Analysis of et1-Ref genomic clones

The et1-Ref genomic library had been screened by M. Ahrend (Diploma thesis, 1998) using the 2.5 kb et1 genomic fragment probe, which lead to the identification of the et1 locus. However, two different classes of clones were isolated from the library. The Class I contained fifteen clones, which on analysis were found to represent the et1 locus. The complete sequence of the et1 gene was found to be missing from this locus. The sequence of the et1 probe hybridising to the et1-Ref genomic clones represented the gene upstream region.

In the second class, three clones were present, which were different from the Class I clones in their restriction pattern as well as pattern of hybridisation with the 2.5 kb et1 probe. In addition, unlike the Class I clones, they also showed a hybridisation signal with the et1 cDNA, c9.1 (Ahrend, 1998). This information indicated that they belonged to another locus. One probable explanation for the hybridisation with c9.1 was a deletion and translocation of the et1 gene, complete or in part, to another

position during the X-ray irradiation event. On the other hand, the presence of a second copy or a gene homologue, which is often the case in Zea mays, was also possible. The latter possibility was also supported by the Southern analysis carried out on LC genomic DNA, where additional bands, not belonging to et1, were detected. Hence, these three genomic clones were further sequenced and analysed.

In order to characterise these three clones, viz., et1-R 1.1, et1-R 10.2, and et1-R 18.1, they were first analysed by new hybridisation experiments with the et1 cDNA and the 2.5 kb et1 genomic fragment probes. Using restriction fragment and hybridisation analyses, a restriction map of the clones was constructed. Based on their restriction maps, two clones, et1-R 10.2 and et1-R 18.1, were found to overlap each other (Fig. 3.13, next page) and the third clone, et1-R 1.1, did not to belong together with the other two. Moreover, all three clones showed a restriction pattern different from that of et1 from the wild type alleles. This indicated the presence of possibly two different loci in addition to the deleted et1 locus. This, however, also supported the hypothesis that apart from the second copy of et1, a translocated et1 gene fragment might also be present at another position in the et1-Ref genome. In order to clarify their identities, these clones were analysed further.

Fig. 3.13: The restriction pattern of the Class II genomic λ clones, et1-R 10.2 and et1-R 18.1, obtained from an et1-Ref line and representing a new gene, zmzr1. The λ clones are shown in the 5´–> 3´ orientation of the gene. The λ arms are represented as grey boxes at the ends of the clones.

From top to bottom, the first clone is λ et1-R 10.2, followed by a restriction map of the zmzr1 gene obtained with the help of both the clones, and finally the clone λ et1-R 18.1. The subclones prepared from these two genomic clones are indicated below each of their restriction maps. After cloning fragment 1 from λ etR 10.2, the fragments 2, 3 and 4 were further subcloned from it, which were then sequenced completely. Similarly, after subcloning fragment 5 from λ etR 18.1, the fragments 6 and 7 were again subcloned from it and sequenced. The common restriction pattern in both the clones is represented in the middle of the two λ clones, showing the structure of the new gene zmzr1. The coloured blocks represent the four exons in the gene. The bold line represents the region sequenced.

The scale bar on the right represents a length of 1kilobase. Abbreviations: λ, the phage λ arms;

restriction enzymes: B Bam HI, D DraII, E Eco RI, H HindIII, N NsiI, S SalI, St SstI, X XhoI, Xb XbaI.

For sequencing the regions of hybridisation, first, some subfragments of the three clones were chosen for subcloning experiments (Table 3.2). These subfragments, as shown in Fig.3.13, were sequenced and evaluated for homology to et1.

6 7

5

1kb λ et1-R 10.2

H B D H D D St E B H B S S

S

1

3 4 2

λ λ

λ et1-R 18.1

H B H St N

B

E N S

S H

λ λ

Xb Xb

H B H St

B

S.No. Clone (fragment) Length (Restriction Site) Vector (Restriction Site) 1. et1-R 1.1 (1) ~ 2kb (SalI) pZErO-II (XhoI)

2. et1-R 1.1 (2) ~ 1.3 kb (SalI/ PstI) pZErO-II (XhoI/ PstI) 3. et1-R 1.1 (3) ~ 0.7 kb (SalI / PstI) pZErO-II (XhoI/ PstI) 4. et1-R 10.2 ~ 5.5 kb (SstI/ SalI) pZErO-II (SstI/ XhoI) 5. et1-R 10.2 (1) ~ 4.5 kb (SalI/SstI) pZErO-II (XhoI/ SstI) 6. et1-R 10.2 (2) ~ 2 kb (SalI/ DraII) pZErO-II (XhoI/ DraII) 7. et1-R 10.2 (3) ~ 1.2 kb (DraII) pZErO-II (DraII) 8. et1-R 10.2 (4) ~ 1 kb (DraII) pZErO-II (DraII) 9. et1-R 18.1 (5) ~ 4 kb (SalI/ Bam HI) pZErO-II (SalI/ Bam HI) 10. et1-R 18.1 (6) ~ 2 kb (NsiI) pZErO-II (NsiI) 11. et1-R 18.1 (7) ~ 1.5 kb (NsiI) pZErO-II (NsiI)

Table3.2: Subcloning of the three et1-Ref λ genomic clones into the plasmid vector, pZErO-II for sequencing. The first column represents the λ clones from which the fragments were subcloned.

If they were given a number (Fig. 3.13), these are shown in parentheses. The second column shows the approximate size of the fragments and in parentheses their restriction digested ends. The third column shows the cloning vector and in parentheses the site of cloning.

Sequencing of the et1-Ref subclones revealed, as also indicated by their restriction maps, that the two genomic clones, et1-R 10.2 and et1-R 18.1, contained overlapping regions (Fig. 3.13) which were completely homologous. Therefore, it indicated that these two clones belonged to one locus, while the genomic λ clone et1-R 1.1 probably belonged to another locus. These two classes of clones were then analysed separately.

Sequence analysis of the et1-R 1.1 λ clone revealed the presence of only a part of the et1 gene. However, the λ clone also contained a Mu8 element upstream of the start codon at the same position as the et1-m3 mutant allele. A comparison of the 2kb Sal I sequence of the et1-R 1.1 λ clone to that of et1-m3/+(B73) wild type alleles (da Costa e Silva et al., 2001) revealed that on leaving out the Mu8 sequence from

et1-R 1.1, the sequence was completely identical to the wild type 2.5 kb et1 sequence. The restriction map of one of the et1-m3 mutant clones, λ 5.1 (da Costa e Silva et al., 2001), was also found to be similar to that of et1-R 1.1 (Appendices A and C). In addition, on carrying out sequence analysis, the sequences of both their λ clone ends were found to be the same (data not shown).

In an attempt to isolate a λ clone belonging to λ et1-R 1.1 locus, containing a complete et1 sequence, an et1-Ref genomic library was screened using c9.1 and Mu8-specific probes. However, no clones hybridising with both the probes could be isolated. The clones isolated with c9.1 were found to belong to the same class as the other two et1-Ref clones, et1-R 10.2 and et1-R 18.1.

Thus, in order to clarify the origin of λ et1-R 1.1, a Mu-primer based AIMS experiment was performed using genomic DNA from LC as a negative control and that from et1-m3/et1-Ref and m15/Ref mutants as positive controls. The homozygous et1-Ref and et1-et1-Ref/+(B73) genomic DNA were taken as test samples. The AIMS blots were hybridised with the LC et1 cDNA, c9.1, as well as the 2.5 kb et1 fragment probes. No Mu associated amplified bands in et1-Ref samples hybridised to either of the probes (data not shown), whereby, if the λ et1-R 1.1 clone had originated from et1-Ref genome, both the probes should have shown hybridisation signals with et1-Ref samples. Only the positive controls, et1-m3/et1-Ref and et1-m15/et1-Ref, showed two bands each of the expected size.

Based on these analyses, the et1-R 1.1 clone was no longer regarded as an et1-Ref clone, but a contaminant, which might be coming from the et1-m3 genomic library.

Thus, the λ clones et1-R 10.2 and et1-R 18.1 were classified as Class II et1-Ref clones and will be referred to as such from here onwards.

The sequence analysis of these Class II et1-Ref clones, i.e., et1-R 10.2 and et1-R 18.1, and their comparison to the available sequence data from the wild type et1 genomic clones and the et1 cDNA clones showed the presence of an ORF and a putative gene with high homology to the et1 gene (Fig. 3.14). The homology was however, mainly restricted to the coding region. Just like et1, four exons and three introns could be identified from the sequence. Although the average value of homology was only 60% in this region, the exons were much more homologous than

the introns. Comparison of the exons of both the genes showed 78 to 79 % homology. The second intron in this gene was found to be much larger than that of et1, so that about 45% of et1 intron sequence in this region was gapped. Although the remaining sequences were largely homologous, a number of small differences were observed between the two sequences throughout the coding region.

The upstream proximal region of the gene showed almost no homology to that of et1 from either LC or B73. Similarly, only a little homology could be observed between the two downstream flanking sequences of the genes, which decreased further on moving farther downstream.

Therefore, comparison of the restriction patterns and the sequence analyses of the two genes indicated that they were two different genes showing high homology to each other. The gene represented by the Class II clones was named as zmzr1 (Zea mays zinc ribbon 1) and will be referred to as such from here onwards. Whether the gene was functional still remained to be studied.

Fig. 3.14: The genomic structure of the new gene, zmzr1, obtained through sequencing of Class II genomic λ clones et1-R 10.2 and et1-R 18.1 from et1-Ref line, and its comparison to et1.

The zmzr1 gene is aligned with the et1 gene from LC and B73 lines. The genes are represented in the 5´–> 3´ orientation. The exons are represented as darker boxes and intron boxes with lighter colours.

Their lengths in base pairs (bp) have been shown either below and above the exons or within the intron boxes. The exons have been labelled using roman numerals within the boxes.

LC et1 B73 I

I

II II

III III

IV

173 IV

164 75

88 670

129 145

281

75 670 145

zmzr1

215 79 1167

129 154

I 70 II III 279 IV

200 bp Introns

: Exons

: