FINE STRUCTURE OF GENE Seymour Benzer (1950-60) Bead Theory: The gene is the fundamental unit of Structure Change & Function Benzer: if a gene were a sequence of bases it could be divisible at many different locations

Benzer studied of the rII region of T4: (rII mutant causing rapid lysis of E. coli than wild type) Two types of traits: plaque morphology Host range property Permissive host E. coli B; all (rII- & rII+phages grow. Restrictive host E. coli K12; rII+ recombinants grow.

Recombinants of two rII mutants of T4.

Recombination frequency Gene order is determined by frequency of recombinants. If recombination rate is high, genes are far apart. If recombination rate is low, genes are close together.

Intragenic Recombination Recombination between two mutants to give a wild type (non-mutant) form of the gene. O X Mutant 1 Mutant 2 Wild type Double Mutant

Summary of fine structure mapping of rII region in T4

Seymour Benzer’s conclusion: Benzer studied 3000 rII mutants showing nucleotide deletions at different levels of subdivision & determined that the rII region is sub-divisible into >300 mutable sites by series of nested analyses (ANOVA) and comparisons. hypothesis #1: gene is fundamental unit of change.. ? is wrong.. Nucleotide pair is the fundamental unit of change/mutation (muton)

Seymour Benzer’s conclusion: Benzer’s recombination frequency between some pairs of these was as low as 0.02. The T4 genome has 160,000 base pairs of DNA extending over ~1,600 centimorgans (cM). So 1 cM ≅ 100 base pairs So 0.02 cM represents a pair of adjacent nucleotides. hypothesis #2: gene is fundamental unit of structure…indivisible by crossing over ? is wrong.. Nucleotide pair is the fundamental unit of structure (recombination: recon)

Complementation The production of the wild-type phenotype by a cell that contains two mutant genes. The deficiency of one homologue is supplied by the normal allele of the other

complementation or recombination ? protein level, mixing of gene products no change in genotypes relative to parents requires 1 generation of crosses to investigate recombination... DNA level results in new genotypes relative to parents requires 2 generations of crosses to investigate

GENE = UNIT OF FUNCTION? hypothesis #3: gene is fundamental unit of function... parts of genes cannot function in complementation tests ?

GENE = UNIT OF FUNCTION? hypothesis #3: gene is fundamental unit of function... parts of genes cannot function in complementation tests ?... Correct a unit of DNA sequence that codes for a single polypeptide or protein Cistron: Term coined by Benzer for the smallest genetic unit that does NOT show genetic complementation when two different mutations are in trans position; but shows wild-type phenotype when the same mutations are in cis.

(human genes ~30,000 & proteins ~1,20,000 ???) GENE = UNIT OF FUNCTION? Alternative Splicing ? ? ? (human genes ~30,000 & proteins ~1,20,000 ???) One Gene / One Enzyme One Gene / One Polypeptide “One Gene / One set of connected transcripts The definition of a gene by Gerstein et al. [2007] as ‘a union of genomic sequences encoding a coherent set of potentially overlapping functional products’ allows genes to have an overlapping sequence, to be alternatively spliced and to exert functions other than protein coding...

GENE = UNIT OF FUNCTION? Gene definition: After ENCODE (Encyclopedia of DNA Elements) PROJECT Gene definition: Definition 1860s–1900s: Gene as a discrete unit of heredity Definition 1910s: Gene as a distinct locus Definition 1940s: Gene as a blueprint for a protein Definition 1950s: Gene as a physical molecule Definition 1960s: Gene as transcribed code Definition 1970s–1980s: Gene as open reading frame (ORF) Definition 1990s–2000s: Annotated genomic entity, Definition (Gerstein et al. 2007) The gene is a union of genomic sequences encoding a coherent set of potentially overlapping functional products. TIME LINE OF GENE DEFINITION

FINE STRUCTURE OF GENE Seymour Benzer (1950-60) Revised Bead Theory: The nucleotide pair is the fundamental unit of Structure Change & The gene is the fundamental unit of Function New Terms: cistron (unit of function), recon (unit of recombination) and muton (unit of mutation).