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Eucaryotic Eucaryotic recombinationrecombination
RecombinationRecombination– process in which one process in which one
or more nucleic acid or more nucleic acid molecules are molecules are rearranged or rearranged or combined to produce combined to produce a new nucleotide a new nucleotide sequencesequence
In eucaryotes, In eucaryotes, usually occurs as the usually occurs as the result of result of crossing-crossing-overover during meiosis during meiosis
Figure 13.1
Bacterial Bacterial Recombination: Recombination: General PrinciplesGeneral Principles
Several types of recombinationSeveral types of recombination– General recombinationGeneral recombination
can be reciprocal or nonreciprocalcan be reciprocal or nonreciprocal
– Site-specific recombinationSite-specific recombination– Replicative recombinationReplicative recombination
Reciprocal general Reciprocal general recombinationrecombination Most common type of Most common type of
recombinationrecombination A reciprocal exchange between A reciprocal exchange between
pair of homologous chromosomespair of homologous chromosomes Results from DNA strand Results from DNA strand
breakage and reunion, leading to breakage and reunion, leading to crossing-overcrossing-over
Nonreciprocal general Nonreciprocal general recombinationrecombination Incorporation of Incorporation of
single strand of single strand of DNA into DNA into chromosome, chromosome, forming a stretch forming a stretch of of heteroduplex heteroduplex DNADNA
Proposed to occur Proposed to occur during bacterial during bacterial transformationtransformation
Figure 13.3
Site-specific Site-specific recombinationrecombination Insertion of nonhomologous DNA Insertion of nonhomologous DNA
into a chromosomeinto a chromosome often occurs during viral genome often occurs during viral genome
integration into host chromosomeintegration into host chromosome– enzymes responsible are specific for enzymes responsible are specific for
virus and its hostvirus and its host
Site Specific Site Specific RecombinationRecombination
If the two sites undergoing If the two sites undergoing recombination are oriented in the recombination are oriented in the same direction, this may result in a same direction, this may result in a deletiondeletion
InversionsInversions
Recombination at inverted repeats Recombination at inverted repeats causes and inversioncauses and inversion
Replicative Replicative recombinationrecombination Accompanies replication of Accompanies replication of
genetic materialgenetic material Used by genetic elements that Used by genetic elements that
move about the genomemove about the genome
Horizontal gene Horizontal gene transfertransfer
Transfer of genes from one mature, Transfer of genes from one mature, independent organism (donor) to independent organism (donor) to another (recipient)another (recipient)
ExogenoteExogenote– DNA that is transferred to recipientDNA that is transferred to recipient
EndogenoteEndogenote– genome of recipientgenome of recipient
MerozyogoteMerozyogote– recipient cell that is temporarily diploid as recipient cell that is temporarily diploid as
result of transfer processresult of transfer process
Bacterial PlasmidsBacterial Plasmids Small, double-stranded, usually circular Small, double-stranded, usually circular
DNA moleculesDNA molecules Are Are repliconsreplicons
– have their own origin of replicationhave their own origin of replication– can exist as single copies or as multiple can exist as single copies or as multiple
copiescopies CuringCuring
– elimination of plasmidelimination of plasmid– can be spontaneous or induced by can be spontaneous or induced by
treatments that inhibit plasmid replication treatments that inhibit plasmid replication but not host cell reproductionbut not host cell reproduction
Bacterial plasmids…Bacterial plasmids…
EpisomesEpisomes– plasmids that can exist either with plasmids that can exist either with
or without integrating into or without integrating into chromosomechromosome
Conjugative plasmidsConjugative plasmids– have genes for pilihave genes for pili– can transfer copies of themselves to can transfer copies of themselves to
other bacteria during conjugationother bacteria during conjugation
Fertility FactorsFertility Factors
conjugative conjugative plasmidsplasmids
e.g., F factor e.g., F factor of of E. coliE. coli
many are many are also also episomesepisomes
Figure 13.5
Resistance FactorsResistance Factors
R factors (plasmids)R factors (plasmids) Have genes for resistance to Have genes for resistance to
antibioticsantibiotics Some are conjugativeSome are conjugative usually do not integrate into usually do not integrate into
chromosomechromosome
Col plasmidsCol plasmids
Encode colicin Encode colicin – kills kills E. coliE. coli– a type of a type of bacteriocinbacteriocin
protein that destroys other bacteria, protein that destroys other bacteria, usually closely related speciesusually closely related species
Some are conjugativeSome are conjugative Some carry resistance genesSome carry resistance genes
Other Types of Other Types of PlasmidsPlasmids
Virulence plasmidsVirulence plasmids– carry virulence genescarry virulence genes
e.g., genes that confer resistance to host e.g., genes that confer resistance to host defense mechanismsdefense mechanisms
e.g., genes that encode toxinse.g., genes that encode toxins Metabolic plasmidsMetabolic plasmids
– carry genes for metabolic processescarry genes for metabolic processes e.g., genes encoding degradative e.g., genes encoding degradative
enzymes for pesticidesenzymes for pesticides e.g., genes for nitrogen fixatione.g., genes for nitrogen fixation
Transposable ElementsTransposable Elements
TranspositionTransposition– the movement of pieces of DNA around the movement of pieces of DNA around
the genomethe genome Transposable elements Transposable elements
(transposons)(transposons)– segments of DNA that carry genes for segments of DNA that carry genes for
transpositiontransposition Widespread in bacteria, eucaryotes Widespread in bacteria, eucaryotes
and archaeaand archaea
Types of transposable Types of transposable elementselements
Insertion sequencesInsertion sequences (IS elements) (IS elements)– Contain only genes encoding enzymes Contain only genes encoding enzymes
required for transpositionrequired for transposition TransposaseTransposase
Composite transposons( Tn)Composite transposons( Tn)– Carry genes in addition to those needed Carry genes in addition to those needed
for transpositionfor transposition– Conjugative transposonsConjugative transposons
Carry transfer genes in addition to transposition Carry transfer genes in addition to transposition genesgenes
IS sequencesIS sequences
Insertion elements are Insertion elements are mobile genetic elementsmobile genetic elements that that occasionally insert into chromosomal sequences, often occasionally insert into chromosomal sequences, often disrupting genesdisrupting genes . .
Insertion elements are characterized by Insertion elements are characterized by inverted terminal repeatsinverted terminal repeats . These terminal repeats . These terminal repeats likely are recognition sites for an enzyme responsible likely are recognition sites for an enzyme responsible for the insertion. for the insertion.
Mobility of the element depends only on the element Mobility of the element depends only on the element itself; it is an itself; it is an autonomousautonomous element. Thus, it must carry element. Thus, it must carry the coding ability for the transposase recognizing the the coding ability for the transposase recognizing the inverted terminal repeats. inverted terminal repeats.
The direct repeats externally flanking the inverted The direct repeats externally flanking the inverted repeats are not part of the insertion sequence. repeats are not part of the insertion sequence. Instead, they are chromosomal sequences that Instead, they are chromosomal sequences that become duplicated upon insertion, with one copy at become duplicated upon insertion, with one copy at each end; this is called each end; this is called target-site duplicationtarget-site duplication. .
Characteristics of IS Characteristics of IS elementselements The majority of IS elements are between 0.7 and l.8 kb in The majority of IS elements are between 0.7 and l.8 kb in
size and the termini tend to be l0 to 40 base pairs in length size and the termini tend to be l0 to 40 base pairs in length with perfect or nearly perfect with perfect or nearly perfect repeatrepeats. s.
These sequences also tend to have RNA termination signals These sequences also tend to have RNA termination signals as well as nonsense codons in all three reading frames and as well as nonsense codons in all three reading frames and are therefore polar. are therefore polar.
Typically they encode one large open reading frame of 300 Typically they encode one large open reading frame of 300 to 400 amino acids and by definition the protein encoded by to 400 amino acids and by definition the protein encoded by this reading frame is involved in the transposition event. this reading frame is involved in the transposition event.
Two exceptions to the size range given above should be Two exceptions to the size range given above should be noted: The first, }; is 5.7 kb and the other, IS101, is a scant noted: The first, }; is 5.7 kb and the other, IS101, is a scant 0.2 kb in size. Although there are exceptions, insertion 0.2 kb in size. Although there are exceptions, insertion sequences tend to be present in a small number of copies in sequences tend to be present in a small number of copies in the genome. the genome.
For example, IS1 is present in 6 to l0 copies in For example, IS1 is present in 6 to l0 copies in E. coliE. coli chromosome while IS2 and 3 are typically present in about chromosome while IS2 and 3 are typically present in about five copies. five copies.
IS actionsIS actions
Insertion sequences mediate a variety of Insertion sequences mediate a variety of DNA rearrangements. One of the first DNA rearrangements. One of the first recognitions of this fact was the involvement recognitions of this fact was the involvement of insertion sequences in the integration of F of insertion sequences in the integration of F and R plasmids into the host chromosome. and R plasmids into the host chromosome. This event gives rise to Hfr strains. This event gives rise to Hfr strains.
The initial DNA rearrangement mediated by The initial DNA rearrangement mediated by IS elements is the "insertional duplication" IS elements is the "insertional duplication" that they tend to generate at the site of that they tend to generate at the site of insertion. insertion.
IS1 generates an 8 or 9 base pair duplication IS1 generates an 8 or 9 base pair duplication while IS2 generates a 5 base pair duplication. while IS2 generates a 5 base pair duplication.
TransposonsTransposons As defined above, a As defined above, a transposontransposon is a mobile genetic is a mobile genetic
element containing additional genes unrelated to element containing additional genes unrelated to transposition functions. In general, there are known transposition functions. In general, there are known to be two general classes:to be two general classes:
Class l or "compound Tns" encode drug resistance Class l or "compound Tns" encode drug resistance genes flanked by copies of an IS in a direct or genes flanked by copies of an IS in a direct or indirect repeat. A direct repeat exists when the two indirect repeat. A direct repeat exists when the two sequences at either end are oriented in the same sequences at either end are oriented in the same direction while an indirect (or inverted) repeat exists direction while an indirect (or inverted) repeat exists when they are in opposite directions. In this class of when they are in opposite directions. In this class of transposons, the IS sequence supplies the transposons, the IS sequence supplies the transposition function. transposition function.
The second class of transposons are known as The second class of transposons are known as "complex" or Class 2. With these, the element is "complex" or Class 2. With these, the element is flanked by short (30-40 bp) indirect repeats with the flanked by short (30-40 bp) indirect repeats with the genes for drug resistance and transposition encoded genes for drug resistance and transposition encoded in the middle (see figure of Tn3 belowin the middle (see figure of Tn3 below). ).
Preferential sites for Preferential sites for transpositiontransposition Class 1Class 1 GCTNAGC - Not AT richGCTNAGC - Not AT rich Sites found approximately every 100 Sites found approximately every 100
bases in the E. coli genomebases in the E. coli genome
Class 2Class 2 AT rich regions are preferable sitesAT rich regions are preferable sites Homology at ends of regionHomology at ends of region
The transposition The transposition eventevent Usually transposon replicated, Usually transposon replicated,
remaining in original site, while remaining in original site, while duplicate inserts at another siteduplicate inserts at another site
Insertion generates direct repeats Insertion generates direct repeats of flanking host DNAof flanking host DNA
Effects of transpositionEffects of transposition
Mutation in coding regionMutation in coding region-deletion of genetic material-deletion of genetic material
Arrest of translation or Arrest of translation or transcriptiontranscription
Activation of genesActivation of genes Generation of new plasmidsGeneration of new plasmids
– resistance plasmidsresistance plasmids
Figure 13.13
The U-tube experiment
demonstrated thatdirect cell to cellcontact wasnecessary
after incubation,bacteria plated onminimal media
no prototrophs
R1 plasmid
RTF = resistance transfer factor
a conjugativeplasmid
sources ofresistance genesare transposons
Bacterial ConjugationBacterial Conjugation
transfer of transfer of DNA by direct DNA by direct cell to cell cell to cell contactcontact
discovered discovered 1946 by 1946 by Lederberg and Lederberg and TatumTatum
FF++ x F x F–– Mating Mating
FF++ = donor = donor– contains F factorcontains F factor
FF–– = recipient = recipient– does not contain F factordoes not contain F factor
F factor replicated by rolling-circle F factor replicated by rolling-circle mechanism and duplicate is mechanism and duplicate is transferredtransferred
recipients usually become Frecipients usually become F++
donor remains Fdonor remains F++
F factorF factor
The F factor can exist in three different states: The F factor can exist in three different states: F+ refers to a factor in an autonomous, F+ refers to a factor in an autonomous,
extrachromosomal state containing only the genetic extrachromosomal state containing only the genetic information described above. information described above.
The "Hfr" (which refers to "high frequency The "Hfr" (which refers to "high frequency recombination") state describes the situation when recombination") state describes the situation when the factor has integrated itself into the chromosome the factor has integrated itself into the chromosome presumably due to its various insertion sequences. presumably due to its various insertion sequences.
The F' or (F prime) state refers to the factor when it The F' or (F prime) state refers to the factor when it exists as an extrachromosomal element, but with the exists as an extrachromosomal element, but with the additional requirement that it contain some section of additional requirement that it contain some section of chromosomal DNA covalently attached to it. A strain chromosomal DNA covalently attached to it. A strain containing no F factor is said to be "F-". containing no F factor is said to be "F-".
FF++ x F x F–– mating mating
In its extrachromosomal In its extrachromosomal state the factor has a state the factor has a molecular weight of molecular weight of approximately 62 kb approximately 62 kb and encodes at least 20 and encodes at least 20 tra genes. It also tra genes. It also contains three copies of contains three copies of IS3, one copy of IS2, and IS3, one copy of IS2, and one copy of a À one copy of a À sequence as well as sequence as well as genes for incompatibility genes for incompatibility and replication. and replication.
Hfr ConjugationHfr Conjugation
Hfr strainHfr strain– donor having F factor integrated into donor having F factor integrated into
its chromosomeits chromosome both plasmid genes and both plasmid genes and
chromosomal genes are chromosomal genes are transferredtransferred
FF Conjugation Conjugation FF plasmid plasmid
– formed by formed by incorrect incorrect excision from excision from chromosomechromosome
– contains contains 1 1 genes from genes from chromosomechromosome
FF cell can cell can transfer Ftransfer F plasmid to plasmid to recipientrecipient
Figure 13.15a
integrated F factor
chromosomal gene
Tra YTra Y
Characterization of the Characterization of the Escherichia coli F factor traY Escherichia coli F factor traY gene product and its binding gene product and its binding sitessites
WC Nelson, BS Morton, EE Lahue WC Nelson, BS Morton, EE Lahue and SW Matson and SW Matson Department of Biology, University of Department of Biology, University of North Carolina, Chapel Hill 27599. North Carolina, Chapel Hill 27599.
Tra GenesTra Genes
Tra YTra Y gene codes for the protein binds gene codes for the protein binds to the Ori Tto the Ori T
Initiates the transfer of plasmid across Initiates the transfer of plasmid across the bridge between the two cellsthe bridge between the two cells
Tra ITra I Gene is a helicase responsible for Gene is a helicase responsible for the conjugationthe conjugation
strand-specific transesterification strand-specific transesterification (relaxase) (relaxase)
Conjugative ProteinsConjugative Proteins
Key players are the proteins that Key players are the proteins that initiate the physical transfer of initiate the physical transfer of ssDNA, the conjugative initiator ssDNA, the conjugative initiator proteins proteins
They nick the DNA and open it to They nick the DNA and open it to begin the transferbegin the transfer
Working in conjunction with the Working in conjunction with the helicases they facilitate the transfer helicases they facilitate the transfer of ss RNA to the F- cellof ss RNA to the F- cell
DNA TransformationDNA Transformation
Uptake of naked DNA molecule Uptake of naked DNA molecule from the environment and from the environment and incorporation into recipient in a incorporation into recipient in a heritable formheritable form
Competent cellCompetent cell– capable of taking up DNAcapable of taking up DNA
May be important route of genetic May be important route of genetic exchange in natureexchange in nature
DNA bindingprotein
nuclease – nicks and degrades onestrand
competence-specificprotein
Streptococcus pneumoniae
Artificial Artificial transformationtransformation Transformation done in laboratory Transformation done in laboratory
with species that are not normally with species that are not normally competent (competent (E. coliE. coli))
Variety of techniques used to Variety of techniques used to make cells temporarily competentmake cells temporarily competent– calcium chloride treatmentcalcium chloride treatment
makes cells more permeable to DNAmakes cells more permeable to DNA
TransductionTransduction
Transfer of bacterial genes by Transfer of bacterial genes by virusesviruses
Virulent bacteriophagesVirulent bacteriophages– reproduce using lytic life cyclereproduce using lytic life cycle
Temperate bacteriophagesTemperate bacteriophages– reproduce using lysogenic life cyclereproduce using lysogenic life cycle
LambdaLambda
In order for the lambda prophage In order for the lambda prophage to exist in a host to exist in a host E. coliE. coli cell, it cell, it must integrate into the host must integrate into the host chromosome which it does by chromosome which it does by means of a means of a site-specific site-specific recombination reactionrecombination reaction. .
Attachment siteAttachment site
The The E. coliE. coli chromosome contains one site at which chromosome contains one site at which lambda integrates. The site, located between the lambda integrates. The site, located between the galgal and and biobio operons, is called the operons, is called the attachment siteattachment site and is and is designated designated attB attB since it is the attachment site on the since it is the attachment site on the bbacterial chromosome. acterial chromosome.
The site is only 30 bp in size and contains a conserved The site is only 30 bp in size and contains a conserved central 15 bp region where the recombination reaction central 15 bp region where the recombination reaction will take place. will take place.
he structure of the recombination site was determined he structure of the recombination site was determined originally by genetic analyses and is usually originally by genetic analyses and is usually represented as represented as BOB'BOB', where , where BB and and B'B' represent the represent the bacterial DNA on either side of the conserved central bacterial DNA on either side of the conserved central element element
Recombination siteRecombination site
The bacterioThe bacteriopphage recombination site - hage recombination site - attPattP - is more complex. It contains the - is more complex. It contains the identical central 15 bp region as identical central 15 bp region as attBattB. .
The overall structure can be The overall structure can be represented as represented as POP'POP'. However, the . However, the flanking sequences on either side of flanking sequences on either side of attPattP are very important since they are very important since they contain the binding sites for a number contain the binding sites for a number of other proteins which are required for of other proteins which are required for the recombination reaction. The the recombination reaction. The PP arm arm is 150 bp in length and the is 150 bp in length and the P'P' arm is arm is 90 bp in length. 90 bp in length.
IntegrationIntegration
Integration of bacteriophage lambda requires Integration of bacteriophage lambda requires one phage-encoded protein - one phage-encoded protein - IntInt, which is , which is the the integraseintegrase - and one bacterial protein - - and one bacterial protein - IHFIHF, which is , which is IntegrationIntegration HostHost FactorFactor. .
Both of these proteins bind to sites on the Both of these proteins bind to sites on the PP and and P'P' arms of arms of attPattP to form a complex in to form a complex in which the central conserved 15 bp elements which the central conserved 15 bp elements of of attPattP and and attBattB are properly aligned. are properly aligned.
The integrase enzyme carries out all of the The integrase enzyme carries out all of the steps of the recombination reaction, which steps of the recombination reaction, which includes a short 7 bp branch migration.includes a short 7 bp branch migration.
Enzymes and Enzymes and RecombinationRecombination There are two major groups of enzymes that carry out site-There are two major groups of enzymes that carry out site-
specific recombination reactions; one group - known as the specific recombination reactions; one group - known as the tyrosine tyrosine recombinaserecombinase family family - consists of over 140 proteins. - consists of over 140 proteins.
These proteins are 300-400 amino acids in size, they contain These proteins are 300-400 amino acids in size, they contain two conserved structural domains, and they carry out two conserved structural domains, and they carry out recombination reactions using a common mechanism recombination reactions using a common mechanism involving a the formation of a covalent bond with an active involving a the formation of a covalent bond with an active site tyrosine residue. site tyrosine residue.
The strand exchange reaction involves staggered cuts that The strand exchange reaction involves staggered cuts that are 6 to 8 bp apart within the recognition sequence. are 6 to 8 bp apart within the recognition sequence.
All of the strand cleavage and re-joining reactions proceed All of the strand cleavage and re-joining reactions proceed through a series of transesterification reactions like those through a series of transesterification reactions like those mediated by type I topoisomerases.mediated by type I topoisomerases.
Excision of Excision of bacteriophagesbacteriophages Excision of bacteriophage lambda requires Excision of bacteriophage lambda requires
two phage-encoded proteins: two phage-encoded proteins: IntInt (again!) and (again!) and XisXis, which is an , which is an excisionaseexcisionase. .
It also requires several bacterial proteins. It also requires several bacterial proteins. In addition to In addition to IHFIHF, a protein called , a protein called FisFis is is
required. required. All of these proteins bind to sites on the All of these proteins bind to sites on the PP and and
P'P' arms of arms of attLattL and and attRattR forming a complex forming a complex in which the central conserved 15 bp in which the central conserved 15 bp elements of elements of attLattL and and attRattR are properly are properly aligned to promote excision of the prophage.aligned to promote excision of the prophage.
Generalized Generalized TransductionTransduction Any part of bacterial genome can Any part of bacterial genome can
be transferredbe transferred Occurs during lytic cycleOccurs during lytic cycle During viral assembly, fragments During viral assembly, fragments
of host DNA mistakenly packaged of host DNA mistakenly packaged into phage headinto phage head– generalized transducing particlegeneralized transducing particle
Specialized Specialized TransductionTransduction
also called also called restricted transductionrestricted transduction carried out only by temperate carried out only by temperate
phages that have established phages that have established lysogenylysogeny
only specific portion of bacterial only specific portion of bacterial genome is transferredgenome is transferred
occurs when prophage is occurs when prophage is incorrectly excisedincorrectly excised
Mapping the GenomeMapping the Genome
locating genes on an organism’s locating genes on an organism’s chromosomeschromosomes
mapping bacterial genes mapping bacterial genes accomplished using all three accomplished using all three modes of gene transfermodes of gene transfer
Hfr mappingHfr mapping used to map relative location of used to map relative location of
bacterial genesbacterial genes based on observation that chromosome based on observation that chromosome
transfer occurs at constant ratetransfer occurs at constant rate interrupted mating experimentinterrupted mating experiment
– Hfr x FHfr x F-- mating interrupted at various mating interrupted at various intervalsintervals
– order and timing of gene transfer order and timing of gene transfer determineddetermined
E. coliE. coli genetic map genetic map
gene locations gene locations expressed in expressed in minutes, minutes, reflecting time reflecting time transferredtransferred
made using made using numerous Hfr numerous Hfr strainsstrains
Figure 13.23
Transformation Transformation mappingmapping used to establish gene linkageused to establish gene linkage expressed as frequency of expressed as frequency of
cotransformationcotransformation if two genes close together, if two genes close together,
greater likelihood will be greater likelihood will be transferred on single DNA transferred on single DNA fragmentfragment
Generalized Generalized transduction mappingtransduction mapping used to establish gene linkageused to establish gene linkage expressed as frequency of expressed as frequency of
cotransductioncotransduction if two genes close together, if two genes close together,
greater likelihood will be carried greater likelihood will be carried on single DNA fragment in on single DNA fragment in transducing particletransducing particle
Specialized Specialized transduction mappingtransduction mapping provides distance of genes from provides distance of genes from
viral genome integration sitesviral genome integration sites viral genome integration sites viral genome integration sites
must first be mapped by must first be mapped by conjugation mapping techniquesconjugation mapping techniques
Recombination and Recombination and Genome Mapping in Genome Mapping in VirusesViruses
viral genomes can also undergo viral genomes can also undergo recombination eventsrecombination events
viral genomes can be mapped by viral genomes can be mapped by determining recombination determining recombination frequenciesfrequencies
physical maps of viral genomes can physical maps of viral genomes can also be constructed using other also be constructed using other techniquestechniques
Recombination Recombination mappingmapping
recombination recombination frequency frequency determined determined when cells when cells infected infected simultaneouslsimultaneously with two y with two different different virusesviruses
Figure 13.24
Physical mapsPhysical maps
heteroduplex mapsheteroduplex maps– genomes of two different viruses genomes of two different viruses
denatured, mixed and allowed to denatured, mixed and allowed to annealanneal regions that are not identical, do not regions that are not identical, do not
reannealreanneal
– allows for localization of mutant allows for localization of mutant allelesalleles
Physical maps…Physical maps…
restriction endonuclease mappingrestriction endonuclease mapping– compare DNA fragments from two compare DNA fragments from two
different viral strains in terms of different viral strains in terms of electrophoretic mobilityelectrophoretic mobility
sequence mappingsequence mapping– determine nucleotide sequence of determine nucleotide sequence of
viral genomeviral genome– identify coding regions, mutations, identify coding regions, mutations,
etc.etc.