4/07/2017

IIT JAM BIOTECH COACHING


Dr. Charu S 
Molecular Biology

CSIR NET- DEC’14 (PART C)

79.  A gene producing red pigment was placed near centromeres of fission yeast and thus subjected to position effect variegation and produced white colonies. A screen for mutants that increased the red pigment production was undertaken. Which of the following genes, when mutated, is likely to produce this genotype?
1. Histone deacetylase                       2.  Histone acetylase
3.  RNA polymerase                         4. TATA binding factor

Solution:
In this experiment, the position of a red pigment producing gene is altered by placing it near the centromere.  The change in position seems to have inactivated the gene, since it can’t produce the red pigment, resulting in the formation of white colonies.

This is called position effect. It is defined as a change in the level of gene expression brought about by a change in the position of the gene relative to its normal chromosomal environment.  Position effect variegation means the some cells may or may not produce the red pigment, giving the variegation effect. 

Position effect may be due to various reasons. In this case, it is clearly due to placing the gene near the centromere. The red pigment gene has been inactivated by heterochromatization, when placed near the centromere.

DNA in eukaryotic cells is packaged into protein/DNA complexes called chromatin.  The basic unit of chromatin is thenucleosome, which is composed of ~ 146 base pairs (bp) of DNA wrapped around an octamer of the four core histones (H2A, H2B, H3, and H4).  The linker histone H1, interacts with DNA links between nucleosomes The core histones have amino-terminal tails which undergo extensive post-translational modifications (PTMs).  

Based on organization of chromatin, the cell nucleus contains regions of darkly staining, tightly wound heterochromatin and the lightly staining, more open euchromatin, which is more accessible to the transcriptional apparatus. Euchromatin contains most of the genes, and replicates earlier in S phase. Heterochromatin replicates later in S phase. It may be divided into facultative and constitutive heterochromatin.  The centromeric and telomeric regions of chromosomes consist of constitutive heterochromatin and contain mainly repetitive DNA. Facultative heterochromatin may alternate between transcriptionally active and inactive state.

The lysine residues on the histone tails can be either acetylated by histone acetyltransferase enzymes (HATs), or deacetylated by histone deacetylases (HDACs). Lysine residues have a net positive charge that allows it to bind tightly to the negatively charged DNA to condense nucleosomes. Acetylation removes the positive charges and reduces the affinity between histones and DNA. Thus deacetylation of histone tails is associated with heterochromatization and acetylation with the formation of euchromatin.  


In the above experiment, red mutants are selected from the white cells which showed position effect, . In these mutants, the heterochromatization of the red pigment gene should be reversed. Mutants for the histone acetylase gene should loose their ability to acetylate histones. Deacetylation would reverse the inactivation of the red pigment gene.   

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