Update on RelA ProjectCloning of amplified relA

T

TpGEM-T Easy

Insert amplified product in both orientations

PCR

Update on RelA ProjectCloning of amplified relA

pUR351b pUR351a

Update on RelA Project Controlled overexpression of Ptac-relA

pUR351a

SalI

ScaI

SalI SmaI

Update on RelA Project Controlled overexpression of Ptac-relA

relA

Status

• relA fragment in agarose

• pRL502a digestion completed

pUR354 (KT)

Update on RelA Project Controlled overexpression of PEm-relA

pUR351a

SacI

ScaI

NsiIClaI

Update on RelA Project Controlled overexpression of Ptac-relA

pUR351a

SacI

ScaI

pBluescriptKS

SacI

EcoRV PstI

Update on RelA Project Controlled overexpression of Ptac-relA

pBluescriptKS

PstI

ClaI

Status

• First attempt failed

• Second attempt transformed

pUR355 (Karen)

Update on RelA Project Controlled production of antisense relA

pUR351a

SacISacISmaI DpnI

(93 bp)

DpnI

(1072 bp)

Update on RelA Project Controlled production of antisense relA

SacIDpnI DpnI

Status

• DpnI fragment isolated

• pRL502a digestion completed

pUR357 (Julie)

Update on RelA Project Construction of relA insertional mutant

pUR351b

PvuII

HpyCH4IVNsiI ClaI

SacI

Update on RelA Project Construction of relA insertional mutant

pUR351b

PvuII

HpyCH4IV

SacI

pBluescriptKS

SacI

SmaI

PvuII

PvuII

(2476 bp)(2597 bp)

AprApr

BglI

Update on RelA Project Construction of relA insertional mutant

HpyCH4IVmatches ClaI

pBluescriptKS

PstImatches NsiI

AprStatus

• 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, P30

• 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 254:562-565• 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 crescentusCell cycle-regulated differentiation

Caulobacter crescentusCell 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 21 Sheffery & Newton (Caulobacter periodicity) Continue constructs; ligate

Fri Mar 23 Quon et al (Caulobacter master regulator)

Mon Mar 26 Continue Quon et al; Organismal databases

Wed Mar 28 Two presentations

Fri Mar 30 Two presentations