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Griffiths AJF, Gelbart WM, miller JH, et al. Modern Genetic Analysis. New York: W. H. Freeman; 1999.


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Conjugation is just the combination of two compatible bacter cells. Happen twogenotypes together and enabling them to conjugate is the indistinguishable of making across in eukaryotes. Our discussion of conjugation will facility on the gut bacteriumEscherichia coli (E. Coli). Conjugation and also gene deliver inE. Coli are thrust by a circular DNA plasmid called the fertility aspect or sex factor(F), which is discovered in some however not all cells. Hence to understand how tomake a cross in E. Coli, we need to understand the nature ofF.


The impressive Properties of the F Plasmid

Cells carrying the F plasmid are designated F+, and also thoselacking it room F−. The F plasmid contains approximately100 genes, which provide the plasmid several essential properties:

1.

The F plasmid can replicate its own DNA, enabling the plasmid to bemaintained in a separating cell population (Figure 9-3a).

2.

Cells carrying the F plasmid encourage the synthetic of pili (singular,pilus) on the bacterial cabinet surface. Pili are minuteproteinaceous tubules that enable the F+ cells to affix toother cells and maintain contact with them; that is, to conjugate (Figure 9-3b).

3.

F+ and also F− cells can conjugate. When conjugationoccurs, the F+ cells deserve to act as F donors. The F plasmid DNAreplicates and the newly synthesized copy the the one F molecule istransferred to the F− recipient (Figure 9-3c). However, a copy that F always remainsbehind in the donor cell. The recipient cabinet becomes convert intoF+, because it now has a circular F genome. Thetransfer the the F plasmid native F+ come F− is rapid,so the F plasmid can spread choose wildfire throughout a population fromstrain to strain.

4.

F+ cells are usually inhibited indigenous making contact with otherF+ cells; therefore the F plasmid is not moved fromF+ to F+.

5.

Sometimes F carries within its genome one or more IS (insertion-sequence)elements (see chapter 13).An IS element is a cell phone segment that DNA the moves fromplace to ar within the hold chromosome or between chromosome andplasmid. The visibility of a specific IS facet both in the plasmidand in the chromosome affords a website at whichhomologous crossing-over occasionally occurs. A crossover between thetwo circular DNAs leads to the integration of the plasmid right into thebacterial chromosome, as shown in figures 9-4 below and also 9-5a ~ above the complying with page. Once thisintegration occurs, F have the right to drive the deliver of the whole hostchromosome into the recipient cell, along with its own combined F DNA(Figure 9-5b).


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Figure 9-5

The transfer of E. Coli chromosomal mite mediated byF. (a) Occasionally, the elevation F factor combines through theE. Coli chromosome. (b) as soon as the integrated Ftransfers to another E. Coli cell throughout conjugation,it carries along any type of E. Coli DNA (more...)


This last process, the connected transfer that F and host genes, has actually someinteresting features. First, in any population of cell containing the F factor,F will combine into the chromosome just in a small portion of cells (Figure 9-5c). These few cells have the right to nowtransfer chromosomal alleles come a second strain. The transfer is detectablebecause donor and also recipient alleles recombine to develop genetic recombinantsthat can be identified. Indeed, the monitoring of recombinants led to theinitial exploration of gene deliver by conjugation (see genetics in procedure 9-1 on page277).


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It is possible to isolation the rarely cells in i m sorry the F factor is incorporated intothe organize chromosome from the bacterial population and to cultivate pure strainsderived from these cells. In such strains, every cell donates chromosomalalleles during F transfer, therefore the frequency that recombinants because that these strains ismuch greater than that is because that cells in the initial population, where the F factoris not integrated in many cells. Therefore, strains v an integrated F factorare termed high frequency of recombination (Hfr) strains todistinguish lock from typical F+ strains, i m sorry contain just a fewrare Hfr cells and also thus screen only a short frequency that recombination for thestrain together a whole. Due to the fact that they carry chromosomal markers efficiently, Hfrstrains room the ones offered for hereditary mapping, as we candlestick see later on.

6.

The integrated F aspect occasionally leaves the chromosome of one Hfr celland moves back to the cytoplasm, in part rare situations carrying a few hostchromosomal genes in addition to it (Figure9-5d). This modified F, referred to as F′ (pronounced “Fprime”), have the right to now deliver these certain host gene to a recipient(F−) cell in an transmittable manner, in the same way that Fis spread. Thus, the recipient cell now contains two duplicates of the samegene—one residents copy on its bacter chromosome and one copy top top thenewly moved cytoplasmic F′ factor.


Recombination in between Donor and also Recipient DNA

Conjugation allows genes indigenous two different parental cell to come with each other inthe very same cell and also hence provides an opportunity for recombination come occur.Hence mapping analysis is possible.

All conjugations (“crosses”) are by definition of the form Hfr(donor) × F− (recipient). After cell union, the Hfr chromosomereplicates in a strange manner that reels the end a single-stranded DNA molecule,which is then transferred linearly into the F− cell. The replicationand transfer start at a particular point at one next of the combined F, calledthe origin (O). gene close come the beginning are transferred first.The combined F aspect would be transferred last; however, in mostconjugations, the chromosomal transfer procedure stops prior to F beginning (Figure 9-6).


Figure 9-6

Transfer of single-stranded fragment of donor chromosome, andrecombination with recipient chromosome. Note: doublecrossovers can happen in any kind of location; those shown are examples.


Once inside the F− cell, the direct single-stranded DNA molecule actsas a polymerization template and is converted into a DNA double helix. Thislinear donor fragment is the exogenote, and the resides F−chromosome is the endogenote. Together a complimentary molecule, the exogenote cannot replicateand will come to be lost, but because exogenotes and also endogenotes room homologous,crossing-over can take place in between them. A solitary crossover between a linearmolecule (the exogenote) and also a circular one (the endogenote) would create asingle lengthy molecule that would certainly be inviable. However, twocrossovers would combine a part of the donor genome right into the recipient. That isin this method that recombination takes location (Figure 9-6). (Note that, although such integrative exchanges deserve to beconsidered to be double crossovers in the formal genetic sense, in ~ the DNA levelthe system is a single integration event in i beg your pardon a long donor segmentreplaces the indistinguishable segment in the recipient.)

Gene transfer and recombination carry out the vital to mapping the bacterialchromosome. There room two key methods: mapping by interrupted conjugation,which to produce a low-resolution map of big parts that the genome, and also mapping byrecombinant frequency, which produces a higher-resolution map the a smallerregion.


Mapping by Interrupted Conjugation

In mapping through interrupted conjugation, the Hfr and F− cells room mixed,and conjugation proceeds. Then, at solved times, the F− cells aresampled to determine which donor alleles have entered. This sampling isaccomplished by using a kitchen blender to different the join cells, resultingin interrupted conjugation. After ~ separation, the Hfr cells are selectivelykilled, and the remaining F− cells, the exconjugants,are experiment to see which of the donor alleles have entered and also stably recombinedwith the endogenote. The time at which miscellaneous donor allelesfirst show up in the exconjugants space calculated. If a donorallele a+ enters the recipient in ~ 5 minute afterunion and allele b+ enters at 8 minutes, then thetwo genes are claimed to it is in 3 minute apart on the chromosome. The map devices inthis case are minutes. Choose the maps based on crossover frequencies, theselinkage maps are purely hereditary constructions. Return the lot of DNAcorresponding come a minute is currently known, when the an approach was very first devised thiswas no the case.

Let’s analyze a common cross in i m sorry the order and also map place of the genesunder study space not known. In this specific cross, the genes through which theparents differ will certainly be azi (resistance or sensitivity to sodiumazide), festival (ability or i can not qualify to make use of galactose together anenergy source), lac (ability or i can not qualify to make use of lactose asan energy source), and also ton (resistance or sensitivity tobacteriophage T1). A streptomycin-sensitivity allele(strs) in the Hfr and a streptomycin-resistanceallele (strr) in the receiver are used toselectively kill the Hfr cell after conjugation. Selective killing isaccomplished by adding streptomycin come the mixture of cell after interruptingthe conjugation. That is beneficial if such an Hfr “contraselecting” alleleenters close come last, since then the will only rarely get in the F−;in other words, it should be close to the combined F factor. Thus theposition that the contraselected gene must have actually been established in previousexperiments. The parental of the overcome under factor to consider here are as follows,where the unmapped genes are written in alphabet order:

The outcomes of the interrupted-mating experiment are shown in number 9-7. Theazir gene is the very first to it is in detected, enteringat 8 minutes, complied with by tonr,lac+, and gal+ inthat order. Therefore not only is gene stimulate on the chromosome map established,but map ranges in minutes likewise are obtained, as displayed in number 9-8.


Figure 9-7

Interrupted-mating shortcut experiments with E.coli. F− cells the arestrr are crossed through Hfr cellsthat are strs. The F− cellshave a variety of mutations (indicated through the genetic markersazi, ton, lac, and also gal) thatprevent then (more...)


Figure 9-8

Chromosome map based upon Figure9-7. A linkage map have the right to be created for the E.coli chromosome native interrupted-mating studies, byusing the moment at i beg your pardon the donor alleles very first appear ~ mating.The devices of street are provided in minutes; arrowhead (more...)


Note, from figure 9-7, that allelestransferred at an early stage are discovered in a high portion of F− exconjugants,but the late alleles are found in only a little proportion. The reason for thisdifference is one of two people that transport spontaneously stop or the the chromosomebreaks. However, this an outcome does not influence the time-of-entry calculations.

The loved one positions that the azi, ton, lac, andgal gene were established in our experiment. However, thechromosomal region containing this loci might be just a small proportion of theentire chromosome. The complete map is obtained from plenty of such interruptedconjugation experiments, in which parental strains heteroallelic because that differentcombinations of genes space used; climate the in its entirety map is pieced together from thecomplete collection of data. In Hfrs of different origin, the combined F variable canbe in different positions and different orientations. Instances of the positionsand orientations of F in different Hfrs are shown in number 9-9.


Figure 9-9

Circularity that the E. Coli chromosome. (a) Throughthe use of different Hfr strains (H, 1, 2, 3, 312) that have actually thefertility element inserted into the chromosome at different pointsand in various directions, interrupted-mating experiments indicatethat (more...)


High-Resolution Mapping through Recombinant Frequency

Interrupted-mating experiments carry out a rough set of gene locations over theentire map. As we learned, the genes room mapped through time of entry. In suchexperiments, the exogenote must incorporate by a double recombination event, butthe mapping an approach is not based on any kind of measurement the recombinant frequencies.However, to carry out a higher-resolution an approach for measuring the size ofsmaller map distances, recombinant frequencies are used.

Suppose that us undertake one experiment come map three genes—met,arg, and leu—by recombinant frequency. Come measurerecombination in between these genes, us must set up a merozygote that isheterozygous for every three. This can be accomplished if we can create whichgene enters critical by an interruptedconjugation analysis. The Hfr allele of thelast-entering gene is selected amongst the F− exconjugants. Then,knowing the we have selected the critical gene, we recognize that the various other two mustalso have been in the merozygote. If we know from interrupted-conjugationexperiments that the gene order is met first followed byarg and also then leu, the merozygote in across of Hfrmet+ arg+ leu+ × F−met arg leu must have been together follows:

The critical gene to enter is leu+;therefore we pick initially for leu+ exconjugantsby plating them on tool containing no leucine yet containing methionine andarginine. Currently we can proceed to calculate map street in the standard way byusing a map unit same to a recombinant frequency the 1 percent. In practice,this calculate is done by measure the proportion of the totalleu+ exconjugants that likewise carriesarg+ or met+ orboth or neither. The recombination occasions needed to develop these recombinantgenotypes are shown in number 9-10. Weknow that a double crossover have to have occurred to integrateleu+: one crossover is atthe left that the leu gene, but the other deserve to be in variouspositions at the right. Thus the genotype the arises indigenous recombinationbetween leu and arg will certainly beleu+ arg− met−;so the percent of bacteria with this genotype in theleu+ exconjugants will give us our recombinantfrequency worth for the leu-to-arg interval.The leu+ exconjugants emerging from recombinationbetween met and arg will certainly beleu+ arg+ met−.The percent of bacteria through this leu+subgenotype will provide the recombinant frequencies and also hence the map distancesbetween the genes.


Figure 9-10

Mapping through recombination in E. Coli. After ~ a cross,selection is produced the leu+ marker,which is donated late. The beforehand markers(arg+ andmet+) might or may not be inserted,depending top top the website where recombination between the Hfr fragmentand the (more...)


In the cross simply described, theleu+ arg− met+recombinants would certainly require 4 crossovers rather of 2 (Figure 9-10d). These recombinants would be relativelyrare.

Let us think about some data from this cross. The percentages that the three maingenotypes derived after testing leu+ exconjugantsare:

From these results, we have the right to conclude the the leu–arg street is4 map units and also that the arg–met distance is 9 map units.


MESSAGE

Time-of-entry measurements in interrupted conjugation have the right to generate abroad-scale map that the bacterial chromosome. Recombinant frequenciesamong exconjugants have the right to be provided in fine-scale mapping.


F determinants Carrying bacter Genes

Occasionally, the incorporated F factor of one Hfr strain exits from the bacterialchromosome. Usually this event is a clean cut regenerating an intact Fplasmid. However, as portrayed in Figure9-5a, in part cases, the excision occasion is no a specific reversal ofthe initial insertion, and also a part of the bacter chromosome is incorporatedinto the liberated plasmid. Figure 9-11shows organization of a surrounding lac gene right into the plasmid, butthe an exact gene integrated depends on wherein the F aspect had originallyintegrated in the details Hfr. Such plasmids carrying bacterial gene arecalled F′. They are called for the gene that they carry:F′-lac, together in the instance illustrated in number 9-11, or F′-gal,F′-trp, and also so forth. An F′ have the right to be obtained by spring forrapid contagious transfer that a gene the is typically transferred so late on thechromosome the the details Hfr stress, overload used.


Figure 9-11

Origin and also reintegration that the F′ factor—in this case, F′lac. (a) F is inserted in an Hfr strain betweenthe ton and also lac+alleles. (b,c) Abnormal “outlooping” and also separation that F wake up toinclude the lac locus, (more...)


If one F′ plasmid is transferred upon conjugation through an F− strain,the recipients created are stable merozygotes, delivering acomplete bacter genome add to a donor fragment ~ above the autonomously replicatingplasmid. The process of producing a merozygote by an F′ element is referred to as sexduction orF-duction. secure partial diploids are valuable inbacterial genetics since they deserve to be supplied for hereditary studies typically possibleonly in a diploid cell, such together determination of dominance. Because that example, if alac+ donor is provided to create anF′-lac+ plasmid and also this plasmid istransferred come an F− recipient the carries the allelelac−, climate the partial diploid isheterozygouslac+ / lac−,and this cells can be supplied to determine which allele is dominant(lac+ transforms out to be dominant in thiscase).

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