Update on RelA Project Cloning of amplified relA T T pGEM-T Easy Insert amplified product in both orientations PCR

Update on RelA Project Cloning of amplified relA pUR351b pUR351a

Update on RelA Project Controlled overexpression of P tac -relA pUR351a SalI ScaI SalISmaI

Update on RelA Project Controlled overexpression of P tac -relA relA Status relA fragment in agarose pRL502a digestion completed pUR354 (KT)

Update on RelA Project Controlled overexpression of P Em -relA pUR351a SacI ScaI NsiI ClaI

Update on RelA Project Controlled overexpression of P tac -relA pUR351a SacI ScaI pBluescriptKS SacI EcoRVPstI

Update on RelA Project Controlled overexpression of P tac -relA pBluescriptKS PstI ClaI Status First attempt failed Second attempt transformed pUR355 (Karen)

Update on RelA Project Controlled production of antisense relA pUR351a SacI SmaI DpnI (93 bp) DpnI (1072 bp)

Update on RelA Project Controlled production of antisense relA SacIDpnI Status DpnI fragment isolated pRL502a digestion completed pUR357 (Julie)

Update on RelA Project Construction of relA insertional mutant pUR351b PvuII HpyCH4IV NsiI ClaI SacI

Update on RelA Project Construction of relA insertional mutant pUR351b PvuII HpyCH4IV SacI pBluescriptKS SacI SmaI PvuII (2476 bp) (2597 bp) Ap r BglI

Update on RelA Project Construction of relA insertional mutant HpyCH4IV matches ClaI pBluescriptKS PstI matches NsiI Ap r Status First try failed Second try transformed pUR352 (Carrie)

Update on  E Background (Trempy et al; Stragier et al)  E made first as a precursor protein, P 30 spoIIGA + sigE expression in vegetative cells gives  E Sporulation-dependent expression requires SpoIIE SpoIIE also required for sporulation-dependent division Questions Is SpoIIGA the processing protease? Does processing require cell division?

Update on  E News from  F Margolis et al (1991). Science 254:  F required for expression of forespore genes But spoIIAC (encodes  F ) expressed prior to septation Is  F active only in forespore? In both cells? Method Fuse  F -dependent gene to lacZ Use antibody to ß-gal and fluorescence microscopy

Update on  E News from  F Result  F -dependent genes turned on only in forespore True even in case where nonsporulation gene used. Conclusion  F active only in forespore BUT  F required for activity of  E in mother cell! See Figure 5 of Losick & Stragier

Update on all sporulation  ’s Question What controls the activity of the five sporulation  ’s? Sigma Factors  H (spo0H/sigH)Karen  E (spoIIG/sigE) --  F (spoIIA/sigF)Julie  G (spoIIIG/sigG)Carrie  K (spoIIIC/sigK)KT Presentations Wed, March 28 and Fri, March 30 (article by Friday, March 23; meetings to be scheduled)

Caulobacter crescentus Cell cycle-regulated differentiation

The phenomenon: Sheffery and Newton (1981) Cell 24:49-57 Regulation of periodic protein synthesis in the cell cycle: Control of initiation and termination of flagellar gene expression To be discussed Wednesday, March 21 Focus: Fig 2 (Carrie), Fig 3 (KT), Fig 4 (Julie), Fig 5 (Karen)

Coming Attractions Wed Mar 21Sheffery & Newton (Caulobacter periodicity) Continue constructs; ligate Fri Mar 23Quon et al (Caulobacter master regulator) Mon Mar 26Continue Quon et al; Organismal databases Wed Mar 28Two presentations Fri Mar 30Two presentations