3 3 3 Codon usage
Plate 3. 2 Computerised prediction of the secondary structure of the crnA
encoded polypeptide. The straight lines represent 6-sheets and the position of a-helices are indicated by a zig-zag line. The blue diamonds are representative of the hydrophobic domains. In contrast, red circles are indicative of particularly hydrophilic areas.
protein, as predicted by the rules of G am ier, O sguthorpe and Robson (1978) (UWGCG program ), does not clearly show the ten m em brane- spanning a-helices (Plate 3.2). According to this model the secondary structure of the crnA encoded polypeptide is composed mainly of fi-sheet structures. This is a reasonable interpretation due to the relative abundance of the AAs valine, isoleucine, phenlyalanine and tryptophan, all favouring the form ation of 6 -sheets. Further support for this m odel is
obtained from the observation that m any glycine residues reside in the trarism em brane domains. As explained, these may disrupt the a-helical structure. However, the com puter model does not predict the structure the protein m ay assum e w hen inserted into a m em brane and further work is necessary before a true prediction can be made (4.7.3).
3. 4. 0. REGULATION OF crnA GENE EXPRESSION
Regulation of gene expression at the level of mRNA accumulation m ay be studied by examining mRNA levels on N orthern blots (1.3.1) The expression of the crnA gene w as found to be regulated at this level allowing an investigation into the m echanism of regulation.
The crnA cDNA hybridises to two messages, one of 1.1 kb and the other of 1.8 kb, m easured using Hindlll cut X DNA m olecular w eight m arkers. A quantity of 20 pg of total RNA isolated from nitrate grow n mycelia is required for the detection of both messages (Fig 3.6). A reduced am ount leads to the complete loss of the smaller m essage although the 1.8 kb mRNA may be detected w ith 5 pg of RNA. The 1.8 kb message, show n to have greater abundance than the 1.1 kb mRNA, is considered to be that of the crnA mRNA as its size compares well with that predicted from the sequencing data (3.3.2). However, the smaller transcript is of unknow n origin.
•*«2
A B c D E
1.8 kb 1.1 kb-
Figure 3.6 Northern blot of A.nidulans total RNA isolated from wild type grown in MM with 10 mM nitrate. Lane A; 5 pg, lane B; 10 pg, lane C; 20 pg, lane D; 50 pg, lane E; 100 pg. RNA was electrophoresed on a 1.2% formaldehyde denaturing agarose gel, transferred to nylon membrane and hybridised to the hexaprime labelled crnA cDNA fragment from pSTA1500.
i
i
An initial investigation into the two messages was conducted by increasing the post-hybridisation washing temperature from 55^ C to 65® C in 0.1 X SSC and 0.1% SDS. Both transcripts disappeared at the same rate suggesting that the nucleotide sequence of the two messages is equally similar to the probe DNA (data not shown). To examine the possibility that the smaller message may be from a second nitrate transport gene (1.4.2) Northern and Southern blots of crnA deletion mutants (Fig 1.6) were hybridised against the crnA cDNA. Total genomic A.nidulans DNA for Southern blotting was digested with endonuclease Sail and electrophoresed through a 0.8% agarose gel. The hybridisation profile (Fig 3.7) of the wild type, lane A and crnA-niiA-niaD A507 strain, lane B, is identical to that obtained by previous workers for the crnA g e n e (Johnstone et al, 1990). The larger band of 3.8 kb, fragment SI (Fig 1.6) is
unaltered in the mutant, in contrast to the 1.8 kb fragment, S2 (Fig 1.6) ^ showing an increase in size. That no other band is detected implies that
the A.nidulans genome does not include two nitrate transport genes of similar sequence. In support of this, the Northern blot (Fig 3.7) of total RNA from nitrate grown mycelia of the strains; wild type, lane A, A507, lane B, and A506 (Fig 1.6), lane C shows that, in contrast to the wild type, both the 1.8 kb and 1.1 kb transcripts are absent in the deletion mutants. The Northern blot was stripped of radioactivity and re-probed with the
A.nidulans actin gene (Fig 3.7 iii) demonstrating equal loading and 4
transfer of RNA. The actin gene is expressed in all these strains and is therefore a good internal control (2,5.7). These results not only demonstrate that the A507 deletion extends into the upstream regulatory sequences of the crnA gene, i.e. into S2 (Fig 1.6), but also imply that the 1.1 kb message is either transcribed from within the region of the crnA gene or it is an artefact (4.2.0). It is clear from the sequencing data that only one open reading frame, that of the crnA gene itself, exists on either strand
i) A___B ii) A B C 23.0— * 9.4— 6.1 4.3— 1 . 8 kb # 1 . 1 kb 2.3— 2.0— m iii) 1 . 8 kb
Figure 3-7 i) Southern blot of A.nidulans D N A isolated from the strains, lane A; wild type and lane B; A507. 20 pg of DNA was digested to completion with Sal I, electrophoresed on a
0 .8 % agarose gel, transferred to nylon membrane and hybridised to
the hexaprime labelled cDNA fragment from pSTAlSOO.
ii) and iii) Northern blot of A. nidulans
total RNA isolated from the strains, lane A; wild type, lane B; A506, lane C; A507. 20 pg of RNA was electrophoresed on a 1.2% form aldehyde denaturing agarose gel, transferred to nylon membrane and hybridised to the hexaprime labelled, ii) crnA cDNA fragment from p ST A1500 and ïn)A.nidulans act A fragments.
between the EcoRl and N ru l sites (Fig 3.3) of pSTA4. Therefore, it is plausible to assume that the 1.1 kb transcript is derived from the crnA
gene itself.