Frecuencia de los juegos en red y rendimiento escolar

TC total RNA 7 2 /1 4 4 12 3 2 X DegMgR F&R

1 X no data - mixed clone cDNA libaries:

K562 (H 5) 6 5 /2 3 3 18 16 16 X E.coli pgm

Placenta (H9) 2 7 /8 3 18 11 10 X E.coli pgm

1 X E.co//proline dehydrogenase {putA) DNA sample:

T.cruzi 2 2 0 /3 7 0 24 4 1 X aspartate aminotransferase {ASAT)

1 X T.cruzi 82kDa surface antigen

1 X no ORF 1 X DegMgR F&R

T.cruzi\Nh\ch showed homology to a variety of eukaryotic cytosolic aspartate aminotransferase genes {ASAl). The nucleotide sequence was 68% identical to mouse, 67% identical to chicken and 65% identical to arabidopsis ASAT over 137 nucleotides. The outer set of nested primers amplified the DNA, with

DegSer116F2 acting as the reverse primer and DegPGMR as the forward primer. ASAT, like PGM, is an isozyme marker for distinguishing between different isolates

of T.cruzi. Therefore, the LSHTM were provided with the clone in order to identify

and characterize the ASAT gene.

4.2.3 AGM SEQUENCE BASED DEGENERATE PRIMER PCR

N-acetylglucosamine phosphomutase (AGM) has been identified in

S.cerevisiae, and shown to possess phosphoglucomutase activity. The yeast

protein is of similar molecular weight to human PGMl and the contains the active site and magnesium binding loop motifs characteristic of the other phosphohexomutases. The distance between these two motifs in AGM is 228 amino acids, which is greater than the 168 amino acids seen in human PGMl. Degenerate primers specific to the AGM protein were designed to investigate the presence of a homologue in the human genome. The strategy employed was the same as used previously (refer to figure 4.4).

4.2.3.1 AGM Degenerate Primers, Template DNA and PCR Conditions

The problem with designing AGM specific degenerate primers was deciding the amino acid sequence upon which they should be based. Without any

homologous sequences for comparison, or information on the secondary structure of the protein, identification of probable conserved regions is more difficult. However, proline, glycine and hydrophobic residues are found to be highly conserved betweeen homologues, as they are generally important for maintaining the secondary structure. Thus, the forward degenerate primer, DegAGMFI covered the magnesium binding loop residues FDGDADR and the reverse primer, DegAGMRI, was based on a hydrophobic region of the protein, DMLAVL (Figure 4.13).

Nested degenerate primer PCR was carried out in an attempt to increase the specificity of the PCR. Since AGM contains both the active site and

magnesium binding site motifs, the degenerate primers DegSerl 16F2 and DegMgR2 were used along with two new AGM specific primers (Figure 4.13). DegAGMF2 was based on a hydrophobic region, GILAV, at the carboxyl end of the protein and was used as an outer primer with DegMgR2. DegAGMR2,

Figure 4 .1 3 Location o f the AGM cDNA degenerate primers. DegAGMFI (DGDADR) DegAGMRI (DMLAV) DegAGMF2 D e g S e rl!6 F 2 (GILAV) ({G/A}SHNP) DegAGMR2 DegMgR2 (GADYV) (GD{G/A/F}DR)

Figure 4 .1 4 Nucleotide sequences of AGM cDNA degenerate primers and annealing temperatures.

Forward Primer Reverse Primer ANNEALING

TEMPERATURE DegAGMFI 5 ' TTYGAYGGNGAYGCNGAYAG 3' DegAGMRI S' ARNACNGCNAGCATRTC S' 4S°C / Tm°C DegAGMF2 5 ’ GAAGCTTCGGNATYYTNGCNGT S' DegMgR2 S' CTCTAGAGCGRTCNVMRTCNCC S' SS°C /T m ° C DegSerl 1 6F2 S' GAAGCTTCGSNWSNAYAAYCC S' DegAGMR2 S' CTCTAGAGACRTARTCNGCNCC S' S8°C / Tm°C

based on hydrophobic residues, GADYV, located 22 amino acids upstream of the magnesium binding loop, was used for the nested PCR with DegSer116F2. A summary of the data showing the nucleotide sequences of the AGM primers and the annealing temperatures used is seen in figure 4.14.

Total RNA from the cell lines K562, JG, ED and TC was reverse transcribed using random hexamer primers. In addition, degenerate PCR was carried out on the K562 and placenta cDNA libraries. The standard degenerate primer PCR programme was used: denaturing at 95°C for 30secs, annealing for 30 secs and elongation at 72°C for 45secs. Degenerate primer PCR consisted of 35 cycles and the reaction mix contained lOnmoles of each primer. Nested degenerate primer PCR consisted of two rounds of 25 cycles, with the reaction mix containing lOOpmoles of each primer.

4.2.3.2 Results

Amplification with DegAGMFI and DegAGMRI of K562 cDNA produced a faint doublet of 420 and 450bp. No bands, however, were produced from the JG and ED cDNA samples (Figure 4.15). A faint band of 325bp was amplified from both of the cDNA libraries. The subsequent transformation results are shown in figure 4.16. In total, 93 recombinant plasmids were analyzed, and of these only 19 appeared to contain inserts of between 250bp and 750bp. Of these 16 were identified as vector rearrangements. None of the remaining three were novel PGM-like sequences; the single clone from K562 was identified as human IBs rRNA, and as found previously, the recombinant plasmids transformed with PCR products from the cDNA libraries contained E.coli sequences.

Nested degenerate primer PCR with the AGM specific primers on the cDNA samples did not increase the specificity of the PCR. None of the bands

amplified by RT-PCR in the first round were enhanced by nested PCR primers in the second round. After both the first and second round of PCR, no products were detected from the cDNA libraries.

4.3 SUMMARY

i) Low stringency PCR was investigated in an attempt to identify PGM2 and

PGM3 cDNA sequences. Under a variety of cycling parameters, only PGM1

was amplified from the control lymphoblastoid cell lines. Hybridization of the PCR products with the HPGM probe identified no related sequences amplified from K562 or the control cell lines.

Figure 4 .1 5 PCR results from experiments using degenerate AGM cDNA primers.

Primers Template DNA Results

DegAGMFI/DegAGMRI

cDNA samples:

K562 pA+ RNA Lane 1 : Faint doublet of 420 &450bp, strong band 1 75bp________________ JG total RNA Lane 2: No bands, low mol, wgt. smear ED total RNA Lane 3: No bands, low mol, wgt. smear Negative control Lane 4: dHgO

cDNA libraries:

K562 (H5) Lane 5: Faint 325bp band