Download E18 Neo 8d 14d 24d 28d 42d 5m 9m A B A B A B A B A B A B A B A

E18
Neo 8d
14d 24d 28d 42d 5m 9m
A B A B A B A B AB AB A B A B
(a)Egr-1
(b)GAPDH
(c)GalK
Figure 1:
Northern blots of RNA extracted from the livers of mice of different ages. E18 = embryonic day
18; Neo = neonatal, d = postnatal day, m = months of age. A and B are samples from two
different littermates.
32
32
(a) Gel probed with P-labelled cDNA complementary to Egr-1 mRNA. (b) Gel probed with P32
labelled cDNA complementary to GAPDH mRNA. (c) Gel probed with P-labelled cDNA
complementary to galactokinase (GalK) mRNA.
7
Information:
Egr-1 is a zinc finger transcription factor. It is known to regulate the
expression of genes that are involved in processes of cell growth,
differentiation and stress responses. It has been suggested that Egr-1 may
have a role in the normal development of the liver. In order to investigate
this possibility, the expression of mRNA encoding Egr-1 was studied in the
liver of mice at different ages. Total RNA was extracted from liver
homogenates, and the same amounts of mRNA from each sample were
analysed by Northern blotting. The resulting data are shown in Figure 1.
Blot (a) shows the gel probed with 32P-labelled cDNA complementary to a
sequence of the Egr-1 mRNA. Blot (b) shows the gel probed with cDNA
complementary to the mRNA for the so-called ‘housekeeping’ gene, GAPDH
(glyceraldehyde phosphate dehydrogenase, a key enzyme in glycolysis), the
level of expression of which is thought to be stable, that is, to remain
constant. Blot (c) shows the gel probed with cDNA complementary to the
mRNA for the liver enzyme galactokinase (GalK), which is involved in the
metabolism of the milk disaccharide, lactose. (Note for background
information only: lactose is converted into glucose and galactose, and then,
in the liver, galactose is phosphorylated by galactokinase, into glucose-6
phosphate
1.Why was the gel probed with GAPDH cDNA? Briefly describe how the level of GAPDH
mRNA varied between different lanes?
2.Describe the changes in Egr-1 mRNA expression in the liver of animals from embryonic
age E18 to 9 months of age.
3.Does the pattern of expression of Egr-1 mRNA support the suggestion that it may play a
role in pre-and/or postnatal liver development? Explain your answer
4.If Egr-1 does act as a transcription factor that influences the expression of genes in the liver,
then it would be predicted that the expression of these genes may change developmentally in
line with changes in expression of Egr-1. One gene that may potentially be regulated by Egr-1
is that encoding the enzyme galactokinase (GalK), which is involved in the metabolism of the
milk disaccharide, lactose, and so is important in neonatal mammals. Does the pattern of
expression of GalK mRNA shown in Figure 1c support the suggestion that its expression may
be regulated by Egr-1? Explain your answer. (
4.Figure 1 only shows analysis of mRNA levels. What experiments, at the level of protein
analysis, would you suggest are needed to support the suggestion that Egr-1 may influence
expression of GalK, and why?