Annual Progress Report on Malting Barley Research March, 2002

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Fig. 3. Expression of Trx-Hth fusion protein genes in E. coli. A: Separation of bacterial proteins by SDS-
PAGE. Soluble protein from induced cultures Lane 1, standards. Proteins from bacteria harboring Trx
(wedge) only (lane 2), Trx-Hth1 (lane 3), and Trx-Hth2 (lane 4; wedge marks Trx-HTH fusion protein). B.
Top: Northern blot using RNA extracted from transformed E. coli and probed with 32 P-labeled Hth1 cDNA.
RNA from the uninduced (lane 1) and IPTG-induced (Lane 2) bacteria harboring Trx-Hth1 and uninduced
(lane 3) and induced (lane4) bacteria harboring Trx-Hth2. B: Bottom: The stripped northern blot was
rehybridized with 32 P-labeled cDNA probes synthesized from bacterial total RNA.
A
B
B
16S
(1.5kb
)
80S
Trx-HTH1 Trx-HTH2
IPTG - + - +
1 2 3 4
8-mer
14-mer
16S
Fig. 4. Analysis of Hth2 mRNA on polyribosomes. A: Leaf polyribosomes from transformant B03 were
separated on a sucrose density gradient. 8-mer refers to mRNA with 8 ribosomes attached. B: Equal
volume fractions were collected, and RNA was applied to a Nytran filter through a slot blot apparatus. The
blot was probed with 32 P-dCTP labeled Hth1 cDNA. C: RNAs from free and membrane-bound (mem)
polyribosomes from leaves of lines B01 and B03 were northern blotted and probed as above. Control lane
(con) is total leaf RNA from line B03. D: Blot for panel C was stripped and reprobed with labeled total
RNA cDNA, as above. Dark band is 18S rRNA; light upper band is 28S rRNA.
C
D
A
kDa 1 2 3 4
9
4 6
7 4
3
3
Trx 0
2
0
B03 B01
free mem free mem con