An In Silico Assay for An In Silico Assay for Apoptosis in YeastApoptosis in Yeast

Lou LietoLou LietoJohn EberthJohn Eberth

IntroductionIntroduction The existence of apoptotic proteins The existence of apoptotic proteins

in yeast would provide information in yeast would provide information on the origin of apoptosis in on the origin of apoptosis in multicellular organismsmulticellular organisms

Homology between Human and Homology between Human and C.elegans apoptotic proteins and C.elegans apoptotic proteins and yeast proteins was examinedyeast proteins was examined

Implications for yeastImplications for yeast

Apoptosis in yeastApoptosis in yeast

Apoptosis in single celled organisms is Apoptosis in single celled organisms is counter-intuitivecounter-intuitive

One would expect that apoptosis in single One would expect that apoptosis in single celled organisms would rather quickly select celled organisms would rather quickly select it’s genome for extinctionit’s genome for extinction

However in single celled organisms that form However in single celled organisms that form colonies it is the survival of the colony rather colonies it is the survival of the colony rather than that of individual members that ensures than that of individual members that ensures the survival of the genomethe survival of the genome

Apoptotic vs. Oncotic Apoptotic vs. Oncotic DeathDeath

ApoptosisApoptosis KaryorhexisKaryorhexis

– Budding- membrane Budding- membrane shrinkageshrinkage

Chromatin clumpingChromatin clumping– Regular cleavage of Regular cleavage of

the DNA by a the DNA by a endonucleaseendonuclease

Lack of InflamationLack of Inflamation

OncosisOncosis KaryolysisKaryolysis

– Blebbing-increased Blebbing-increased membrane permeabilitymembrane permeability

Random DNA breakageRandom DNA breakage InflamationInflamation

– Release of cellular Release of cellular contentscontents

Human and C.elegans Human and C.elegans ProteinsProteins

HUMANHUMAN Caspases 1-9Caspases 1-9 Bcl-2Bcl-2 BaxBax BikBik BadBad P53P53 Cytochrome CCytochrome C TraddTradd FaddFadd FliceFlice

C.elegansC.elegans Ced 1-10Ced 1-10

TechniquesTechniques Human and C. elegans proteins were Human and C. elegans proteins were

blasted against the S. Cerevisiae blasted against the S. Cerevisiae database in Genbankdatabase in Genbank

The Hits were then entered into the The Hits were then entered into the Global Gene Hunter which checks 8 Global Gene Hunter which checks 8 databases for the ORF in questiondatabases for the ORF in question

The results were studied for homology The results were studied for homology and any similar activity to the original and any similar activity to the original search proteinsearch protein

ResultsResults Homology with yeastHomology with yeast Caspases 1-9Caspases 1-9 45% Av.45% Av. Ced 1-10Ced 1-10 52% Av.52% Av. Bcl-2 Bcl-2 64% 64% BaxBax 42%42% BikBik 42%42% BadBad 56%56% P53P53 32% 32% Cytochrome CCytochrome C 76%76% TraddTradd 47%47% FaddFadd 46%46% FliceFlice 44% 44%

Cytochrome CCytochrome C

Human Cytochrome C shared a Human Cytochrome C shared a 76% homology with Yeast 76% homology with Yeast Cytochrome CCytochrome C

This is to be expected considering This is to be expected considering that Cytochrome C plays an that Cytochrome C plays an essential role in electron transport essential role in electron transport in the mitochondrial respiratory in the mitochondrial respiratory chainchain

Global Gene HunterGlobal Gene Hunter Except for Cytochrome C the gene Except for Cytochrome C the gene

hunter either failed to return any hunter either failed to return any information regarding the ORFs in information regarding the ORFs in question or else it identified the question or else it identified the yeast proteins as having a function yeast proteins as having a function dissimilar to that of the search dissimilar to that of the search proteinprotein

What This MeansWhat This Means Human Caspase 1 and Ced 3 have Human Caspase 1 and Ced 3 have

closely related functions yet only closely related functions yet only share a 29 % identityshare a 29 % identity

It is possible that some of the ORFs It is possible that some of the ORFs that are unidentified yet share a that are unidentified yet share a low homology with their search low homology with their search proteins have similar activityproteins have similar activity

Future Areas of Future Areas of Study Study

Apoptosis in yeast needs to be Apoptosis in yeast needs to be identified via morphological or identified via morphological or histological methods, such as the histological methods, such as the TUNEL assayTUNEL assay

The Yeast genome needs better The Yeast genome needs better organization and full identification organization and full identification of protein functions of protein functions