test 1 - Review by Roth field o FTS= filamentous thermal...

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Review by Roth field o FTS= filamentous thermal sensitive o Mutants for filaments at higher temperature, how FTS genes were identified o Looked at filaments for mutants and distinguished differences o Mutant Phenotype o Gene o (picture 1) long multinucleated filaments o no evidence of cytokinesis or septation o ftsZ o (picture 2) long multinucleated filaments with blunt constrictions (blunt pinched in at regular intervals at normal sites where the cells would divide) o Fts A, or fts Q, or fts I (individual mutants) o (picture 3) o Fts K, or envA o mutant only expresses certain phenotype in certain conditions, @30 degrees it doesn’t express it, if its always mutant, cells never divide and you cant find it o 42 degrees they form filaments o These phenotypes tell you about order of events of when the product of these genes are functioning during cell division- which protein functions first in cell division? Fts Z because it hasn’t started dividing (early before others), FTS A,Q, I happen intermediately & FtsK and envA is later stage o Mutant phenotypes help with product level o Fts K’s function is later, but it enters the cell early so its not telling you when it enters Division Site Placement (436-442) o Cell division occurs only at midcell in Wild Type E.coli Can cell division occurs any wehre else in WT e.coli- NO always in middle of wildtype o (picture 4) E.coli min mutant = different phenotype picure Minicell=no chrom., cant replicate Persists because there are some normal cells Division occurs with equal probability at midcell or near one cell pole or other phenotype o Cell division in wild type cell= only divide at middle site so…it must be being blocked at the ends because there are three potential sites o Three proteins identified: Min C, Min D, Min E= proteins control location of cell division Examine the phenotype of various min mutants to determine function of proteins
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Strain Phenotype Wild type (WT) WT, cell division at midcell only MinC mutant Minicell phenotype (1/3, 1/3, 1/3) MinD mutant Minicell phenotype (1/3, 1/3, 1/3) MinE mutant Filamentous growth, no cell division *WT (+ overexpression of minC and minD genes)(everythings there with added stuff) Filamentous growth, no cell division Look at phenotype and know mutants job normally, take away battery you determine function of the battery is to make car start, take away tires the car doesn’t roll so function of tires is to roll car (look at it when its not there to determine function) *= you can overexpress by putting genes behind an inducible promoter to make a lot of genes to make a lot of resulting proteins OR clone genes onto a multicopy plasmid (genes have own promoter but you put so many copies in cell its overexpressed)- same effect o Model for Division site Placement (picture 5) MinE mutant strain (no minE)- min CD acts at all sites to inhibit division (it’s a nonspecific cell division inhibitor; no division occurs so it just keeps replicating and
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