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Unformatted text preview: BIO 320: Lecture 11
Gene function, genetic complementation. 2008 Chapter 7: 7-21, 7-22, 7-24a, 7-25, 7-27, 7-28, 7-29, 7-31 QB: N1-23. The simplest version of this hypothesis predicts that there will be a different gene for every enzyme (protein). However, life is not this simple--for example: 1) One gene (transcription unit) may produce more than one protein. This can occur through differential processing at either the RNA or protein levels. (Two processes that depend on this are the generation of antibody diversity in humans, and the determination of sex in Drosophila). 2) Duplicated genes may produce the same protein (e.g., histone genes are repeated in most eukaryotes). 3) Some enzymes are composed of multiple proteins (i.e. have more than one subunit). 4) Not all proteins are enzymes. 5) Some genes encode RNA molecules (e.g tRNA and rRNA) and not proteins. The one gene/ one enzyme hypothesis DNA gene A gene B RNA protein 5' 3' 5' 3' enzyme A enzyme B The genetic complementation test This approach is used to determine if different mutations are in the same gene. Example 1: Beadle and Tatum isolated mutant strains of Neurospora that were unable to synthesize the amino acid arginine (and therefore cannot grow unless arginine is provided). Consider two mutations, (arg-1 and arg-2 ) that affect arginine biosynthesis
If arg-1 is a mutation in gene A & arg-2 is a mutation in gene B, then... Determine growth requirement of heterokaryon.
A- B + The pathway..... ornithine
enzyme A citrulline
enzyme B fused to generate heterokaryon with both nuclei sharing a common cytoplasm Haploid spores
A + B- A- / A + B+ / B- arginine
NOTE: Mutations in genes A and B can also be distinguished biochemically. A- strains can grow if either citrulline or arginine is provided. B- strains can only grow if arginine is provided. .... if the mutations are in two different genes then the heterokaryon will have functional copies of both enzymes A and B and will be able to synthesize its own arginine If both the arg-1 & arg-2 mutations are in the same gene (A), then...
A- B+ A- / A B+ / B+ A- B + ... heterokaryon does not have a good copy of gene A and is unable to grow without adding arginine Example 2: Benzer rapid lysis mutants of bacteriophage T4 included mutations in three different genes (rI, rIIA and rIIB). Complementation testing of phage mutants:
r- mutant 1 r- mutant 2 +
Mixture of two different phage mutants is spotted onto agar dish seeded with bacteria. COMPLEMENTATION is observed if the mutations are in different genes (e.g. rIIA and IIB). Plaques are wild type, i.e., small and fuzzy. NON-COMPLEMENTATION (i.e., mutant plaque morphology) is observed if the mutations are in the same gene. Complementation testing only works with recessive mutations--why? Loss of function mutants are often recessive. Dominant mutants may arise if the encoded protein is present at a time or place which is abnormal or if the mutation causes the protein to acquire a novel function. Incomplete dominance may arise if two copies of a gene are necessary to provide sufficient protein for a particular phenotype (for example, red pigment in flowers). A gene is a linear sequence of DNA which encodes the linear sequence of amino acids that make a polypeptide. Changes in the DNA sequence of a gene can result in changes in the amino acid sequence of the protein. These changes may disrupt the three dimensional structure of the protein, thereby inactivating it. Because many biological processes are mediated by proteins, either as enzymes or as structural components, inactive proteins often lead to mutant phenotypes. Loss of function mutant alleles are recessive because they can be complemented by the active protein encoded by the wild-type allele present in the same cell. ...
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This note was uploaded on 06/03/2008 for the course BIO 320 taught by Professor N/a during the Spring '08 term at SUNY Stony Brook.
- Spring '08