User:Nkuldell/SAGA swap

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<font color = red> check codon bias Sc vs Sp </font color> [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=Abstract&list_uids=7992504&query_hl=24&itool=pubmed_docsum]
<font color = red> check codon bias Sc vs Sp </font color> [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=Abstract&list_uids=7992504&query_hl=24&itool=pubmed_docsum]
Codon Usage Comparison using GeneArt tool called Graphical Codon Usage Analyzer [http://gcua.schoedl.de/sequential.php]
Codon Usage Comparison using GeneArt tool called Graphical Codon Usage Analyzer [http://gcua.schoedl.de/sequential.php]
-
{| border="1"
 
-
! Ada subunits
 
-
! <i> cerevisiae </i>
 
-
! gene size,chromosome,null p-type
 
-
! <i> pombe </i>
 
-
! gene size,chromosome,null p-type
 
-
! BLAST comparison info
 
-
|-
 
-
|
 
-
| Ada1 (aka HFI1, SUP110, SRM12, GAN1)  [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=ADA1]]
 
-
| 1.467 kb/489 aa, <br> Chr. XVI, viable
 
-
| <b> Nucleotide comparison </b> identifies clone spc05247 <br> <b> Protein comparison </b> identifies  SPBC887.18c [[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=19113284]]
 
-
| <b> Nucleotide comparison </b> <br> 0.167 kb, late log phase mRNA <br> <b> Protein comparison </b> <br> 339 aa, Chr. II, hyptothetical protein NM_001022412.1 
 
-
| <b> Nucleotide comparison<br></b> Identities = 74/114 (64%), E= 0.94 [[http://seq.yeastgenome.org/cgi-bin/blast-fungal.pl?name=YPL254W#AU009688.1]]<br><b> Protein comparison<br></b>  Identities = 72/271 (26%), Positives = 123/271 (45%), Gaps = 26/271 (9%), E = 6.3e-15 [[http://seq.yeastgenome.org/cgi-bin/blast-fungal.pl?name=YPL254W&suffix=prot#NP_596492.1]]
 
-
|-
 
-
|
 
-
| Ada2 (aka SWI8) [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=ADA2]]
 
-
| 1.305 kb/434aa, <br> Chr. IV, viable
 
-
| SPCC24B10.08c [[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=68019844]]
 
-
| 1.314 kb/437aa, Chr. III, hypothetical protein NP_588011.1
 
-
| Identities = 384/645 (59%), E=1e-24 [[http://seq.yeastgenome.org/cgi-bin/blast-fungal.pl?name=YDR448W]]
 
-
|-
 
-
|
 
-
| Ada3 (aka NGG1, SWI7) [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=ADA3]]
 
-
| 2.109 kb/702aa, <br> Chr. IV, viable
 
-
|<b> Nucleotide comparison </b> identifies cosmid c16H5 [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=nucleotide&cmd=search&term=AL022104.2]] <br> <b> Protein comparison </b> identifies SPBC28F2.10c [[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=19112463]]
 
-
|<b> Nucleotide comparison </b> <br> Chr. II <br> <b> Protein comparison </b> <br> 551 aa, Chr. II, Spt-Ada-Gcn5-Acetyltransferase NP_595671
 
-
|<b> Nucleotide comparison<br></b>  Identities = 342/619 (55%), E= 0.001 [[http://seq.yeastgenome.org/cgi-bin/blast-fungal.pl?name=YDR176W#NC_003423.2]]<br><b> Protein comparison<br></b>  Identities = 53/153 (34%), Positives = 84/153 (54%), Gaps = 20/153 (13%), E=1.6e-28[[http://seq.yeastgenome.org/cgi-bin/blast-fungal.pl?name=YDR176W&suffix=prot]]
 
-
|-
 
-
|
 
-
| Gcn5 (aka ADA4, SWI9) [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=GCN5]]
 
-
| 1.32 kb/439aa, <br> Chr. VII, viable
 
-
| SPAC1952.05 [[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=68005592]]
 
-
| 1.365 kb/454aa,Chr. I,  hypothetical protein NM_001020236
 
-
| Identities = 718/1103 (65%)E= 4.7e-79 [[http://seq.yeastgenome.org/cgi-bin/blast-fungal.pl?name=YGR252W]]
 
-
|-
 
-
|
 
-
| Ada5 (aka SPT20) [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=Ada5]]
 
-
| 1.815 kb/604aa, <br> Chr. XV, viable
 
-
| <b> Sequence Comparison</b> identifies SPBC21D10.09c [[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=68012124]]<br><b> Protein Comparison</b> <br>identifies SPAC4D7.10c [[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=19115875]]
 
-
| <b> Sequence Comparison</b> 4.833 kb, Chr. II, hypothetical protein NP_596004.1<br><b> Protein Comparison</b>  <br> 473 aa, Chr. I, hypothetical protein NP_594963
 
-
|  <b> Sequence Comparison</b><br>Identities = 270/488 (55%), <br> E = 0.99 [[http://seq.yeastgenome.org/cgi-bin/blast-fungal.pl?name=YOL148C#NC_003423.2]] <br><b> Protein Comparison</b>  <br> Identities = 15/43 (34%), Positives = 27/43 (62%), <br>E= 0.0002 [[http://seq.yeastgenome.org/cgi-bin/blast-fungal.pl?name=YOL148C&suffix=prot]]
 
-
|}
 
-
{| border="1"
 
-
! Spt subunits
 
-
! <i> cerevisiae </i>
 
-
! gene size, chromosome, null p-type
 
-
! <i> pombe </i>
 
-
! gene size, chromosome, null p-type
 
-
! BLAST comparison info
 
-
|-
 
-
|
 
-
| Spt3 [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=spt3]]
 
-
| 1.014 kb/337aa, <br> Chr. IV, viable
 
-
| SPCC61.02 [[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=68026498]]
 
-
| 0.924 kb/307aa, Chr. III, hypothetical protein NP_588193.1
 
-
| Identities = 194/297 (65%) <br> E=1.6e-36 [[http://seq.yeastgenome.org/cgi-bin/blast-fungal.pl?name=YDR392W#NM_001023183.1]]
 
-
|-
 
-
|
 
-
| Spt7 (aka GIT2) [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=Spt7]]
 
-
| 3.999 kb/1332aa, <br> Chr. II, viable
 
-
| SPBC25H2.11c [[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=68012509]]
 
-
| 2.940 kb/979aa, Chr. II , hypothetical protein NP_596356.1
 
-
|  Identities = 570/979 (58%)<br> E =4.3e-28 [[http://seq.yeastgenome.org/cgi-bin/blast-fungal.pl?name=YBR081C]]
 
-
|-
 
-
|
 
-
| Spt8 [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=Spt8]]
 
-
| 1.809 kb/602aa, <br> Chr. XII, viable
 
-
| SPBC14C8.17c [[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=68012036]]
 
-
| 1.581 kb/526aa, Chr. II, hypothetical protein NP_595920.1
 
-
| Identities = 135/230 (58%)<br> E= 0.053 [[http://seq.yeastgenome.org/cgi-bin/blast-fungal.pl?name=YLR055C]]
 
-
|-
 
-
|
 
-
| Spt20 (aka Ada5) [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=SPT20]]
 
-
| same as Ada5 <br> 1.815 kb/604aa, Chr. XV, viable
 
-
| <b> Sequence Comparison</b> identifies SPBC21D10.09c [[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=68012124]]<br><b> Protein Comparison</b> <br>identifies SPAC4D7.10c [[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=19115875]]
 
-
| <b> Sequence Comparison</b> 4.833 kb, Chr. II, hypothetical protein NP_596004.1<br><b> Protein Comparison</b>  <br> 473 aa, Chr. I, hypothetical protein NP_594963
 
-
|  <b> Sequence Comparison</b><br>Identities = 270/488 (55%), <br> E = 0.99 [[http://seq.yeastgenome.org/cgi-bin/blast-fungal.pl?name=YOL148C#NC_003423.2]] <br><b> Protein Comparison</b>  <br> Identities = 15/43 (34%), Positives = 27/43 (62%), <br>E= 0.0002 [[http://seq.yeastgenome.org/cgi-bin/blast-fungal.pl?name=YOL148C&suffix=prot]]
 
-
|}
 
-
 
-
{| border="1"
 
-
! TAF subunits
 
-
! <i> cerevisiae </i>
 
-
! gene size, chromosome, null p-type
 
-
! <i> pombe </i>
 
-
! gene size, chromosome, null p-type
 
-
! BLAST comparison info
 
-
|-
 
-
|
 
-
| TAF5 (aka TAF90) [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=TAF5]]
 
-
| 2.397 kb/798aa, Chr. II, <font color = red>inviable</font color>
 
-
| SPCC5E4.03c (aka spTAF72) 
 
-
| 643aa
 
-
|
 
-
|-
 
-
|
 
-
| TAF6 (aka TAF60) [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=TAF6]]
 
-
| 1.551 kb/516aa, Chr. VII, <font color = red>inviable</font color>
 
-
| SPCC16C4.18c (aka spTAF50)
 
-
| <font color = red>inviable</font color> 452aa
 
-
| 39% identical [[Image:Macintosh HD-Users-nkuldell-Desktop-ScSpTAFs NAR02.pdf]]
 
-
|-
 
-
|
 
-
| TAF9 (aka TAF17) [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=TAF9]]
 
-
| 0.474 kb/157aa, Chr. XIII, <font color = red>inviable</font color>
 
-
| SPAC12G12.05c
 
-
| 163aa
 
-
|
 
-
|-
 
-
|
 
-
| TAF10 (aka TAF23, TAF25) [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=TAF10]]
 
-
| 0.621 kb/206aa, Chr. IV, <font color = red>inviable</font color>
 
-
| SPBC21H7.02
 
-
| 215aa
 
-
|
 
-
|-
 
-
|
 
-
| TAF12 (aka TAF61, TAF68) [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=TAF12]]
 
-
| 1.620 kb/539aa, Chr. IV, <font color = red>inviable</font color>
 
-
| <b> Nucleotide comparison </b> identifies cosmid clone 1465-1759 [[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=4049533]] <br> <b> Protein comparison </b> identifies SPAC15A10.02
 
-
| <b> Nucleotide comparison </b> Chr. III <br> <b> Protein comparison </b> 450 aa, Chr. I, hypothetical protein NM_001019712.1
 
-
| <b> Nucleotide comparison </b> Identities = 187/313 (59%) E = 1 [[http://seq.yeastgenome.org/cgi-bin/blast-fungal.pl?name=YDR145W]]<br> <b> Protein comparison </b>  Identities = 76/209 (36%), Positives = 117/209 (56%), Gaps = 11/209 (5%), E= 2.2e-37 [[http://seq.yeastgenome.org/cgi-bin/blast-fungal.pl?name=YDR145W&suffix=prot]]
 
-
|}
 
-
 
-
{| border="1"
 
-
! Tra1 subunit
 
-
! <i> cerevisiae </i>
 
-
! gene size, chromosome, null p-type
 
-
! <i> pombe </i>
 
-
! gene size, chromosome, null p-type
 
-
! BLAST comparison info
 
-
|-
 
-
|
 
-
| Tra1 [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=TRA1]]
 
-
| 11.235 kb/3744aa, Chr. VIII, <font color = red>inviable</font color>
 
-
| SPBP16F5.03c [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retrieve&dopt=full_report&list_uids=2541265]]
 
-
| 3655aa, Chr. II, hypothetical protein
 
-
| GeneDB [[http://www.genedb.org/genedb/Search?organism=pombe&name=SPBP16F5.03c]]
 
-
|}
 
-
 
-
{| border="1"
 
-
! other subunits
 
-
! <i> cerevisiae </i>
 
-
! gene size, chromosome, null p-type
 
-
! <i> pombe </i>
 
-
! gene size, chromosome, null p-type
 
-
! BLAST comparison info
 
-
|-
 
-
|
 
-
| Sgf73 [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=sgf73]]
 
-
| 1.974 kb/657aa, Chr. VII , viable
 
-
| <b> Protein comparison</b> identifies SPCC126.04c [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=protein&cmd=search&term=NP_588447.1]]
 
-
| <b> Protein comparison </b> 344aa, Chr. III, hypothetical protein NP_588447
 
-
| <b> Protein comparison </b> identifies best stretch as  Identities = 48/100 (48%), Positives = 60/100 (60%), Gaps = 1/100 (1%) E=1.7e-26 [[Image:Sc Sp Sgf73.pdf]]
 
-
|-
 
-
|
 
-
|Sgf29 [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=sgf29]]
 
-
|0.779 kb/259aa, Chr. III, viable
 
-
|<b> Protein comparison </b> identifies SPBC1921.07c [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=protein&cmd=search&term=NP_596000.2]]
 
-
|<b> Protein comparison </b> <br> 244 aa, Chr. II, hypothetical protein NP_596000
 
-
|<b> Protein comparison </b>  Identities = 70/125 (56%), Positives = 85/125 (68%), Gaps = 5/125 (4%)E=3.1e-31 [[Image:Sp Sc Sgf29p .pdf]]
 
-
|-
 
-
|
 
-
|Sgf11 [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=sgf11]]
 
-
|0.3 kb/99aa, Chr.XVI, viable
 
-
|<b> Protein comparison </b> identifies SPA(C)57A10.14 [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=protein&cmd=search&term=NP_001018231.1]]
 
-
|<b> Protein comparison </b> <br>117 aa, Chr. I, hypothetical protein NP_001018231
 
-
|<b> Protein comparison </b>Identities = 20/51 (39%), Positives = 27/51 (52%), Gaps = 3/51 (5%)E=3.6e-05
 
-
|-
 
-
|
 
-
|Ubp8 [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=ubp8]]
 
-
|1.416 kb/471aa, Chr. XIII, viable
 
-
|<b> Protein comparison </b> identifies SPAC13A11.04c [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=protein&cmd=search&term=NP_592992.1]]
 
-
|<b> Protein comparison </b> <br> 449 aa, Chr. I, hypothetical protein NP_592992
 
-
|<b> Protein comparison </b>  Identities = 115/364 (31%), Positives = 177/364 (48%), Gaps = 50/364 (13%)E=1.8e-44
 
-
|-
 
-
|
 
-
|Sus1 [[http://db.yeastgenome.org/cgi-bin/locus.pl?locus=Sus1]]
 
-
|gene with intron, Chr. II, viable
 
-
|<b> Protein comparison </b> identifies SPBC6B1.12c [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=protein&cmd=search&term=NP_001018822.1]]
 
-
|<b> Protein comparison </b> <br> 108 aa, Chr. II, hypothetical protein NP_001018822
 
-
|<b> Protein comparison </b> <br>  Identities = 24/86 (27%), Positives = 47/86 (54%) E=1.6e-09
 
-
|}
 
===S. cerevisiae vs human SAGA subunits===
===S. cerevisiae vs human SAGA subunits===

Current revision

Contents

Idea

Justification

The genetic and epigenetic requirements for gene expression will need to be considered if logic circuits and genetic programs are to be reliably written for eukaryotic cells. Since chromatin dynamics are an integral part of eukaryotic gene regulation (see as recent example [1][2]) a better understanding of chromatin modifying complexes will be necessary if DNA sequences are to preform, i.e. express, in a predictable way. Ideally a common chromatin remodelling complex could be described that would perform seemlessly in any (or less ambitiously, several) hosts/operating system. Is there a generic complex that could plug into multiple eukaryotic hosts and properly regulate histone acetylation/deacetylation as well as nucleosome positioning over transcribed regions? As a first step in designing such a common chromatin remodeller, the SAGA subunits of S. cerevisiae can be replaced with the S. pombe homologs. Shoot, if Apple can find a way to run a MS operating system, smart yeast should make this SAGA swap possible.

H.s. to S.c. swap of SGF73 at [[3]] Other yeast to S.c. swap of SPT3 at [[4]] [[5]]

Proposal Details

Standard yeast techniques can be used to replace the S. cerevisiae subunits with the homologs from S. pombe and effects on gene expression can be assessed. Specifically it should be possible to integrate a URA3 marker at a gene encoding a SAGA-subunit and then replace that marker wtih the S. pombe gene by transformation and seletion on FOA. The pombe genes would be amplified from a cDNA library, the source of which is still to be determined. Expression of each replaced subunit can be followed by Western if Abs are available or with epitope tags (less desirable), SAGA integrity can be functionally and biochemically assessed, cell-wide effects on gene expression can be followed by array, mutant phenotypes (Spt, drug sens etc...) Single gene replacements of multisubunit are often unsuccessful but activity can be restored with multiple replacements of interacting subunits (check Stan Fields STE12 and MCM1?)

Four distinct classes of genes make up the multisubunit SAGA complex in S. cerevisiae
1. The Ada proteins

  • Ada1, Ada2, Ada3, Gcn5, Ada5

2. The Spt proteins

  • Spt3, Spt7, Spt8, Spt20

3. The TAF proteins

  • TAF5, TAF6, TAF9, TAF10, TAF12

4. The Tra1 protein

  • essential gene
  • target of gene specific activators

Can generic versions of these subcomplexes be described and then used to intelligently specify the chromatin packaging needed to execute a genetic program? Success would provide an existance proof for a re-usable genetic module capable of appropriately remodelling chromatin in N>1 host.

Fact tables

S. cerevisiae SAGA chemistry

summarized from TiBS 05 review Image:Macintosh HD-Users-nkuldell-Desktop-SAGA swap-SAGAunveiled TiBS05.pdf

  • GCN5 has HAT activity
  • Ada2, Ada3 regulate GCN5
  • Ada5 is = Spt20
  • SAGA structural integrity depends on Ada1, Spt7, Spt20, and TAF12
  • interaction with TBP requires Spt8 and Ada3
  • gene specific transcriptional activators Gcn4, VP16 and Gal4 target Tra1
  • histone fold pairs: TAF6 and TAF9, TAF10 and Spt7, TAF12 and Ada1
  • SAGA variants such as SALSA, SLIK without Spt8 and truncation of Spt7/ of questionable functional significance
  • Other SAGA associated subunits that don't fall into one of 4 catagories above but that purify with SAGA: Sgf73, Sgf29, Sgf11, Ubp8 and Sus1 (in S. cerevisiae)which correspond to SPCC126.04c, SPBC1921.07c, SPA(C)57A10.14, SPAC13A11.04c, SPBC6B1.12c (in pombe).

summarized from Dom/Mark/Fred's talks, Winston retreat 06.07.06

  • Structural role: Spt7, Ada1, Spt20
  • Histone fold pairs: Taf6 and Taf9, Taf10 and Spt7, Taf12 and Ada1
  • H3, H2B acetylation: Gcn5, Ada2, Ada3
  • H2B deubiq: Ubp8, perhaps Sgf11
  • TBP recruitment: Spt3, Spt8
  • Intn w/ activators: Tra1
  • Unknown: Sgf73 (though mutations in this subunit confer PAU defects) and Sgf29

Genetic interactions:

  • Spt3 still in SAGA in spt20 deletion strain
  • Spt3 not in SAGA in ada1 deletion or spt7 deletion strain (region withing 873-1125 req'd)
  • Spt7 50aa deletion leads to loss of Spt8 from SAGA
  • Spt8 still associated with Spt7 in ada1, spt20 deletions
  • dstI, sgf73 synthetic lethal (Krogan in Mol Cell 2003 12(6):1565)

S. cerevisiae vs S. pombe SAGA subunits

check codon bias Sc vs Sp [6] Codon Usage Comparison using GeneArt tool called Graphical Codon Usage Analyzer [7]


S. cerevisiae vs human SAGA subunits

1. structures

2. common subunits

subunit cerevisiae gene size, chromosome, null p-type human gene size, chromosome, null p-type BLAST comparison info
Spt7 [[8]] 3.999 kb, Chr. II, viable
TAF6 (aka TAF60) [[9]] 1.551 kb, Chr. VII, inviable hTAF80

3. human specific

TFTC-subunits(from Wu in Mol Cell 04)

  • PAF65-beta
  • Taf2
  • Taf4

TBP Comparisons

subunit cerevisiae pombe human other
TBP SPT15,BTFI [[10]] SPTFIID [[11]] TATA binding factor [[12]] C. albicans [[13]]
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