Genetic fine str. analysis & complementation
- 1. PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY College of Agriculture, Rajendranagar, Hyderabad- 500030 Presented by, Ajay Kumar Chandra RAM/14-97 M.Sc. (Ag) Mol. Biology & Biotechnology
- 2. What …… we going to discuss today? - G. Mendel (1866) - One factor - one character - A.E. Garrod (1909) - one mutant gene – one metabolic block - Beadle and Tatum (1942)- One Gene- One Enzyme - Vernon Ingram (1957) One gene – one polypeptide • IS THE GENE, THE BASIC UNIT OF GENETIC STRUCTURE? BEADS -ON - A STRING CONCEPT: 1). Oliver experiment- in lozenge locus on Drosophila 2). Benzer's experiment on rII locus of Bacteriophage T4 3). Complementation test by Lewis on Drosophila • REVISED BEAD THEORY • The new concept of gene • Organization of a Typical Eukaryotic Gene • Conclusion • Reference
- 3. How has the concept of a gene developed in the minds of geneticists? • G. Mendel (1866) - One factor - one character Inheritance is governed by “characters” or “constant factors” that each controls a phenotypic trait such as flower colour. • A.E. Garrod (1909) - one mutant gene – one metabolic block - Inborn errors of Metabolism - Individual genes can mutate to cause a specific metabolic block. - Concept later elaborated as “one gene-one enzyme”.
- 4. 4 Beadle and Tatum (1942)- One Gene- One Enzyme
- 5. • Each gene controls the synthesis of an enzyme involved in catalyzing the conversion of an intermediate into arginine.
- 6. One gene – one polypeptide • Vernon Ingram (1957) • The hypothesis that a large class of structural genes exists in which each gene encodes a single polypeptide, which may function either independently or as a subunit of a more complex protein. • Originally it was thought that each gene encoded the whole of a single enzyme, but it has since been found that some enzymes and other proteins derive from more than one polypeptide and hence from more than one gene.
- 7. Is the gene, the basic unit of genetic structure?
- 8. BEADS -ON - A STRING CONCEPT The gene is the fundamental unit of 1. Structure... indivisible by crossing over 2. Change... Mutations change alleles from one form to another; there are no smaller units within genes that can change 3. Function... parts of genes cannot function alone in tests of complementation FINE STRUCTURE OF GENESeymour Benzer (1950-60) The genes in a chromosome were considered analogous to beads on a string. Recombination was believed to occur between the beads or genes, not within the gene. The gene was believed to be indivisible.
- 9. RECOMBINATION WITH IN THE GENE The following experiments supported this concept- which are 1) Oliver experiment- in lozenge locus on Drosophila 2) Benzers experiment on rII locus of Bacteriophage T4 3) Complementation test by Lewis on Drosophila
- 10. C.P.Oliver (1940) - Intergenic recombination at lozenge • Mutations in lozenge affect eye shape in Drosophila. • In Drosophila, Lozenge (lz)----smaller, darker & elliptical eyes fly heterozygous for two lozenge mutant alleles (lz1/lz2) (spectacle/glassy eye) will not yield wild type in its progeny, because wild allele is absent. • Two mutations, lzs and lzg, were considered alleles of the same gene because lzs/lzg heterozygotes have lozenge, not wild-type, eyes. • But when lzs/lzg females are crossed to lzs or lzg males, about 0.2% of the progeny are wild-type! • These must result from recombination between lzs and lzg , because the wild-type progeny always had recombinant flanking markers. Also, the frequency of 0.2% is much higher than the reversion rate of the mutations.
- 11. Fine structure of Lozenge locus The results of Green on intragenic recombination in lozenge locus in Drosophila melanogaster demonstrated 14 alleles can be located on four mutational sites separated by distances proportionate to recombination frequencies. Close-linked alleles of this type, which have similar phenotypic effects but still recombine with each other, are considered to occupy a complex locus. Alleles on different mutational sites could be subjected to cis-trans test, & complementation relationships show that all 14 alleles exhibit lack of complementation among each other, so belong to one functional unit. Complex locus can be divided into subloci between which re- combination frequency can be scored. The complex locus lozenge consists of four groups, each containing one or more different alleles. Recombination distances between these groups are on the order of approx. 0.03 to 0.09 percent.
- 12. Fine structure of Lozenge locus Further studies of intergenic recombination in bacteriophage and bacteria showed that recombination occurs between adjacent nucleotide pairs. So the nucleotide, not the gene, is the basic unit of genetic structure. Nucleotide, not the gene
- 13. Two types of traits: plaque morphology Host range property 1. Permissive host E. coli B; all rII- & rII+ phages grow. 2. Restrictive host E. coli K12; rII+ recombinants grow. Benzer , studied rII gene of T4 Bacteriophage to test these hypotheses...
- 14. Recombinants of two rII mutants of T4
- 15. 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 structure…indivisible by crossing over ? is wrong.. ….? - Nucleotide pair is the fundamental unit of structure (recombination: recon)
- 16. Hypothesis -1: gene is fundamental unit of structure... indivisible by crossing over ? GENE = UNIT OF STRUCTURE? O O X Mutant 1 Mutant 2 OO Wild type Double Mutant 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. Recombination between two mutants to give a wild type (non-mutant) form of the gene.
- 17. what is the smallest unit of recombination detectable? phage system can detect 1 mutation in 109 progeny recombination frequency (RF) = 1 x 10–9 smallest distance = 1 x 10–7 cM if 1 cM 5 x 105 bp (on average, in flies) bp are separated by 2 x 10–6 cM phage system ~20x more powerful than needed to detect smallest possible distance. GENE = UNIT OF STRUCTURE? PFU on B = total, PFU on K = ½ of recombinants • Recombination frequency (RF) = 2(PFU on K) / PFU on B 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 i.e, 0.02 cM represents a pair of adjacent nucleotides. Frequency of = 2 x No.of plaques onK12(λ) recombination No. of plaques on B
- 18. Fine structure mapping of rII region in T4
- 19. Hypothesis -2: gene is fundamental unit of change... mutations change alleles from one form to another; there are no smaller mutable units within a gene ? - Nucleotide pair is the fundamental unit of change/mutation (muton) • Same experimental protocol as for Hypothesis -1 GENE = UNIT OF CHANGE? • Mutants from 1 were deletions when they did not ... - recombine with some other mutants - ever revert back to wild type • Deletions used to map positions of mutational sites
- 20. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 GENE = UNIT OF CHANGE? A). all survive on...B only 4 + + 5 4 + + 5 survive on...B only K & B 4 5 + +
- 21. 4 5 4 5 survive on...B only K & B GENE = UNIT OF CHANGE? B). 4 + + 5 4 + + 5 survive on...B only + +
- 22. C). 4 + + 5 4 + + 5 + 5 4 + survive on...B only B only No recombination
- 23. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 D1 D2 D3 D4 D1 D2 D3 D4 A B C D E F G D). Deletion mapping 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
- 24. Deletion mapping of the rII region of Bacteriophage T4 Point mutations (revert to wild type at low frequency) mapped to regions defined by deletion mutations. Point mutations in the same regions that did not recombine with each other were identical sites. Distribution of rII point mutations was not random...
- 25. • Based on the presence or absence of recombinants. In any cross between an unknown point mutation and one of the deletions Figure - Localization of an rII mutation by deletion mapping
- 26. Fine structure mapping of rII region in T4
- 27. Distribution of rII point mutations was not random... Hot spots & Cold spots GENE = UNIT OF CHANGE?
- 28. Poisson distribution predicted missed cold spots observed hot spots (e.g.) GENE = UNIT OF CHANGE? site = nucleotide base pair
- 29. Hypothesis -3: gene is fundamental unit of function... parts of genes cannot function in complementation tests ? GENE = UNIT OF FUNCTION? mutants fell into two functional groups... 2 genes mutations fail to complement others in the same group
- 30. Allelic Complementation test • Diagnostic test for Allelism. • The complementation test was developed by Edward B. Lewis. • A complementation test (sometimes called a "cis-trans" test) can be used to test whether the mutations in two strains are in different genes. • Mutants are crossed to bring two recessive mutations together in heterozygous form. – if phenotype is mutant, mutations are in same gene • they fail to complement • both have loss of function – if phenotype is wild-type, mutations are in different gene • each mutant contributes normal gene at different locus • complementation due to interaction of different proteins. • Heterokaryons used in haploid organisms
- 31. GENE = UNIT OF FUNCTION? 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.
- 32. 32 Complementation test for mutations in different genes
- 33. 33 Detecting recombination between two mutations in the same gene
- 34. 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 Conclusion: 1) Bead theory was not correct. genes are linear sequences of nucleotide pairs . 2) Genetic exchange can take place within a gene and probably between any adjacent base pairs. 3) Some regions of chromosomes mutate at a higher rate than others – Hot spots. 4) The smallest units of mutation and recombination are now known to be correlated with single nucleotide pairs. 5) Complementation testing - are two mutations in the same or different genes?
- 35. The New Concept of Gene Benzer defined the unit of function, recombination and mutation, and coined the terms Cistron, Recon and Muton. Cistron: The unit of function was called cistron, the elements of which exhibit cis-trans phenomenon. A cistron can be divided into many recons. Recon: The smallest unit capable of undergoing recombination is called Recon. A recon is further subdivided into mutons. Mutons:The smallest unit of gene which is capable of mutating is known as Muton. As mutation can take place by single base replacement, a single nucleotide pair is the ultimate limit of muton. Thus, a gene can consist of several cistrons, a cistron can have several recons and a recon can consist several mutons.
- 36. Organization of a Typical Eukaryotic Gene Enhancer (distal control elements) Proximal control elements DNA Upstream Promoter Exon Intron Exon Intron Poly-A signal sequence Exon Termination region Transcription Downstream Poly-A signal ExonIntronExonIntronExonPrimary RNA transcript (pre-mRNA) 5 Intron RNA RNA processing: Cap and tail added; introns excised and exons spliced together Coding segment P P PGmRNA 5 Cap 5 UTR (untranslated region) Start codon Stop codon 3 UTR (untranslated region) Poly-A tail Chromatinchanges Transcription RNA processing mRNA degradation Translation Protein processing and degradation Cleared 3 end of primary transport
- 37. 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” Gene definition: 1860s–1900s: Gene as a discrete unit of heredity 1910s: Gene as a distinct locus 1940s: Gene as a blueprint for a protein 1950s: Gene as a physical molecule 1960s: Gene as transcribed code 1970s–1980s: Gene as open reading frame (ORF) 1990s–2000s: Annotated genomic entity, 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?
- 38. Reference • BOOKS REFFERED: INTRODUCTION TO GENETIC ANALYSIS: Anthony J.F.Griffiths & his groups, 9th edition, W. H. Freeman and Company GENETICS- A CONCEPTUAL APPROACH: Benjamin A. Pierce, fourth edition , south western university, W. H. Freeman and company. MOLECULAR BIOLOGY OF THE GENE: James D. Watson, Tania A. Baker, Stephan P. Bell and others, 5th edition, Pearson- Benjamin Cummings