Seymour Benzer's experiment and complementation test

Complementation test

1. COMPLEMENTATION TEST & SEYMOUR BENZER'S EXPERIMENT COURSE INSTRUCTOR: DR. SANJAY KUMAR STUDENT: AKANKSHA

2. In 1954 Seymour Benzer w orked on a complementation test in Bacteriophages. INTRODUCTION He chose T4 phage which infects E.coli. Among different genes of phage chromosome one was r11 which was selected for the complementation test. Benzer identified two separate loci within the r11 gene (rII+ and rII-)

3. WHAT IS A COMPLEMENTATION TEST? A genetic test used to determine whether two mutations are in the same or different genes. It is also called cis-trans test and used to investigate a specific phenotype (external character) associated with two gene mutation which would be due to one gene alleles or variations of two different genes. Key Terms: Cis: Mutations on the same chromosome. Trans: Mutations on different chromosomes.

4. When conducting a complementation test, there are four important factors to consider: Both mutations must be recessive. the mutations can cause different homozygous phenotypes. A trans-heterozygote may exhibit a more severe phenotype than a homozygote alone. A single mutation might impact multiple gene products within a complex genetic locus. FACTORS

5. Cistron: Synonym for gene; a segment of DNA encoding a polypeptide chain. Prokaryotic mRNA is called polycistronic i.e. single mRNA contains several genes encode for several polypeptide. Whereas in eukaryote, mRNA is monocistronic i.e. each mRNA contains one gene encodes one protein CISTRON, RECON, AND MUTON Recon: Seymour Benzer coined the term, recon for the smallest recombinable unit within a cistron. A region of a gene within which there can be no crossing-over; now known to be a single nucleotide pair.It is the smallest unit capable of recombining genetically. Muton: The smallest unit of mutation. It consists of few nucleotides than a cistron.Benzer coined the term muton to that DNA capable of mutational change. A muton may thus be delimited to a single nucleotide and some part of nucleotide.

6. Plaque of E.coli B Plaque of E.coli K Permissive host EcoliB: All rII+ and rII- phages grow. Restrictive host Ecoli K: Only rII+ Recombinant grows. Plaque Morphology Host range BENZER’S EXPERIMENT

7. Seymour benzer performed 3 seperate test T4 phage (1) T4 phage (2) He took K12 Strain of E.coli and infected it with two types of T4 phages T4 type 1 and T4 Type 2. both type of phages contain mutation in rIIA locus but the position of mutation is different, and their rIIB locus is wild so no mutations found. Phages don't grow. rIIA(Mutation at Position A ) rIIA(Mutation at Position B ) rIIB(Wild) rIIB(Wild) Phages do not grow Ecoli lawn

8. Seymour benzer performed 3 seperate test T4 phage (3) T4 phage (4) He took K12 Strain of E.coli and infected it with two types of T4 phages T4 type 3 and T4 Type 4. both type of phages contain mutation in rIIB locus but the position of mutation is different, and their rIIA locus is wild no mutations found,Phages don't grow. rIIB(Mutation at Position A ) rIIB(Mutation at Position B ) rIIA(Wild) rIIA(Wild) Phages do not grow Ecoli lawn

9. Seymour benzer performed 3 seperate test T4 phage (1) T4 phage (4) In 3rd test, he took the same K12 Strain of E.coli and infected it with two types of T4 phages T4 type 1 and T4 Type 4. T4 type1 Phages contain mutation in rIIA locus and T4 type 4 phage contain mutation in rIIB locus Surprisingly Phages could grow rapidly in this culture which means these two types of phases complement each other rIIA(Mutation at Position A ) rIIB(Mutation at Position B ) rIIB(Wild) rIIA(Wild) Phages grow Ecoli lawn

10. Benzer started with a hypothesis that a gene is a segment of DNA. This hypothesis implies that parts of genes can be mutated separately and recombination can occur inside a gene. He detected crossing over between independent mutations in the same genes implying that the two mutations are indeed in separate base pairs and the crossing over within the genes separates the mutation these results show that Benzer's hypothesis is correct In addition, Benzer defined a gene as a unit of function detectable experimentally as a complementation group CONCLUSION