DETERMINACIÓN DE LOS NIVELES DE TRANSFERRINA, FERRITINA Y

The protein sequence of clone B is 55% identical to E. coli hemB.

1 R H G H C G V L C D H G V D N D L T L T N L G K Q A W . A A Q R C D F IA P S A A M V G Q V Q A IR R R W S H G H C G V L C E H G V D N D A T L E N L G K Q A W A A A A G A D F IA P S A A M D G Q V Q A IR . . . 78 TRGLHRYRYHVYSLVAHFSTLQEI.... .QALDAAGFKDTAIMSYSTKFASSFYGPFR

138

F ig u re 3.13. D ot b lo t analysis of clone B.

a ) D ot blot p ro b ed w ith clone B.

Clone B was labeled with ^2p and used to probe Aspergillus and E. coli genomic DNA.

The hybridisation and washes were carried out at 45°C with a final wash in 2 x SSC. Clone B did not hybridise to either genome although control hybridisation of the probe to itself was detected at 100 and 10 pg DNA.

Lane 1: Probe (clone B - control) DNA at loadings of 100, 10 and 1 pg. Hybridisation is detected at 100 and 10 pg DNA.

Lane 2: E. coli genom ic DNA at 1000, 500, 100 and 10 ng DNA. No hybridisation is

detected.

Lane 3: A s p e r g illu s genom ic DNA at 2000, 1000, 500 and 100 ng DNA. No

b) D ot blot p ro b ed w ith E. coli hem B .

The dot blot was reprobed with E. coli hemB as a positive control.

Lane 1: Probe {hemB - control) DNA at loadings of 500, 100 and 10 pg. H ybridisation is

detected at all loadings.

Lane 2: E. coli genom ic DNA at loadings of 100 and 500 ng DNA. H ybridisation is

detected at both loadings.

Lane 3: Aspergillus genomic DNA at 1000 and 2000 ng DNA. Hybridisation is detected at 2000 ng and very faintly at 1000 ng.

Chapter 3: Enzyme isolation and purification

A final attempt at gene isolation by PCR was carried out using a selection of primers 1-15 with new reaction conditions. In addition, specific primers designed to the 5' and 3' regions of yeast HEM2 called Y1 and Y2 were tried (ECPCR primers are given later in figure 3.16a). The control DNAs were as before except that yeast HEM2 DNA and yeast genomic DNA were also used. Each primer combination was tested with the range of control DNAs at temperatures of 40, 45, 50, 55 and 60°C using 'hot start' in which Taq

polym erase is added at 94°C. This should result in more specific hybridisation as the enzyme cannot amplify any transiently formed duplexes during the initial dénaturation step. Primer pairs giving single bands of the expected size were selected.

In the PCR, the templates used were genomic DNA isolated from cultures o f Ustilago

maydis, Neurospora and Aspergillus. Genomic DNA was used instead o f cDNA libraries

because of concern that amplifying cDNA libraries in a lambda ZAP expression system had caused contamination with E. coli DNA and this may have been the cause of the failure of PCR to amplify the desired gene on previous occasions. PCR was carried out using hot start and the range of hybridisation temperatures described above.

The results shown in figure 3.14 illustrate that the hot start and variable hybridisation tem perature yielded clean reaction products. The yeast H EM 2 prim ers consistently am plified a product o f 975bp from A sp e rg illu s DNA and, whilst this result was unexpected as these primers are non-degenerate, this product was cloned and sequenced. The DNA was digested with EcoRl and H indRl and ligated into pBluescript (pSKt). The resulting plasmid was transformed into DH5a E. coli competent cells.

The resulting clone, called clone C, was sequenced using the KS primer and this revealed the reaction product to be the yeast H E M 2 gene, presum ably arising from the contamination o f reaction mixtures with yeast DNA. Considerable precautions had been taken to avoid such contamination, (setting up reactions in different rooms, running in separate PCR machines, use of filtered tips and dedicated pipettes), and it was therefore decided that PCR was not a useful strategy to pursue as recurring contamination meant there was no realistic chance of amplifying the desired product.

F ig u re 3.14. R esults of hot s ta r t PC R .

A garose gel (1%) analysis o f PCR products obtained using the 'hot start' method is shown. The primers used were Y land Y2, ECPCR primers designed to the HEM2 gene.

Lane 1: Lanes 2-4: 60"C. Lanes 5-7: 60°C. Lanes 8-10: 60°C. Ikb mai'ker

E. coli DNA (1 ng) with Y1 and Y2 and hybridisation at 50°C, 55°C and

E. coli DNA (1 ng) with Y1 only and hybridisation at 50°C, 55“C and

E. coli DNA (1 ng) with Y2 only and hybridisation at 50°C, 55°C and

Lanes 11-13: A sp erg illu s DNA (100 ng) with Y1 and Y2 and hybridisation at 50°C,

55°C and 60°C.

Lanes 14-16: Aspergillus DNA (10 ng) with Y1 and Y2 and hybridisation at 50°C, 55°C

and 60°C.

Lanes 17-19: A spergillus DNA (1 ng) with Y 1 and Y2 and hybridisation at 50°C, 55°C

and 60°C. 1 2 3 4 5 6 7 8 9 10 11 12 13 1415 1617 18 19 kb 3 .0 5 4 /2 .0 3 6 1.636 1.018 0 .5 1 7 / 0 .3 9 4 0.344

Chapter 3: Enzyme isolation and purification

Southern and dot blots were carried out to determine whether the yeast HEM2 gene could be used to isolate other fungal ALADs by direct hybridisation or whether other fungi have ALAD sequences too different to allow their isolation by standard molecular biological techniques. In dot blots, between 3,000 and 10 ng genomic DNA from yeast, E. coli,

N e u r o s p o ra , A sp e rg illu s and U stilago was dotted onto H ybond N hybridisation

m em brane. Sim ilarly, 5|ig genomic DNAs were loaded for the Southern blot. The protocol followed was as described earlier for clone B. Hybridisation was carried out overnight at 45°C or 65°C. Washes were also carried out at room temperatures or 45°C using a range o f buffer concentration between 4 x SSC and 0.3 x SSPE as in table 2.4. The photographs of the dot blot results shown in figure 3.15a illustrate that hybridisation of HEM2 could be seen to:

Yeast DNA at 1,500 and 400ng

E. coli DNA at 1,500, 400 and 100 ng

Aspergillus DNA at 3,000 and 1,500 ng.

Neurospora DNA at 3,000 and 1,500 ng

Ustilago DNA at 3,000 and 1,500 ng

The approximate size of the yeast, Aspergillus, Neurospora and Ustilago genomes are 4.2x10^ bp whereas the E. coli genome is about 4.2x10^ bp. The smaller size of the E. coli genome means that a 10 ng loading of E. coli DNA would be expected to contain the same num ber of sequence repeats as a 100 ng loading of a fungal DNA. The results therefore show that the HEM2 gene hybridises to yeast and E. coli DNA as expected as

the H EM 2 sequence is derived from yeast and is 36% homologous overall and 84%

identical in conserved regions to the E. coli hemB gene. However, the hybridisation to the other fungal DNAs is poor. This suggests that the sequences encoding ALAD in fungi are very different from the H E M 2 sequence. The Southern blot also dem onstrated hybridisation of HEM2 to yeast and to E. coli genomic DNA but no hybridisation to other fungal DNAs (figure 3.15^).

F ig u re 3.15. Dot b lo t an d S o u th ern analysis of H E M 2 DNA.

The possibility of using the yeast HEM2 sequence to isolate another fungal gene encoding

ALAD was investigated using dot {a) and Southern blots {b ). H ybridisation was carried

out at 45°C and the final wash was in 2 x SSC at 45°C.

a) Dot blot.

Lane 1: Y east genom ic DNA at loadings of 1,500; 400 and 100 ng. H ybridisation is detected down to 400 ng.

Lane 2: Aspergillus genomic DNA at loadings of 3,000; 1,500 and 100 ng. Hybridisation is detected down to 1500 ng.

Lane 3: Neurospora genomic DNA at loadings of 3,000; 1,500 and 100 ng. Hybridisation is detected down to 1500 ng.

Lane 4: Ustilago genomic DNA at loadings of 3,000; 1,500 and 100 ng. Hybridisation is

detected down to 1500 ng.

Lane 5: E. coli genom ic DNA at loadings o f 1,500; 400; 100 and 10 ng DNA.

Hybridisation is detected down to 100 ng DNA.

Lane 6: Probe {HEM2) DNA at loadings of 100 and 10 pg. Hybridisation was detected at

both loadings.

b) S o u th e rn blot.

H EM 2 was used to probe restricted genom ic DNA from yeast, E. coli, Aspergillus,

Neurospora and Ustilago.

Lane 1: Yeast genomic DNA (5 |ig). Hybridisation occurs in a smear of bands.

Lane 2: E. coli genomic DNA (5 | L i g ) . Hybridisation occurs in one band.

Lane 3: Aspergillus genomic DNA (5 | L i g ) . No hybridisation occurs.

Lane 4: Neurospora genomic DNA (5 |ag). No hybridisation occurs.

Chapter 3: Enzyme isolation and purification

On the basis of Southern and dot blots as well as PCR, it seems unlikely that it will be possible to isolate another fungal gene encoding ALAD using PCR or hybridisation-based techniques. A lternative experim ental approaches include cloning the gene by complementation of an E. coli hemB mutant (section 2.3.8) or a yeast HEM2 mutant. The alternative would be to purify ALAD protein from a fungus, obtain amino terminal and / or internal sequence information and then design minimally redundant gene-specific probes based on the primary structure. As a preliminary check into the feasibility of this approach, an extract from Ustilago maydis was analysed for ALAD activity. The activity was detectable but very low (approximately one fifth of that found in a similar extract of

E. coli where ALAD is already only 1% o f total cell protein) and the isolation and

purification of a fungal ALAD would therefore be expected to be difficult.