BIO222 Molecular Genetics Lecture Notes PDF

Because learning changes everything. ® BIO222 Molecular genetics 3 Cr. Hrs. = (2 LCT + 0 TUT + 2 LAB + 0 OTH) – SWL = 150 – ECTS = 6 Prerequisite - - - Introduction to Molecular genetics. Regulation of transcription and translation in eukaryotic and prokaryotics....

Because learning changes everything. ® BIO222 Molecular genetics 3 Cr. Hrs. = (2 LCT + 0 TUT + 2 LAB + 0 OTH) – SWL = 150 – ECTS = 6 Prerequisite - - - Introduction to Molecular genetics. Regulation of transcription and translation in eukaryotic and prokaryotics. Genome, Transcriptome, Proteome. Genome structure, stability, and organization. Prokaryotic Genomes. Eukaryotic Genomes. Accessing Genomes. Mapping genomes. Molecular genetics of development. Types of mutations and identification of disease genes. Epigenetics, inherited diseases.  Because learning changes everything. ® Chapter 07 Genetic Transfer and Mapping in Bacteria Genetics: Analysis & Principles EIGHTH EDITION Robert J. Brooker © McGraw Hill LLC. All rights reserved. No reproduction or distribution w ithout the prior w ritten consent of McGraw Hill LLC.  Genetic Transfer and Mapping in Bacteria Eye of Science/Science Source © McGraw Hill 3  7.3 Conjugation and Mapping via Hfr Strains In the 1950s, Luca Cavalli-Sforza discovered a strain of E. coli that was very efficient at transferring chromosomal genes This strain was designated Hfr (for High frequency of recombination) Hfr strains are derived from F+ strains (Figure 7.5a) © McGraw Hill 4  (Figure 7.5a) Integration of an F factor to form an Hfr cell and its subsequent excision to form an F′ (a) When an F factor integrates into the chromosome, it creates an Hfr cell. Access the text alternative for slide images. © McGraw Hill 5  F Factors can be Excised Imprecisely In some cases, the integrated F factor is excised in an imprecise ‫غير دقيق‬fashion (Figure 7.5b) May carry genes that were once found on the bacterial chromosome These types of F factors are called F’ factors (for F prime factors) © McGraw Hill 6  (Figure 7.5b) Integration of an F factor to form an Hfr cell and its subsequent excision to form an F′ (b) When an F factor excises imprecisely, an F’ factor is created Access the text alternative for slide images. © McGraw Hill 7  Conjugation between Hfr and F− Strains 1 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 When the DNA is cut, or nicked, at this site it becomes the starting point for the transfer of the Hfr chromosome to the F- cell Then, a strand of bacterial DNA begins to enter the recipient cell in a linear manner © McGraw Hill 8  Conjugation between Hfr and F− Strains 2 It generally takes about 1.5 to 2 hours for the entire Hfr chromosome to be passed into the F- cell Most conjugations do not last that long Only a portion of the Hfr chromosome gets into the F- cell Chromosomal material from the Hfr cell can recombine with the homologous region on the chromosome of the recipient cell © McGraw Hill 9  Transfer of Bacterial Genes from an Hfr Cell to an F− Cell Genotype of Hfr cell is: lac + → Ability to metabolize lactose pro +→ Ability to synthesize proline Genotype of F- cell is: lac - → Inability to metabolize lactose pro -→ Inability to synthesize proline © McGraw Hill 10  (Figure 7.6) Transfer of bacterial genes from an Hfr cell to an F− cell 1 Access the text alternative for slide images. © McGraw Hill 11  (Figure 7.6) Transfer of bacterial genes from an Hfr cell to an F− cell 2 Access the text alternative for slide images. © McGraw Hill 12  Experiment 7A Interrupted Conjugation Technique Developed by Elie Wollman and François Jacob in the 1950s 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, interrupting conjugations at different times leads to various lengths being transferred The order of genes along the chromosome can be deduced by determining the genes transferred during short conjugations versus those transferred during long conjugations © McGraw Hill 13  Strains for Interrupted Mating 1 The donor (Hfr) strain genetic composition: thr + : Able to synthesize the essential amino acid threonine leu + : Able to synthesize the essential amino acid leucine azi s : Sensitive to killing by azide (a toxic chemical) ton s : 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 s str : Sensitive to killing by streptomycin (an antibiotic) © McGraw Hill 14  Strains for Interrupted Mating 2 ( ) The recipient F− strain had the opposite genotype: thr − : Unable to synthesize the essential amino acid threonine - leu : Unable to synthesize the essential amino acid leucine azi r : Resistant to killing by azide (a toxic chemical) r ton : Resistant to infection by T1 (a bacterial virus) − lac : Unable to metabolize lactose and use it for growth − gal : Unable to metabolize galactose and use it for growth r str : Resistant to killing by streptomycin (an antibiotic) © McGraw Hill 15  The Goal (Discovery-Based Science) 1 Wollman and Jacob already knew that: The thr + and leu + genes were transferred first, in that order Both were transferred within 5 to 10 minutes of mating 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 © McGraw Hill 16  The Goal (Discovery-Based Science) 2 Therefore their main goal was to determine the times at + which genes azi s , ton s , lac + , and gal were transferred The transfer of the str s was not examined Streptomycin was used to kill the donor (Hfr) cell following conjugation ( ) The recipient F- cell is streptomycin resistant © McGraw Hill 17  Achieving the Goal 1. Mix together a large number of Hfr donor and F- recipient cells. 2. After different periods of time, take a sample of cells and interrupt conjugation in a blender. 3. Plate the cells on solid growth medium lacking threonine and leucine but containing streptomycin. Note: The general methods for growing bacteria in a laboratory are described in the Appendix A. 4. Pick each surviving colony, which would have to be thr + leu + str r , and test to see if it is sensitive to killing by azide, sensitive to infection by T1 bacteriophage, and able to metabolize lactose or galactose. © McGraw Hill 18  (Figure 7.7) The use of conjugation to map the order of genes along the E. coli chromosome Access the text alternative for slide images. © McGraw Hill 19  The Data 1 Number of Percentage of Percentage of Percentage of Percentage of Percentage of Minutes That Surviving Surviving Surviving Surviving Surviving Bacterial Cells Bacterial Bacterial Bacterial Bacterial Bacterial Were Allowed to Colonies with Colonies with Colonies with Colonies with Colonies with Conjugate the Following the Following the Following the Following the Following Before Blender Genotypes: Genotypes: Genotypes: Genotypes: Genotypes: Treatment thr +leu+ azis tons lac + gal+ 5* — — — — — 10 100 12 3 0 0 15 100 70 31 0 0 20 100 88 71 12 0 25 100 92 80 28 0.6 30 100 90 75 36 5 40 100 90 75 38 20 50 100 91 78 42 27 60 100 91 78 42 27 *There were no surviving colonies within the first 5 minutes of conjugation. Source: Data from Francois Jacob and Elie Wollman (1961), Sexuality and the Genetics of Bacteria, Academic Press, New York. © McGraw Hill 20  The Data 2 KEY RESULTS: There were no surviving colonies after 5 minutes of mating. After 10 minutes, the thr + leu + genotype was obtained. Next, the azi s gene is transferred, s Next, the ton gene, The lac + gene enters between 15 and 20 minutes, + The gal gene enters between 20 and 25 minutes. © McGraw Hill 21  A Genetic Map was Constructed 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 © McGraw Hill 22  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 The starting point, 0 minutes, is arbitrarily assigned near the thrA gene. Units are minutes Refers to the relative time it takes for genes to first enter an F − recipient during a conjugation experiment © McGraw Hill 23  (Figure 7.8) A simplified genetic map of the E. coli chromosome indicating the positions of several genes Access the text alternative for slide images. © McGraw Hill 24  Interrupted Conjugation Experiment The distance between genes is determined by comparing their times of entry during an interrupted conjugation experiment The approximate time of entry is computed by extrapolating the time back to the x-axis In this example, these two genes are approximately 9 minutes apart along the E. coli chromosome © McGraw Hill 25  (Figure 7.9) Time course of an interrupted E. coli conjugation experiment Access the text alternative for slide images. © McGraw Hill 26