Expression analysis of the zmzr1 gene - Isolation of an etched1 homologue, zmzr1

3. Results

3.4. Isolation of an etched1 homologue, zmzr1

3.4.2. Expression analysis of the zmzr1 gene

hybridisation experiments with LC and et1-Ref genomic DNA, where only a very weak signal was detected. Another possibility, also based on the Northern analysis, was weak expression of the zmzr1 gene.

Therefore, based on the possibility that the zmzr1 cDNA clone, if present, had somehow been missed in the cDNA library screening, another simpler but sensitive experiment was tried. Using zmzr1 specific primers present in the 5´ and 3´

untranslated regions, with a melting temperature ranging between 65°C-70°C, and vector specific primers, the cDNA library was screened through PCR amplification experiments (Fig. 3.15). To select the zmzr1 specific primers, the zmzr1 transcript was conceptually translated from the zmzr1 gene by comparison to the et1 cDNA sequence. From the 5´ end of the deduced cDNA sequence, two gene specific primers were taken, one, Et 44, from the 5´ UTR and another, Et 26, at the start of the putative exon 2. From the 3´ end, three different primers, at three different positions in exon 4, viz., Et 27, Et 45 and Et 46, were taken (Table. 3.3). These experiments were carried out with both et1-Ref as well as LC cDNA libraries simultaneously.

After the first amplification of the samples from the et1-Ref developing kernel cDNA library, a number of strong bands could be observed on an ethidium bromide agarose gel, which, however, did not correspond in length to the bands expected from the zmzr1 gene. The putative bands with lengths in the range expected from the zmzr1 gene were either only faintly visible or, in many cases, were absent. Therefore, in order to check for the presence of the zmzr1 specific cDNA clones, these amplifications were hybridised with the et1 cDNA probe, containing sequences homologous to zmzr1. Only from the et1-Ref cDNA library were positive hybridisation signals in the expected size range detected. In the LC cDNA library no positive amplification products were detected.

In order to analyse the positive et1-Ref cDNA amplifications further, it was necessary to clone these sequences. For this, a larger amount of the PCR product, clearly visible on an ethidium bromide gel, was needed. Therefore, using small aliquots ranging from 0.01 µl to 5 µl from the first PCR, a reamplification (description in 2.2.8.4) of the cDNA clones was carried out using the same primers and the same PCR conditions. In the second round, the zmzr1 specific amplifications were much

stronger in intensity than the other unspecific ones, a number of which were no longer visible in this round (Fig. 3.15).

: :

PCR amplifications with a zmzr1 genomic clone used as a positive control for the reaction.

PCR amplifications with an et1-R developing kernel cDNA library specific to zmzr1.

26-27 26-45 26-46 44-27 44-45 44-46 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

PCR I

26-27 26-45 26-46 44-27 PCR II

Fig. 3.15: (Legend on the next page)

Fig. 3.15: The PCR amplification of zmzr1 specific cDNA obtained from an et1-Ref developing kernel cDNA library. In first round PCR, positive amplifications could be obtained from the cDNA library (lane 2) with the primer combinations: 26-27, 26-45, 26-46, 44-27, 44-45. For each combination, one positive control with the zmzr1 λ genomic clone as template (lane 1) and one negative control with H₂O as template (lane 3) were taken. The amplification as observed on the gel containing 10% of the PCR is shown in (A). The positive amplifications after hybridisation with the et1 cDNA probe are shown in (B). Four different dilutions from each of the five positive PCRs were taken to reamplify (PCR II) the PCR products with the same primer combinations as in PCR I. In PCR II, shown in (C), the combinations 26-27, 26-45, 26-46 and 44-27 could be reamplified. These were isolated and cloned for further analysis.

Similar trials were also carried out using vector specific primers homologous to T3 and T7 promoter sequences, present on either side of the cDNA insert. These primers were used in combination with the zmzr1 specific primers, in order to amplify both the 5´ and 3´ ends and get a complete cDNA sequence. One PCR product containing the 5´end of the cDNA, amplified with the primer pairs T3 and Et 27, was cloned and sequenced, (Fig. 3.16, Table 3.3 and 3.17). However, no 3´-end amplifications could be obtained.

Fig. 3.16: (Legend on the next page) cDNA 1 :

cDNA 2 : cDNA 3 : cDNA 4 :

Et 26 Et 46

T 3 Et 27

Et 44 Et 27

Et 26 Et 27

zmzr1 cDNA vector

cDNA primer start/stop codons vector primer Adapter sequence

Fig. 3.16: A schematic representation of the amplified zmzr1 cDNAs obtained from the et1-Ref developing kernel cDNA library. The four amplified cDNA clones as described in Table 3.3 are represented schematically in the 5´–>3´ orientation. The topmost clone represents the putative cDNA present in the cDNA library, from which the four cDNA clones could be amplified using zmzr1 specific primers. The names and positions of primers with respect to the cDNA are shown below each. The start and stop codons are shown as vertical blue bars.

Table 3.3: Four positive clones could be isolated from four independent amplification events of the et1-Ref dT primer developing kernel cDNA library. These were then cloned and, after sequencing, compared to the zmzr1 genomic clones.

The sequence analysis of cDNA clones isolated from the et1-Ref developing kernel cDNA library revealed the presence of a zmzr1 transcript. The cDNA contained the same open reading frame as that deduced from the comparison of the zmzr1 gene to the et1 cDNA. As can be seen in Fig. 3.17 (next page), the complete ORF could be amplified from the cDNA library. However, the length of the complete 3´ UTR could not be determined and the amplified 5´UTR of the cDNA could also possibly be incomplete. Moreover, from the PCR amplification of 5´UTR, obtained using the T3 and Et 27 primers, a truncated PCR product was obtained, where a small part of the ORF was absent. The amplified sequence was otherwise homologous to the other amplified cDNA sequences (Fig. 3.17, next page).

S.no. cDNA clone Primer Pair Region of amplification length of cDNA 1. cDNA1 T3, Et 27 5´UTR (Exon 1) – Exon 4 433 bp 2. cDNA2 Et 44, Et 27 Exon 1 – Exon 4 480 bp 3. cDNA3 Et 26, Et 27 Exon 2 – Exon 4 297 bp 4. cDNA4 Et 26, Et 46 Exon 2 – 3´ UTR (Exon 4) 439 bp