Update on RelA Project Cloning of amplified relA

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Update on RelA ProjectCloning of amplified relA
PCR
Insert amplified product in both orientations
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Update on RelA ProjectCloning of amplified relA
pUR351b
pUR351a
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Update on RelA Project Controlled overexpression
of Ptac-relA
ScaI
pUR351a
SalI
SalI
SmaI
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Update on RelA Project Controlled overexpression
of Ptac-relA
pUR354 (KT)
Status

• relA fragment in agarose
• pRL502a digestion completed

relA
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Update on RelA Project Controlled overexpression
of PEm-relA
ScaI
pUR351a
SacI
ClaI
NsiI
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Update on RelA Project Controlled overexpression
of Ptac-relA
ScaI
SacI
pUR351a
EcoRV
PstI
SacI
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Update on RelA Project Controlled overexpression
of Ptac-relA
PstI
pUR355 (Karen)
Status

• First attempt failed
• Second attempt transformed

ClaI
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Update on RelA Project Controlled production of
antisense relA
pUR351a
DpnI
SacI
DpnI
SacI
SmaI
(93 bp)
(1072 bp)
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Update on RelA Project Controlled production of
antisense relA
pUR357 (Julie)
Status

• DpnI fragment isolated
• pRL502a digestion completed

SacI
DpnI
DpnI
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Update on RelA Project Construction of relA
insertional mutant
PvuII
pUR351b
HpyCH4IV
NsiI
ClaI
SacI
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Update on RelA Project Construction of relA
insertional mutant
PvuII
BglI
Apr
Apr
PvuII
pUR351b
PvuII
HpyCH4IV
(2476 bp)
SacI
(2597 bp)
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Update on RelA Project Construction of relA
insertional mutant
pUR352 (Carrie)
HpyCH4IVmatches ClaI
Apr
Status
pBluescriptKS

• First try failed
• Second try transformed

PstImatches NsiI
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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?

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Update on ?ENews from ?F

• Margolis et al (1991). Science 254562-565
• ?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

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Update on ?ENews 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
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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)
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Caulobacter crescentusCell cycle-regulated
differentiation
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Caulobacter crescentusCell cycle-regulated
differentiation
The phenomenon Sheffery and Newton (1981) Cell
2449-57 Regulation of periodic protein synthesis
in the cell cycleControl 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)
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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