David L' Herrin Painter 2'24 2'18 lab 4713843 herrinmail'utexas'edu

1
David L. HerrinPainter 2.24 (2.18
lab)471-3843herrin_at_mail.utexas.edu

• A link to this presentation can be found at
  http//www.esb.utexas.edu/herrin/bio344/

2
Lab Projects

• I. Light-regulated splicing of the psbA introns
• psbA gene in Chlamydomonas has 4 group I introns
  that self-splice in vitro, although poorly at 25?C

3
Mobile
ORFs are not essential maturases. I2 and I4 ORFs
are homing endonucleases.
4
psbA gene encodes the D1 polypeptide of PSII.
Strongly up-regulated by light.
5
Lab Projects

• I. Light-regulated splicing of the psbA introns
• Splicing of psbA introns in vivo is slow in the
  dark (in cells growing photoautotrophically),
  increase at least 6-10-fold within 15 min of
  light administration
• Light stimulation does not require protein
  synthesis but does require electron transport
• Mechanism? Probably mediated by proteins.

6
Genetic approach to get trans-acting factors

• Use suppressor genetics to identify genes that
  promote splicing of psbA and rRNA introns
• Identified at least 2 and probably 3 nuclear
  genes (css1-css3) that can suppress deficiencies
  in the rRNA and psbA4 introns
• Currently trying to clone css1 using an arrayed
  cosmid library

7
The genetic model organism, Chlamydomonas
reinhardtii
All 3 genomes can be transformed.

• Nuclear genome
• 14 haploid chromosomes
• 100,000 KB
• Molecular map
• Draft genome sequence at 8X coverage

8
Lab Projects

• II. Transcriptional regulation by the circadian
  clock

9
Circadian Rhythms

• Oscillate with a period of 24 hours
• Phase determined by light-dark cycle
• Once entrained, continue in constant conditions

10
A Circadian System
11
Relative transcription rates
12
Plastid (Chloroplast) RNA polymerases in vascular
plants
Sigma subunit directs RNA polymerase to promoter
Fig. 6.31 in Buchanan et al.
13
Lab Projects

• II. Transcriptional regulation by the circadian
  clock
• Chlamydomonas has 1 sigma factor-like gene
  (sig1)!!

14
Sig1 mRNA shows a circadian rhythm, peaking a few
h before the peak in chloroplast transcription.
LD ? LL
15
II. Transcriptional regulation by the circadian
clock

• Role of the putative sigma factor for the
  chloroplast (prokaryotic-like) RNA polymerase in
  circadian control of chloroplast transcription
• Approaches
• Biochemistry
• In vivo molecular and cellular analyses
• Microarrays