper phage, the M13 phage sequences direct replicationand packaging of the DNA into phage particles, allowingisolation of single-stranded DNA.

Yeast: Eukaryotic Unicellular OrganismsYeast, like bacteria, is a unicellular organism; however,yeast is characterized as eukaryotic because cellularfunctions are compartmentalized into distinct organelles,such as the nucleus, the endoplasmic reticulum and the

mitochondria. As with bacteria, scientists study a numberof different yeast species, many of them pathogenic. Someyeast species are easy to grow, replicate quickly and areeasy to manipulate genetically. Yet, because yeasts areeukaryotic, they can also be used as a tool to study complexcellular functions such as the cell cycle, chromosomesegregation, transcription, intracellular signalling andprotein modication. Many processes that occur in largereukaryotic cells, such as human cells, can be more readilystudied in yeast. The most commonly used species are thebudding yeast Saccharomyces cerevisiae (also known asbrewers or bakers yeast) and the ssion yeast Schizosac-charomyces pombe . This discussiondeals with S . cerevisiae ,

the most commonly used yeast in molecular biologyresearch.

Yeast Genes and GenomeS . cerevisiae contains 16 chromosomes. During mitotic celldivision each of these chromosomes must be replicated andthen segregated. S . cerevisiae is called a budding yeastbecause, as the cell divides, the parent cell puts out a smallbud. One set of the duplicated chromosomes is segregatedto this bud which then pinches off and becomes a separatedaughter cell.

Yeast promotersThe promoters of yeast and other eukaryotes are morecomplex than prokaryotic promoters. Eukaryotic promo-ters often contain a TATA box sequence near the sitewhere transcription initiates, but must also have other,more distant, sequences to direct RNA polymerase tobegin transcription at the proper location and to controlhow often transcription initiates. These are called up-

stream regulatory sequences and are targets of nuclearproteins (transcriptional regulators) that regulate theactivity of RNA polymerase at the promoter. The functionof these sequences is often studied by cloning the promoter(including the regulatory sequences) next to a reportergene, an enzyme for which activity can be easily assayed.The cloned promoter is mutated to determine whatsequences are important for activation and suppressionof gene expression. Much of our knowledge of eukaryotegene regulation is a result of such yeast studies.

Yeast genomeIn 1996, S . cerevisiae earned the distinction of being therst eukaryote to have its genome entirely sequenced, atotal of 12 million bases. The sequence and muchsupporting data are available on the Internet (see FurtherReading), providing an extremely valuable resource toyeast geneticists and to genetic studies. The sequence datahas been analysed to determine where putative gene codingregions and repeated regions of DNA are located. A largenumber of probable gene sequences are of unknownfunction. A major component of the yeast genome projectis developing strategies to determine the cellular functionof these genes.

Yeast GeneticsYeasts generally exist as haploid organismsof twodifferentmatingtypes,termeda and a in S . cerevisiae . Twostrainsof opposite mating type can be cultured together to form adiploid cell. The diploid cell normally undergoes rapidmeiosis and sporulation, producing four haploid spores.During this process, genetic information is randomlyshuffled so that each spore produced contains some geneticinformation from each parent. Yeast strains commonlyused in research have been altered so that both haploidsand diploids can be cultured for genetic analysis.

Yeast plasmidsYeast plasmids, like bacterial plasmids, also have amultiple cloning site, a selectable marker, and often, butnot always, an origin of replication. The selectable markermay be a drug effective against yeast cells, but is mostcommonly an auxotrophic marker. Yeast does not haveany essential amino acids and can synthesize all the aminoacids from carbon and nitrogen supplied in growthmedium. Mutations in enzymes needed to synthesizecertain amino acids, called auxotrophic markers, areimportant selection tools for yeast geneticists. Forexample, a yeast strain carrying a mutation in a gene forsynthesizing histidine ( his3) cannot grow on media lackinghistidine. However, if a plasmid containing the missing



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gene ( HIS3 ) is transformed into the yeast cell, theindividual cells that obtain the plasmid can now growand form a colony on media lacking histidine. Severaldifferent common auxotrophic markers exist and thus agiven yeast strain can harbour several plasmids at once.Since the auxotrophic marker is not essential to cell growthin rich medium, these plasmids can also easily be removedfrom the cell, in a process called plasmid shuffling.

Using various auxotrophic markers, plasmids have beenconstructed as tools for studying the function of yeastgenes and cellular processes. As with E . coli , plasmids canbe easily transformed into yeast. Yeast plasmids can existas extrachromosomal DNA, replicating as an independentunit, if they contain an origin of replication. Yeastmolecular biologists also use integrating plasmids orsimply transform a fragment of DNA containing aselectable marker. The DNA being transformed will insertinto the chromosome that has DNA sequences matchingthose on the transformed DNA, in a process calledhomologous recombination. This characteristic of yeasthas proven to be a very powerful tool, as researchers caneasily replace a wild-type yeast gene with any mutation, orremove it completely, to see the effects on the cell. Yeastplasmids also allow for selection of important mutations instudying gene function. Many mutations are introducedinto a cloned gene, which is then transformed into yeast,and the resulting colonies selected for a particularphenotype. The plasmid from the selected colonies can beisolated and transformed into E . coli for DNA sequencingto determine which mutations affect gene function. Theability to introduce and select for specic mutations inyeast genes and to move plasmids easily in and out of yeastfor genetic studies has resulted in huge advances in ourunderstanding of eukaryotic cellular processes.

Cloning large pieces of DNASometimes it is desired to have a very large piece of DNAcloned into a yeast cell. This can be accomplished byconstructing a yeast articial chromosome, or YAC. AYAC contains an auxotrophic marker and a centromericand a telomeric sequence found on a yeast chromosome,but the rest of the DNA can be from another organism.Whereas plasmids are generally not much bigger than10 000 bp, YACs can be millions of base pairs, allowing

huge pieces of DNA to be cloned into yeast at once. Sincemany yeast and human genes are functionally related,YACs can be used to study large human genes in yeast.

Expressing genes in yeastYeast plasmids are also used to express large quantities of aspecic protein for research and the biotechnologyindustry. For example, the hepatitis B virus vaccine iscommercially produced in yeast. A foreign gene to be

expressed can be cloned next to an inducible yeastpromoter, like the process used for expressing proteins inbacteria. Some advantages of using yeast cells for proteinproduction are that the expressed protein will containcarbohydrate modications specic to eukaryotic cells, ismore likely to be folded correctly, and will be free of potentially toxic bacterial cell components.

Tissue Culture CellsTissue culture allows scientists to study the individual celltypes of larger multicellular organisms (both plants andanimals) by growing cells individually in a ask. As withmicroorganisms, the cells will divide if given the appro-priate environment and nutrients. The growth require-mentsof cells of larger eukaryotes are not assimple asthoseof microorganisms, so special media and environments are

required. For example, in growing mammalian cells, theenvironment that a cell normally experiences in the wholeorganism must be duplicated in the laboratory. In ourbodies, our cells are kept at a very constant temperature,supplied a very precise amount of oxygen, and bathed inblood or plasma that is kept at a very specic pH. Specialincubators and media that mimic these conditions arerequired for growing mammalian cells. The media, inaddition to carbon and nitrogen sources, must also containother growth factors and serum components. These aregenerally supplied by bovine (cow) serum that is added tothe media. In our bodies, our skin and our immune systemkeep bacteria and yeast out of our blood and tissues. In

tissue culture, the media and cells are in direct contact withthe environment of the laboratory. Work with thesecultures must be done in sterile cabinets, called laminarow hoods. The persons culturing the cells must be suretheir hands are scrubbed of all microorganisms beforeworking with the cultures. Without these precautions, thefaster replicating microorganisms will overrun the culture.

Primary lines and transformed linesCells thatare normally part of a multicellular organism areprogrammed to stop dividing at a certaincell density andtodie at a specic time. Thus, when cells are moved from anorganism into tissue culture, they normally will grow to acertain cell density and then die off. In order to maintaincells in culture over long periods of time, cells must beimmortalized or transformed. Tumour or cancer cells havealready acquired this characteristic. If placed into cultureand given a fresh supply of media every few days they canbe grown indenitely. Scientists often use chemicals orvirusesto immortalize certaincell types in thelaboratorysothat they can be studied over long periods of time. As withmicroorganisms, cultured cells can be transformed with


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cloned DNA to study the effects of gene expression oncellular processes.

Tissue culture is a routine procedure, and a central partof biomedical research, biotechnology and pharmaceuticalproduction. Many drugs, vaccines, monoclonal antibodiesand other substances are produced from cell cultures.

Shuttle Vectors: Plasmids that canReplicate in Two Different HostsRegardless of the organism being studied, plasmid DNAtobe transformedinto anycell type is primarilyprepared in E .coli. These plasmids, called shuttle vectors, must have theability to be selected and to replicate in two differentorganisms, E . coli and the recipient host cell. A shuttlevector has the basic components of an E . coli plasmid, but

in addition must have a selectable marker appropriate tothe host cell and, if applicable, an origin of replicationrecognized by the host cell. For example, a yeast shuttlevector has the major components of a bacterial vector(replication of origin, selectable marker and multiplecloning site), and also contains a yeast replication originand a yeast selectable marker (such as HIS3 , discussedpreviously). Scientists working with a given organism mustmake themselves familiar with the appropriate selectionsand shuttle vectors available for that organism.

SummaryResearchers studying a particular aspect of molecularbiology in any organism use the bacterium E. coli focloning, replicating and manipulating DNA. Yeasts,namely S. cerevisiae and Schizosaccharomyces pombe , aralso commonly used for selecting specic genetic mutantsuseful in studying eukaryotic cellular process. The knowl-edge gained from studying cloned genes in yeast leads to agreater understanding of cellular processes in morecomplex eukaryotic organisms. Specialized yeast andbacterial expression vectors are used in the biotechnologyindustry to produce large quantities of puried proteins.Shuttlevectors allow cloned DNAto be easily replicated inbacteria and then transformed into another host cell.

Further ReadingAtlas RM (1997) Methods for studying microorgansims, pp. 6376;

Genetic mutation, recombination and mapping, pp. 319356;

Industrialmicrobiologyand biotechnology,pp. 820-843.In: Principleof Microbiology . Dubuque, IA: WC Brown.Glazier AN and Nikaido H (1995) Microbial Biotechnology . New York

Freeman.Micklos DA and Freyer GA (1990) DNA Science . Cold Spring Harbor:

ColdSpring Harbor Laboratory Pressand CarolinaBiological SupplyCompany.

Primrose SB (1991) Molecular Biotechnology . Cambridge, MA: Black-well Scientic.

Stanford University Saccharomyces Genome Database. [http://genome-www.stanford.edu/Saccharomyces].

Watson JD,Hopkins NH,Roberts JW,Steitz JA andWeiner AM(1987)Yeasts as the E. coli of eukaryotic cells; Recombinant DNA at work.In: Molecular Biology of the Gene , chaps 1819, pp. 550618. MenloPark, CA: Benjamin/Cummings.


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