1. PowerPoint Presentation Materials to accompany
Genetics: Analysis and Principles
Robert J. Brooker
CHAPTER 6
GENETIC TRANSFER AND
MAPPING IN BACTERIA
AND BACTERIOPHAGES
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2. INTRODUCTION
Bacteria and viruses account for a quarter to a third of human deaths worldwide. Their impact on health is a major reason for studying them.
Like eukaryotes, bacteria often possess allelic differences that affect their cellular traits
However, these allelic differences (such as different sensitivity to antibiotics) are between different strains of bacteria because
HOW ??? They do this.
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3. INTRODUCTION genetic transfer
In this process, a segment of bacterial DNA is transferred from one bacterium to another
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4. 6.1 GENETIC TRANSFER AND MAPPING IN BACTERIA
Like sexual reproduction in eukaryotes, genetic transfer in bacteria enhances genetic diversity
Transfer of genetic material from one bacterium to another can occur in three ways:
Conjugation
Involves direct physical contact
Transduction
Involves viruses
Transformation
Involves uptake from the environment
See Table 6.1
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5. 6-5

7. movie

8. Conjugation
Genetic transfer in bacteria was discovered in 1946 by Joshua Lederberg and Edward Tatum
They were studying strains of Escherichia coli that had different nutritional growth requirements
Auxotrophs cannot synthesize a needed nutrient
Prototrophs make all their nutrients from basic components
One auxotroph strain was designated bio– met– phe+ thr+
It required one vitamin (biotin) and one amino acid (methionine)
It could produce the amino acids phenylalanine and threonine
The other strain was designated bio+ met+ phe– thr–
Had the opposite requirements for growth
Their experiment is described in Figure 6.1
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9. 6-7
Figure 6.1

10. The genotype of the bacterial cells that grew on the plates has to be bio+ met+ phe+ thr+
Lederberg and Tatum reasoned that some genetic material was transferred between the two strains
Either the bio– met– phe+ thr+ strain got the ability to synthesize biotin and methionine (bio+ met+)
Or the bio+ met+ phe– thr– strain got the ability to synthesize phenylalanine and threonine (phe+ thr+)
The results of this experiment cannot distinguish between the two possibilities
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11. He used an apparatus known as U-tube
Bernard Davis later showed that the bacterial strains must make physical contact for transfer to occur
He used an apparatus known as U-tube
It contains at the bottom a filter which has pores that were
Large enough to allow the passage of the genetic material
But small enough to prevent the passage of bacterial cells
Davis placed the two strains in question on opposite sides of the filter
Application of pressure or suction promoted the movement of liquid through the filter
Refer to Figure 6.2
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12. Figure 6.2
Nutrient agar plates lacking biotin, methionine, phenylalanine and threonine
No colonies
Thus, without physical contact, the two bacterial strains did not transfer genetic material to one another
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13. Many, but not all, species of bacteria can conjugate
The term conjugation now refers to the transfer of DNA from one bacterium to another following direct cell-to cell contact
Many, but not all, species of bacteria can conjugate
Moreover, only certain strains of a bacterium can act as donor cells
Those strains contains a small circular piece of DNA termed the F factor (for Fertility factor)
Strains containing the F factor are designated F+
Those lacking it are F–
Plasmid is the general term used to describe extra-chromosomal DNA
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14. These genes play a role in the transfer of DNA
Plasmids, such as F factors, which are transmitted via conjugation are termed conjugative plasmids
These plasmids carry genes required for conjugation
These genes play a role in the transfer of DNA
They are thus designated tra and trb followed by a capital letter
Figure 6.3
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15. Refer to Figure 6.4a for the molecular details of conjugation
The first step in conjugation is the contact between donor and recipient cells
This is mediated by sex pili (or F pili) which are made only by F+ strains
These pili act as attachment sites for the F– bacteria
Refer to Figure 6.4b
Once contact is made, the pili shorten
Donor and recipient cell are drawn closer together
A conjugation bridge is formed between the two cells
The successful contact stimulates the donor cells to begin the transfer process
Refer to Figure 6.4a for the molecular details of conjugation
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16. 6-14 Figure 6.4 Together, these form the conjugation bridge
a complex of proteins encoded by the F factor that span both inner and outer membranes
Together, these form the conjugation bridge
Accessory proteins of the relaxosome are released
One protein, relaxase, remains bound to the end of the T-DNA
Protein complex encoded by the F factor
Transferred DNA
Figure 6.4
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18. 6-15

19. F’ factors can be transferred through conjugation
The result of conjugation is that the recipient cell has acquired an F factor
Thus, it is converted from an F– to an F+ cell
The F+ cell remains unchanged
In some cases, the F factor may carry genes that were once found on the bacterial chromosome
These types of F factors are called F’ factors
F’ factors can be transferred through conjugation
This may introduce new genes into the recipient and thereby alter its genotype
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20. Hfr Strains
In the 1950s, Luca Cavalli-Sforza discovered a strain of E. coli that was very efficient at transferring chromosomal genes
He designated this strain as Hfr (for High frequency of recombination)
Hfr Strains Contain an F Factor Integrated into the Bacterial Chromosome
An episome is a segment of DNA that can exist as a plasmid and integrate into the chromosome
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21. Hfr Strains Can Transfer a Portion of the Bacterial Chromosome to Recipient Cells

23. William Hayes demonstrated that conjugation between an Hfr and an F– strain involves the transfer of a portion of the Hfr bacterial chromosome
The origin of transfer of the integrated F factor determines the starting point and direction of the transfer process
The cut, or nicked site is the starting point that will enter the F– cell
Then, a strand of bacterial DNA begins to enter in a linear manner
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24. The F– cell does pick up chromosomal DNA
It generally takes about hours for the entire Hfr chromosome to be passed into the F– cell
Most matings do not last that long
Only a portion of the Hfr chromosome gets into the F– cell
Since the nick is internal to the integrated F factor, only part of the plasmid is transferred and the F– cells does not become F+
The F– cell does pick up chromosomal DNA
This DNA can recombine with the homologous region on the chromosome of the recipient cell
This may provide the recipient cell with new combination of alleles
Refer to Figure 6.5b
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25. 6-20 Figure 6.5b Transfer of bacterial genes by an Hfr strain
F– cell received short segment of the Hfr chromosome
It has become lac+ but remains pro–
lac+  Ability to metabolize lactose
lac–  Inability
pro+  Ability to synthesize proline
pro–  Inability
Therefore, the order of transfer is lac+ – pro+
F– cell received longer segment of the Hfr chromosome
It has become lac+ AND pro+
Figure 6.5b
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26. Experiment 6A Interrupted Mating Technique
Conjugation Experiments Can Map Genes Along
the E. coli Chromosome
The rationale behind this mapping strategy
The time it takes genes to enter the recipient cell is directly related to their order along the bacterial chromosome
The Hfr chromosome is transferred linearly to the F– recipient cell
Therefore, interrupted mating at different times would lead to various lengths being transferred
The order of genes along the chromosome can be deduced by determining the genes transferred during short matings vs. those transferred during long matings
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27. Wollman and Jacob started the experiment with two E. coli strains
The donor (Hfr) strain had the following genetic composition
thr+ : Able to synthesize the essential amino acid threonine
leu+ : Able to synthesize the essential amino acid leucine
azis : Sensitive to killing by azide (a toxic chemical)
tons : Sensitive to infection by T1 (a bacterial virus)
lac+ : Able to metabolize lactose and use it for growth
gal+ : Able to metabolize galactose and use it for growth
strs : Sensitive to killing by streptomycin (an antibiotic)
The recipient (F–) strain had the opposite genotype
thr– leu– azir tonr lac – gal – strr
r = resistant
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28. Wollman and Jacob already knew that
The thr+ and leu+ genes were transferred first, in that order
Both were transferred within 5-10 minutes of mating
Therefore their main goal was to determine the times at which genes azis, tons, lac+, and gal+ were transferred
The transfer of the strs was not examined
Streptomycin was used to kill the donor (Hfr) cell following conjugation
The recipient (F– cell) is streptomycin resistant
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29. Testing the Hypothesis
The chromosome of the donor strain in an Hfr mating is transferred in a linear manner to the recipient strain
The order of genes along the chromosome can be deduced by determining the time various genes take to enter the recipient cell
Testing the Hypothesis
Refer to Figure 6.6
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30. Figure 6.6
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31. Percent of Surviving Bacterial Colonies with the Following Genotypes
The Data
Minutes that Bacterial Cells were Allowed to Mate Before Blender Treatment
Percent of Surviving Bacterial Colonies with the Following Genotypes
thr+ leu azis tons lac gal+ 5 –– 10
100 12 3 15 70 31 20 88 71 25 92 80 28
0.6 30 90 75 36 40 38 50 91 78 42 27 60
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32. Percent of Surviving Bacterial Colonies with the Following Genotypes
Interpreting the Data
After 10 minutes, the thr+ leu+ genotype was obtained
Minutes that Bacterial Cells were Allowed to Mate Before Blender Treatment
Percent of Surviving Bacterial Colonies with the Following Genotypes
thr+ leu azis tons lac gal+ 5 –– 10
100 12 3 15 70 31 20 88 71 25 92 80 28
0.6 30 90 75 36 40 38 50 91 78 42 27 60
There were no surviving colonies after 5 minutes of mating
The azis gene is transferred first
It is followed by the tons gene
The lac+ gene enters between 15 and 20 minutes
The gal+ gene enters between 20 and 25 minutes
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33. From these data, Wollman and Jacob constructed the following genetic map:
They also identified various Hfr strains in which the origin of transfer had been integrated at different places in the chromosome
Comparison of the order of genes among these strains, demonstrated that the E. coli chromosome is circular
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34. The E. coli Chromosome
Conjugation experiments have been used to map more than 1,000 genes on the E. coli chromosome
The E. coli genetic map is 100 minutes long
Approximately the time it takes to transfer the complete chromosome in an Hfr mating
Refer to Figure 6.7
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35. Arbitrarily assigned the starting point
Units are minutes
Refer to the relative time it takes for genes to first enter an F– recipient during a conjugation experiment
Figure 6.7
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36. The distance between genes is determined by comparing their times of entry during an interrupted mating experiment
The approximate time of entry is computed by extrapolating the time back to the origin
Figure 6.8
Therefore these two genes are approximately 9 minutes apart along the E. coli chromosome
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37. Plasmids
Plasmids occur naturally in many strains of bacteria and a few eukaryotic cells such as yeast
Range in size from a few thousand to 500,000 bp
Carry from one to hundreds of genes
Different plasmids will have one to 100 copies per cell
Have their own origins of replication which are strong or weak
Plasmids can provide a growth advantage to the cell
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38. Plasmids Plasmids fall into five different categories: 6-33
1. Fertility plasmids-allow bacteria to mate to each other
2. Resistance plasmids-confer resistance to antibiotics or toxins
3. Degradative plasmids-enable the digestion of unusual substances
4. Col-plasmids-encode colicines which are proteins that kill other bacteria
5. Virulence plasmids-turn a bacterium into a pathogenic strain
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39. Transduction
Transduction is the transfer of DNA from one bacterium to another via a bacteriophage
A bacteriophage is a virus that specifically attacks bacterial cells
It is composed of genetic material surrounded by a protein coat
It can undergo two types of cycles
Lytic
Lysogenic
Refer to Figure 6.9
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40. It will switch to the lytic cycle
Prophage can exist in a dormant state for a long time
Virulent phages only undergo a lytic cycle
Temperate phages can follow both cycles
The lytic and lysogenic reproductive cycles of certain bacteriophages. Some bacteriophages, such as temperate phages, can follow both cycles. Other phages, known as virulent phages, can follow only a lytic cycle.

41. Transduction Phages that can transfer bacterial DNA include
P22, which infects Salmonella typhimurium
P1, which infects Escherichia coli
Both are temperate phages
Figure 6.10 illustrates the process of transduction
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42. Any piece of bacterial DNA can be incorporated into the phage
This type of transduction is termed generalized transduction
Figure 6.10
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43. They used an experimental strategy similar to that of Figure 6.1
Transduction was discovered in 1952 by Joshua Lederberg and Norton Zinder
They used an experimental strategy similar to that of Figure 6.1
They used two strains of the bacterium Salmonella typhimurium
One strain, designated LA-22, was phe– trp– met+ his+
Unable to synthesize phenylalanine or tryptophan
Able to synthesize methionine and histidine
The other strain, designated LA-2, was phe+ trp+ met– his–
Able to synthesize phenylalanine and tryptophan
Unable to synthesize methionine or histidine
Their experiment is described next
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44. 6-39 phe– trp– met+ his+ phe+ trp+ met– his–
Nutrient agar plates lacking the four amino acids
Genotypes of surviving bacteria must be
phe+ trp+ met+ his+
Therefore, genetic material had been transferred between the two strains
~ 1 cell in 100,000
was observed to grow
However, Lederberg and Zinder obtained novel results when repeating the experiment using the U-tube apparatus
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45. Nutrient agar plates lacking the four amino acids
phe– trp– met+ his+
phe+ trp+ met– his–
Nutrient agar plates lacking the four amino acids
Colonies
No colonies
Genotypes of surviving bacteria must be
phe+ trp+ met+ his+
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46. In this case, the LA-2 strain contained a prophage (such as P22)
Therefore, some agent was being transferred from LA-2 to LA-22 through the filter
Norton and Zinder conducted the same experiment with filters of different pore sizes
They found out that the filterable agent was less then 0.1mm in diameter
They correctly concluded that the filterable agent was a bacteriophage
In this case, the LA-2 strain contained a prophage (such as P22)
The prophage switched to the lytic cycle
Packaged a segment of DNA containing the phe+ and trp+ genes
Passed through the filter and injected the DNA into the LA-22 strain
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47. Transformation
Transformation is the process by which a bacterium will take up extracellular DNA released by a dead bacterium
It was discovered by Frederick Griffith in 1928 while working with strains of Streptococcus pneumoniae
There are two types
Natural transformation
DNA uptake occurs without outside help
Artificial transformation
DNA uptake occurs with the help of special techniques
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48. Transformation
Natural transformation occurs in a wide variety of bacteria
Bacterial cells able to take up DNA are termed competent cells
They carry genes that encode proteins called competence factors
These proteins facilitate the binding, uptake and subsequent corporation of the DNA into the bacterial chromosome
The steps of bacterial transformation are presented in Figure 6.12
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49. A region of mismatch caused by sequence differences between the two alleles
By DNA repair enzymes
Figure 6.12
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52. Transformation
Sometimes, the DNA that enters the cell is not homologous to any genes on the chromosome
It may be incorporated at a random site on the chromosome
This process is termed nonhomologous or illegitimate recombination
Some bacteria preferentially take up DNA of bacteria from the same or related species
Directed by DNA uptake signal sequences
9 or 10 bp long
repeated 1-2,000 times throughout genome
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53. Horizontal Gene Transfer
Horizontal gene transfer is the transfer of genes between two different species
Vertical gene transfer is the transfer of genes from mother to daughter cell or from parents to offspring
A sizable fraction of bacterial genes are derived from horizontal gene transfer
Roughly 17% of E. coli and S. typhimurium genes during the past 100 million years
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54. Horizontal Gene Transfer
The types of genes acquired through horizontal gene transfer are quite varied and include
Genes that confer the ability to cause disease
Genes that confer antibiotic resistance
Genes that give the ability to degrade toxins
Horizontal gene transfer has dramatically contributed to the phenomenon of acquired antibiotic resistance
Bacterial resistance to antibiotics is a serious problem worldwide
In many countries, nearly 50% of Streptococcus pneumoniae strains are resistant to penicillin
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58. 6.2 INTRAGENIC MAPPING IN BACTERIOPHAGES
Viruses are not living
They rely on a host cell for existence and replication
However, they have unique biological structures and functions, and therefore have traits
We will focus our attention on bacteriophage T4
Its genetic material contains several dozen genes
These genes encode a variety of proteins needed for the viral cycle
Refer to Figure 6.13 for the T4 structure
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59. 6-55 Figure 6.13 Contains the genetic material
Used for attachment to the bacterial surface
Figure 6.13
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60. In the 1950s, Seymour Benzer embarked on a ten-year study focusing on the function of the T4 genes
He conducted a detailed type of genetic mapping known as intragenic or fine structure mapping
The difference between intragenic and intergenic mapping is:
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61. Plaques
A plaque is a clear area on an otherwise opaque bacterial lawn on the agar surface of a petri dish
It is caused by the lysis of bacterial cells as a result of the growth and reproduction of phages
Figure 6.14
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62. Plaques are visible with the naked eye
Some mutations in the phage’s genetic material can alter the ability of the phage to produce plaques
Thus, plaques can be viewed as traits of bacteriophages
Plaques are visible with the naked eye
So mutations affecting them lend themselves to easier genetic analysis
An example is a rapid-lysis mutant of bacteriophage T4, which forms unusually large plaques
Refer to Figure 6.15
This mutant lyses bacterial cells more rapidly than do the wild-type phages
Rapid-lysis mutant forms large, clearly defined plaques
Wild-type phages produce smaller, fuzzy-edged plaques
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63. Benzer studied one category of T4 phage mutant, designated rII (r stands for rapid lysis)
It behaved differently in three different strains of E. coli
In E. coli B
rII phages produced unusually large plaques that had poor yields of bacteriophages
The bacterium lyses so quickly that it does not have time to produce many new phages
In E. coli K12S
rII phages produced normal plaques that gave good yields of phages
In E. coli K12(l) (has phage lambda DNA integrated into its chromosome)
rII phages were not able to produce plaques at all
As expected, the wild-type phage could infect all three strains
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64. Complementation Tests
Benzer collected many rII mutant strains that can form large plaques in E. coli B and none in E. coli K12(l)
But, are the mutations in the same gene or in different genes?
To answer this question, he conducted complementation experiments
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65. Figure 6.16 shows the possible outcomes of complementation experiments involving coinfection of plaque formation mutants
Figure 6.16
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66. A cistron is equivalent to a gene
Benzer carefully considered the pattern of complementation and noncomplementation
He determined that the rII mutations occurred in two different genes, which were termed rIIA and rIIB
Benzer coined the term cistron to refer to the smallest genetic unit that gives a negative complementation test
So, if two mutations occur in the same cistron, they cannot complement each other
A cistron is equivalent to a gene
However, it is not as commonly used
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67. Function of protein A will be restored
At an extremely low rate, two noncomplementing strains of viruses can produce an occasional viral plaque, if intragenic recombination has occurred
rII mutations
Viruses cannot form plaques in E. coli K12(l)
rII mutations
Function of protein A will be restored
Therefore new phages can be made in E. coli K12(l)
Viral plaques will now be formed
Figure 6.17
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68. Figure 6.18 describes the general strategy for intragenic mapping of rII phage mutations
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69. r103
r104
Both rII mutants and wild-type phages can infect this strain
rII mutants cannot infect this strain
Number of wild-type phages produced by intragenic recombination
Total number of phages
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70. The data from Figure 6.18 can be used to estimate the distance between the two mutations in the same gene
The phage preparation used to infect E. coli B was diluted by 108 (1:100,000,000)
1 ml of this dilution was used and 66 plaques were produced
Therefore, the total number of phages in the original preparation is
66 X 108 = 6.6 X 109
or 6.6 billion phages per milliliter
The phage preparation used to infect E. coli k12(l) was diluted by 106 (1:1,000,000)
1 ml of this dilution was used and 11 plaques were produced
Therefore, the total number of wild-type phages is
11 X 106
or 11 million phages per milliliter
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71. In this experiment, the intragenic recombination produces an equal number of recombinants
Wild-type phages and double mutant phages
However, only the wild-type phages are detected in the infection of E. coli k12(l)
Therefore, the total number of recombinants is the number of wild-type phages multiplied by two
2 [wild-type plaques obtained in E. coli k12(l)]
Frequency of recombinants =
Total number of plaques obtained in E. coli B
2(11 X 106)
6.6 X 109
Frequency of recombinants =
= 3.3 X 10–3 =
In this example, there was approximately 3.3 recombinants per 1,000 phages
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72. Homoallelic mutations
As in eukaryotic mapping, the frequency of recombinants can provide a measure of map distance along the bacteriophage chromosome
In this case the map distance is between two mutations in the same gene
The frequency of intragenic recombinants is correlated with the distance between the two mutations
The farther apart they are the higher the frequency of recombinants
Homoallelic mutations
Mutations that happen to be located at exactly the same site in a gene
They are not able to produce any wild-type recombinants
So the map distance would be zero
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73. Deletion Mapping
Benzer used deletion mapping to localize many rII mutations to a fairly short region in gene A or gene B
He utilized deletion strains of phage T4
Each is missing a known segment of the rIIA and/or rIIB genes
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74. Let’s suppose that the goal is to know the approximate location of an rII mutation, such as r103
E. coli k12(l) is coinfected with r103 and a deletion strain
If the deleted region includes the same region that contains the r103 mutation
No intragenic wild-type recombinants are produced
Therefore, plaques will not be formed
If the deleted region does not overlap with the r103 mutation
Intragenic wild-type recombinants can be produced
And plaques will be formed
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75. Mutation must be in region contained in BP242 but not in PT1
Mutation must be in region contained in BP242 but not in PT1. This corresponds to A4 in the rIIA gene
Figure 6.19
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76. As described in Figure 6.19, the first step in the deletion mapping strategy localized rII mutations to seven regions
Six in rIIA and one in rIIB
Other strains were used to eventually localize each rII mutation to one of 47 regions
36 in rIIA and 11 in rIIB
At this point, pairwise coinfections were made between mutant strains that had been localized to the same region
This would precisely map their location relative to each other
This resulted in a fine structure map with depicting the locations of hundreds of different rII mutations
Refer to Figure 6.20
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77. Contain many mutations at exactly the same site within the gene
Figure 6.20
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78. Intragenic mapping studies were a pivotal achievement in our early understanding of gene structure
Some scientists had envisioned a gene as being a particle-like entity that could not be further subdivided
However, intragenic mapping revealed convincingly that this is not the case
It showed that
Mutations can occur at different parts within a single gene
Intragenic crossing over can recombine these mutations, resulting in wild-type genes
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