•What are the genes conserved between yeast and humans?

•How are these genes regulated?

•Why is this information valuable?

Issues addressed will include:

Phylogenetic Tree

Photosynthetic Bacteria

Plants

Insects

N. crassa

Baker’s yeast

Human

Aerobic Respiration

C6H12O6 + 6 O2 + 38 ADP +38 Pi

6 CO2 + 44 H2O + 38 ATP

Human & Yeast same pathway

19 times more efficient

Anaerobic Respiration

Humans:

C6H12O6 + 2 ADP +2 Pi 2 Lactate + 2 H+ + 2 H20 + 2 ATP

Yeast:

C6H12O6 + 2 ADP +2 Pi

2 Ethanol + 2 CO2 + 2 H20 + 2 ATP

Human/Yeast homologues involved in respiratory processes

Eif5-a TIF51A-B

SOD SOD1

ANT2 AAC3

TUPLE1 TUP1

COX15 COX15

HUMAN YEAST FUNCTION

Aerobic and anaerobic in yeast; promotes formation of first peptide bond in translation

Superoxide dismutase; provides protection from oxygen toxicity

ADP/ATP transporter in mitochondria

involved in glucose repression

cytochrome c oxidase

Plurality: 2.00 Threshold: 4 AveWeight 1.00 AveMatch 2.91 AvMisMatch -2.00

PRETTY of: pileup.msf{*} December 7, 1998 19:34 ..

1 50pileup.msf{heif} ~maddldFEt gDAGASATfP MQCSALRKNG FVVlKGwPCK IVeMSaSKTGpileup.msf{yeif} msdeehtFEn aDAGASATyP MQCSALRKNG FVViKGrPCK IVdMStSKTG Consensus -------FE- -DAGASAT-P MQCSALRKNG FVV-KG-PCK IV-MS-SKTG

51 100pileup.msf{heif} KHGHAKVHLV giDIFTGKKy EDicPSTHNm dVPnirRndf QLigIqDGYLpileup.msf{yeif} KHGHAKVHLV tlDIFTGKKl EDlsPSTHNl eVPfvkRsey QLldIdDGYL Consensus KHGHAKVHLV --DIFTGKK- ED--PSTHN- -VP---R--- QL--I-DGYL

101 150pileup.msf{heif} SLlqdsGEvp eDlrlPEGdL GkeieqkyDc GeeiliTvlS AMtEEAAvaipileup.msf{yeif} SLmtmdGEtk dDvkaPEGeL GdsmqaafDe GkdlmvTiiS AMgEEAAisf Consensus SL----GE-- -D---PEG-L G-------D- G-----T--S AM-EEAA---

151pileup.msf{heif} Kamak~~pileup.msf{yeif} Keaprsd Consensus K------

Plurality: 2.00 Threshold: 4 AveWeight 1.00 AveMatch 2.91 AvMisMatch -2.00

PRETTY of: pileup.msf{*} December 7, 1998 19:47 ..

1 50pileup.msf{hsod} matkAVcVLK GDgpVqGiin FEQkesngPv kVwgsIkGlt egl.hGFHvHpileup.msf{ysod} ~mvqAVaVLK GDagVsGvvk FEQasesePt tVsyeIaGns pnaerGFHiH Consensus ----AV-VLK GD--V-G--- FEQ-----P- -V---I-G-- -----GFH-H

51 100pileup.msf{hsod} EFGDnTaGCt SAGPHFNPls rkHGgPkDEe RHVGDlGNVt aDkdGVAdvSpileup.msf{ysod} EFGDaTnGCv SAGPHFNPfk ktHGaPtDEv RHVGDmGNVk tDenGVAkgS Consensus EFGD-T-GC- SAGPHFNP-- --HG-P-DE- RHVGD-GNV- -D--GVA--S

101 150pileup.msf{hsod} ieDSvIsLsG dhciiGRtlV vHekaDDLGK GgnEEStKTG NAGsRlACGVpileup.msf{ysod} fkDSlIkLiG ptsvvGRsvV iHagqDDLGK GdtEESlKTG NAGpRpACGV Consensus --DS-I-L-G -----GR--V -H---DDLGK G--EES-KTG NAG-R-ACGV

151pileup.msf{hsod} IGiaqpileup.msf{ysod} IGltn Consensus IG---

Gap Weight: 12 Average Match: 2.912 Length Weight: 4 Average Mismatch: -2.003

Quality: 97 Length: 88 Ratio: 1.115 Gaps: 1 Percent Similarity: 41.379 Percent Identity: 31.034

Match display thresholds for the alignment(s): | = IDENTITY : = 2 . = 1

ytup.seq x htup.seq December 8, 1998 15:57 ..

. . . . . 468 RIWDIENRKIVMILQGHEQDIYSLDYFPSGDKLVSGSGDRTVRIWD.LRT 516 :: .:| : | ||. | |: . : | | | | | || ||. .: 70 KLANVEQWRCVSILRNHSGDVMDVAWSPHDAWLASCSVDNTVVIWNAVKF 119 . . . 517 GQCSLTLSIEDGVTTVAVSPGDGKYIAAGSLDRAVRVW 554 : || |. |||||. . ||..:|| 120 PEILATLRGHSGLVKGLTWDPVGKYIASQADDRSLKVW 157

Plurality: 2.00 Threshold: 4 AveWeight 1.00 AveMatch 2.91 AvMisMatch -2.00

PRETTY of: pileup.msf{*} December 7, 1998 19:49 ..

1 50pileup.msf{yaac2} mssnaqvktp lppapApkkE sNFlIDFLMG GVSAAvAKTA ASPIERVKLLpileup.msf{yaac3} ~~~~~~~~~~ ~mssdAkqqE tNFAInFLMG GVSAAIAKTA ASPIERVKiLpileup.msf{hant2} ~~~~~~~~~~ ~~~~~mtdaa vsFAkDFLaG GVaAAIsKTA vaPIERVKLL Consensus ---------- -----A---E -NFAIDFLMG GVSAAIAKTA ASPIERVKLL

51 100pileup.msf{yaac2} IQNQDEMlKQ GTLDrKYaGI lDCFKRTAtQ EGVISFWRGN TANVIRYFPTpileup.msf{yaac3} IQNQDEMiKQ GTLDKKYsGI vDCFKRTAKQ EGlISFWRGN TANVIRYFPTpileup.msf{hant2} lQvQ.hasKQ iTaDKqYkGI iDCvvRipKe qGVlSFWRGN lANVIRYFPT Consensus IQNQDEM-KQ GTLDKKY-GI -DCFKRTAKQ EGVISFWRGN TANVIRYFPT

101 150pileup.msf{yaac2} QALNFAFKDK IKaMF..GFK KEEGYaKWFA GNLASGGAAG ALSLLFVYSLpileup.msf{yaac3} QALNFAFKDK IKlMF..GFK KEEGYgKWFA GNLASGGAAG ALSLLFVYSLpileup.msf{hant2} QALNFAFKDK yKqiFlgGvd KrtqfwryFA GNLASGGAAG AtSLcFVYpL Consensus QALNFAFKDK IK-MF--GFK KEEGY-KWFA GNLASGGAAG ALSLLFVYSL

151 200pileup.msf{yaac2} DyARTRLAAD sKSSKKGGAR QFNGLiDVYK KTLKSDGvAG LYRGFlPSVVpileup.msf{yaac3} DFARTRLAAD aKSSKKGGAR QFNGLtDVYK KTLKSDGIAG LYRGFmPSVVpileup.msf{hant2} DFARTRLAAD v..gKaGaeR eFrGLgDclv KiyKSDGIkG LYqGFnvSVq Consensus DFARTRLAAD -KSSKKGGAR QFNGL-DVYK KTLKSDGIAG LYRGF-PSVV

201 250pileup.msf{yaac2} GIVVYRGLYF GMYDSLKPLl LTGSLeGSFL ASFLLGWVVT TGASTCSYPLpileup.msf{yaac3} GIVVYRGLYF GMfDSLKPLv LTGSLdGSFL ASFLLGWVVT TGASTCSYPLpileup.msf{hant2} GIiiYRaaYF GiYDtaKgm. Lpdpknthiv iSwmiaqtVT avAgltSYPf Consensus GIVVYRGLYF GMYDSLKPL- LTGSL-GSFL ASFLLGWVVT TGASTCSYPL

251 300pileup.msf{yaac2} DTVRRRMMMT SGQA...VKY dGAfDCLRKI VAaEGVGSLF KGCGANILRGpileup.msf{yaac3} DTVRRRMMMT SGQA...VKY nGAiDCLkKI VAsEGVGSLF KGCGANILRspileup.msf{hant2} DTVRRRMMMq SGrkgtdimY tGtlDCwRKI ardEGgkafF KGawsNvLRG Consensus DTVRRRMMMT SGQA---VKY -GA-DCLRKI VA-EGVGSLF KGCGANILRG

301 323pileup.msf{yaac2} VAGAGVISMY DQLQMILFGK KFKpileup.msf{yaac3} VAGAGVISMY DQLQMILFGK KFKpileup.msf{hant2} mgGAfVlvlY Deikkyt~~~ ~~~ Consensus VAGAGVISMY DQLQMILFGK KFK

251300pileup.msf{hant2} acCcaTTTA. ...tgGTttg gTttTtTttt TTGttAgAtggaGtcTcaCtpileup.msf{yaac3} gaCggTTTAc ctgaaGTgat aTacTgTgcc TTGagAaAcaatGagTagCg Consensus --C--TTTA- -----GT--- -T--T-T--- TTG--A-A-- --G--T--C-

301350pileup.msf{hant2} ctGtTAccCA AgctGgAggg cAgTggcgCg aTctcagCTcAcTGcaaccTpileup.msf{yaac3} acGcTAagCA AcaaGaAaca aAcTttgcCa tTaatttCTtAaTGggtggT Consensus --G-TA--CA A---G-A--- -A-T----C- -T-----CT- A-TG-----T

351400pileup.msf{hant2} ccaccTcCcG gtgcaagcgA ttCTcCTGCC TCAggctTCtgAgGAGctggpileup.msf{yaac3} gtgagTgCgG ccatcgctaA aaCTgCTGCC TCAccaaTCgaAaGAGtcaa Consensus -----T-C-G ---------A --CT-CTGCC TCA----TC- -A-GAG----

401450pileup.msf{hant2} GActacaggC atgcgcCAcc ATGctcaGcT aAtttttGtAttTTTAGtagpileup.msf{yaac3} GAtcttgatC caaaatCAag ATGaaatGaT cAagcaaGgAacTTTAGata Consensus GA-------C ------CA-- ATG----G-T -A-----G-A --TTTAG---

451500pileup.msf{hant2} AgAtagggTt CGcTATgtcg gCcaGGcTgG TcTCgAacttctgaCctcagpileup.msf{yaac3} AaAagtatTc CGgTAT.... .CgtGGaTtG TtTCaAgagaactgCaaagc Consensus A-A-----T- CG-TAT---- -C--GG-T-G T-TC-A---- ----C-----

501550pileup.msf{hant2} gtGAttcACT cgccTtagcc TccCaAaGtg cTgggattAcaGgcATgaGcpileup.msf{yaac3} aaGAgggACT aataTccttt TggCgAgGaa aTactgccAatGttATtcGt Consensus --GA---ACT ----T----- T--C-A-G-- -T------A- -G--AT--G-

551600

Due to complex intron exon structure, nucleotide homology appears lower than protein.

Protein sequence homology shows conservation of the proteins involved in aerobic and anaerobic processes

Most of the genes involved in respiration are regulated at a transcriptional level

These regulators include:

YEAST HUMAN

ROX1-represses most known anaerobic genes

consensus binding

site:YYATTGTTCTC

represses:HEM13, TIF51-b, ERG11, ANB1, COX5, AAC3

O2 and Heme- induces

ROX1; represses: DAN1

HAP1-induces many anaerobic genes including: ERG11, TIF51-a, CYC7, HEM13,

HAP2/3/4-induces: COX genes

HIF1-hypoxia inducing factors

activates many of the enymes in anaerobic glycolysis

targets include: ALD-A, PGK1, enolase1, LDH-A, PFKL

other genes that show HIF1 binding include: TH, VEGF, GLUT1

WHERE ARE THE OTHER REGULATORS?

WHERE ARE THE OTHER REGULATORS?

Regulatory consensus sites

Repressor sites:

Rox1: HEM13, TIF51BYYYATTGTTCTC

ERG11AAACGAUYAAACGAAAAACGAAACGAAACGAAACTAA

ANB1AAGGAACGAACAA

COX5BAACGAACGA

Consensus

Activator sites:

Hem13UAS(GGAGTCG)2

ERG11GCCCGGGAATTACCGGGGGC

TTACGTACTCGCATGTATT

HIF1 Regulates ALD-A, PGK1, ENOLASE1, LDH-A, PFK L, PGD1

Binds TH VEGF, GLUT1TACGTGCT

GRBOXANT2 ATGATT

TIF51B GTTCGTTGCCT

YYATTGTTCTCYYATTGTTCTC CATTGTTCATTGTT

CCATTGTTCCCATTGTTC

Future projects:

• Look for repressors of human anaerobiosis, possibly like ROX1, as the ANT2 regulatory sequence is similar to the ROX1 consensus site; need more sequences from human sequencing projects for this as well as the next one

•Look for yeast inducers of anaerobic genes that may be like HIF1 genes in humans

•Further studies of expressing yeast proteins in mammalian systems; and human proteins in yeast systems

Why Yeast?

•Easy to manipulate

•The entire genome is sequenced

•Can make an entirely aerobic or anaerobic system

Why study aerobic and anaerobic respiration?

Initial topic of interest

• Lactate buildup during exercise

– athletes and athletics

– release of lysosomes, cell death, soreness

WWW Search for lactate buildup

• Yahoo - 12393 hits

• Excite - 19858 hits

Some examples

Devices used to monitor lactate levels

• Accusport Lactate Analyzer

• Endurex Excel dietary supplement

Horses also addressed

• Vita-Flex Lactanase

Secondary topic of interest

Diseases

• Cancer

• HIV

• Influenza

all anaerobic

Our purpose

Elucidation

Data mining