Biology Reference
In-Depth Information
•
The opposite changes in glycolytic and respiratory
TL
(See Fig. 12.4)
induced by glucose-derepression are due to opposite changes in
TD
, thus
establishing an instance of degradational control in contrast to the well-
known transcriptional control.
•
There are 9 mechanisms underlying underlying the changes in
TL
(See
Fig. 12.3).
12.2. Materials and Data Acquisition
12.2.1.
Glucose-Galactose Shift Experiments
The
S. cerevisiae
yeast strain BQS252 was grown overnight at 28 degrees Cel-
sius in YPD medium (2% glucose, 2% peptone, 1% yeast extract) to exponential
growth phase (
OD
600
=0
.
5) [10]. Cells were recovered by centrifugation, re-
suspended in YPGal medium (2% galactose, 2% peptone, 1% yeast extract), and
allowed to grow in YPGal medium for 14-15 hours after the glucose-galactose
shift. Cell samples were taken at 0 (denoted as
t
0
), 5 (
t
1
), 120 (
t
2
), 360 (
t
3
), 450
(
t
4
) and 850 (
t
5
) minutes after the glucose-galactose shift. The
t
5
sampling time
corresponds to the exponential growth phase in YPGal medium. Two different
aliquots were taken from the cell culture at each sampling time. One aliquot was
processed to measure
TR
according to the genomic run-on protocol (see the next
section), and the other was processed to measure
TL
using the same DNA arrays
recovered after
TR
measurements.
12.2.2.
Measuring Transcription Rates (
TR
) Using the Genomic Run-on
(
GRO
)Method
The experimental details of the genomic run-on procedures are given in Garcia-
Martinez
et al.
[10]. This technique is a scaled-up version of the usual nuclear
run-on method [13]. Lysed cells contain transcription complexes stalled on the
DNA template due to lack of ribonucleotides. Transcription is re-initiated
in vitro
by adding new nucleotides, one of which is radiolabled (
e.g.
,[
α
33
P]UTP). After
allowing transcription to finish, one can determine via autoradiography the den-
sity of RNA polymerases for each gene. Assuming a constant speed for RNA
polymerase II molecules this density allows us to estimate the transcription rates
in the cells of interest. By comparing the amount of gene-specific radiolabeled
RNA synthesized in one nuclei preparation with another, it is possible to estimate
the extent of the RNA polymerase densities and, therefore, transcription rates in
the cells of interest. The
TR
data utilized in the
TL-TR
plots shown in Fig. 12.1
areavailableat
http://scsie.uv.es/chipsdna/chipsdna-e.html
datos
.
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