TSEP22 Genomic Analysis

3.7.2.1. Analysis of the TSEP22 Cosmid Clones

The mouse genomic DNA cosmid clones 2112 and 1277 showed different restriction patterns with BamHI. Therefore, a PCR analysis approach was designed in order to identify the TSEP22 gene sequence present in the cosmid clones. This approach was based on the different sequence blocks found in the cDNA clones (Fig. 3.31.). According to this, five different PCR products could be identified: the primer combination P1-36RRT amplified from sequence B to the 3´region of sequence E. P2-36RRT amplified from the 3´region of sequence A2 to the 3´of E. P3-36RRT comprised from the 5´region of A2 to end of E. P4-36RRT amplified from sequence D to E and P5- amplified a small part in the middle of sequence E (Fig. 3.34.).

Fig. 3.34. Distribution of the PCR primers along the TSEP22 cDNA sequence.

Here is shown the localisation of the different primers in the TSEP22 cDNA sequence designed to analyse the mouse genomic DNA cosmid clones. The arrows indicate the location and direction of the primers in the cDNA sequence.

After performing the five different PCR reactions (P1, P2, P3; P4 and P5) with the DNA of the two cosmid clones (1277 and 2112), it was found that the cosmid 2112 contained almost the complete sequence corresponding to the cDNA clone O07 which had the longest cDNA sequence (Fig. 3.35).

Only a short sequence on 3´region of E was absent. As long as the PCR products P1, P2 and P3 were longer around 100 bp of expected, an intron of 103 bp between the sequence blocks C and D could be deduced by sequencing. The cosmid 1277 proved to have only a part of sequence E.

P1P2P3P4

M P1P2P3P4 M

Cosmid 1277 Cosmid 2112 Cosmid 1277 Cosmid 2112

1,0

Fig. 3.35. PCR analysis of the cosmids 1277 and 2112.

In this figure, the agarose gels with the PCR products of the cosmids 1277 and 2112 are shown. A: PCR products obtained with the forward primers P1 to P4 and the reverse primer 36T35´ with both cosmids.

It can be observed that these primer combinations yield some bands of around 0.6 and 0.5 kb in cosmid 2112 and no bands in cosmid 1277. When the primer pair 5F/P5R are used, a 0.1 kb band can be seen with the cosmid 1277, whereas no band is detectable with the cosmid 2112. This PCR product corresponds to the size of the cDNA meanwhile the length of the PCR products obtained with the forward primers P1, P2 and P3 were bigger. This is due to a 103 bp intron found by sequencing between the sequence blocks C and D (B).

3.7.2.2. Southern blot Analysis and Copy Gene Determination

A restriction analysis of TSEP22 in mouse genomic DNA was performed and compared with the restriction patterns obtained with the cosmid clones. For this, 20 µg of mouse male genomic DNA were restricted with BamHI, EcoRI, and XbaI, separated in an horizontal gel and blotted to a nitrocellulose membrane. The filter was hybridized with the 657 bp TSEP22 cDNA at 65°C. The band pattern obtained in the mouse genomic DNA Southern blot was compared to those of the mouse cosmid clones (Fig. 3.36.). In this case, the genomic DNA restriction pattern can be explained by the addition of the bands from both cosmid clones. Even though, there are some differences observed in the BamHI restriction, like the 6 kb band present in the genomic DNA that cannot be found in the cosmid clones.

Also, a 12 kb band of the cosmid clone 2112 is not present in the genomic DNA pattern.

An incomplete digestion explains the unclear bands obtained in the genomic DNA restriction with XbaI.

Fig. 3.36. TSEP22 Southern blot of mouse genomic and cosmid DNA.

The figure shows the films of the Southern blot with mouse genomic DNA and from cosmid 1277 and 2112 hybridised with the TSEP22 cDNA. It can be observed that the signals present in the cosmids correspond only partially to the signal pattern of the genomic Southern. Nonetheless, the pattern of both restricted cosmids taken together explains the pattern of the genomic DNA, only with two exceptions:

the BamHI 6 kb band present in the genomic DNA cannot be found in the cosmid clones, as well as a 12 kb band of the cosmid clone 2112 is not present in the genomic DNA pattern.

3.7.2.3. TSEP22 Chromosomal Localisation

The chromosomal localisation of the mouse TSEP22 gene was performed by Fluorescence In Situ Hybridisation (FISH) with help from Dr. B. Gläser (Institut für Humangenetik, Göttingen). For this purpose, the cosmid 2112 was used as a probe. Mouse TSEP22 was localized in the telomeric region of chromosome 12 (Fig. 3.37.). This region is synthenic to the human chromosome 2pter-25. When this human region was analysed in search for diseases and genes involved with reproduction or spermatogenesis, nothing was found.

Fig. 3.37. Chromosomal localisation of the TSEP22 gene.

The picture shows the FISH hybridisation to localise the TSEP22 mouse gene using the cosmid 2112 as a probe. The TSEP22 gene was located to mouse chromosome 12 in the telomeric region. No genes involved in reproduction or spermatogenesis could be found in this region.

3.7.2.4. TSEP22 Genomic Structure

The TSEP22 genomic structure had been attempted to elucidate by subcloning the cosmid fragments resulting from a digestion with different endonucleases such as BamHI, EcoRI and PstI, into the pZERO 2.0 vector. Nonetheless, the sequencing of the genomic subclones was abandoned, because two mouse genomic clones (GI: AC073562 and AC079272) were found after a comparison in the sequence database BLAST of the NCBI (http://www.ncbi.nlm.nih.gov/BLAST/). These genomic clones contained the complete gene, for this reason, the sequencing of the pZERO genomic subclones was stopped. The mouse genomic clones published in Genbank localized to the chromosome 12pter

(sequence in Appendix). The transcribed sequence was comprised in a genomic sequence of 16 kb distributed in three exons separated by two introns of 103 and 13,160 bp, respectively (Fig. 3.38.). The different cDNA sequences arise from alternative splicing of the hnRNA from the exons as well as from internal exon sequences, which presented inner acceptor-donor splicing sites. An exception is the sequence for the sequence block B, which had the sequence of the first 24 bp of TSEP22. In this case, no acceptor-donor splicing site was found. Three GC boxes that lie at the positions 21, 40 and 492 of the first exon were found and can probably be involved in the transcription regulation of the TSEP22 gene.

Fig. 3.38. Genomic structure of the TSEP22 gene.

The scheme A represents the genomic structure of the TSEP22 gene deduced from the cDNA sequence together with the database information. The restriction sites of BamHI (B), EcoRI (E), HindIII (H), PstI (P) and XbaI (X) are drawn as a reference. The first intron is 103 bp long and the second intron is 13.16 kb. B and C: It is schematically represented how the hnRNA can originate the 5 different TSEP22 cDNAs.