Expression analysis of σ1 isoforms

Upon cloning and characterisation of σ1A and σ1B, both genes were reported to be similarly expressed in a number of tissues (Peyrard et al., 1998; Takatsu et al., 1998). No data were available concerning the expression ofσ1C, which was identified during the second year of this study by genome sequencing projects. To examine if the three genes are ubiquitously expressed, we detected the respective RNA by PCR amplification of cDNA as well as by northern blot hybridisation.

Total RNA was isolated from various tissues (2.2.1.5) and converted to cDNA by reverse transcription with an oligo-dT-primer (see 2.2.1.6). Transcripts for σ1-adaptins were amplified by use of specific oligonucleotide primers selected from the untranslated region of the mRNA. To verify the integrity of the cDNA preparation, hypoxanthine-phosphoribosyltransferase (hprt) was amplified as a control. 20 µl of each PCR sample were loaded on a 2% agarose gel (fig. 3.3).

Figure 3.3:Detection of σ1-adaptins by PCR. σ1A,σ1B and σ1C cDNA were amplified from total cDNA of different tissues and loaded on an agarose gel. Amplification of the housekeeping gene hprt served as a control for cDNA quality (last row). The approximate size of the PCR products is indicated. For primer sequences see 2.1.8.

The PCR forσ1A produced a 500 bp band from all cDNA preparations which were tested (fig. 3.3, first row). In contrast, the σ1B PCR was positive from cDNA of most tissues, but no band or only a very faint band was visible in kidney and spleen samples. Apart from the expected band at 500 bp, an additional product was seen at about 600 bp in the samples of skeletal muscle, heart and thymus. This may correspond to another σ1B mRNA form generated by alternative splicing.

Similar results as for σ1B were obtained for σ1C. Whereas the expected band of about 500 bp could be amplified from the majority of cDNA preparations, the level of σ1C cDNA seemed too low for amplification from testis, skeletal muscle, heart and fat tissue. An additional band at about 350 bp was produced from cDNA of liver and thyroid gland.

These data indicate that σ1A mRNA is expressed in all the tissues examined at a detectable level, whileσ1B andσ1C transcripts are abundant only in certain tissues.

Since the amplification by PCR does not reflect the absolute levels of RNA, and its efficiency can be influenced bye.g., chemical contaminants of the RNA preparation, it was necessary to confirm the results by northern blot.

For preparation of northern blots, 10 µg RNA were separated by agarose gel elec-trophoresis and transferred to a nylon membrane (2.2.1.8). The northern blots were hybridised with radioactively labelled DNA fragments comprising exons 2-4 ofσ1A, -B and exons 3-5 of σ1C, respectively (fig. 3.4). Despite the homologous

organisa-3.1 The σ1-adaptin family

Figure 3.4:Northern blot hybridisation with probes for σ1A, -B and C.

10 µg RNA were loaded except for ventral (4 µg) and intrascapular fat (8µg). The position of 18S (1.9 kb) and 28S rRNA (4.7 kb) is indicated.

apparent size. The σ1A probe detected an RNA transcript smaller than 1.9 kb. In one out of four experiments, there was also a signal for a larger RNA species ofσ1A.

Hybridisation with the σ1C probe labelled an RNA species larger than 4.7 kb. In contrast, σ1B was always seen as two bands from fibroblasts, one above 4.7 kb and one between 1.9 and 4.7 kb. Severalσ1B RNA species of different size were detected in testis, while the size ofσ1A andσ1C RNA appeared similar to the other tissues.

All the northern blots were hybridised with hprt cDNA as a control probe to compare hybridisation signals between tissues and on different membranes. Accordingly, each signal was standardised to the hprt signal (fig. 3.5).

As confirmed by their hybridisation pattern, σ1-adaptin genes are expressed in a tissue-specific manner. Whereas all the three genes were detected at a comparable level in e.g., mouse embryonic fibroblast or lung RNA, testis and brain apparently contained mainly σ1A and -B transcripts. σ1A and σ1C mRNA were dominant over σ1B in intestine and σ1C was the most abundant isoform in thyroid gland. In contrast, the signal forσ1B was stronger than the others in skeletal muscle and heart.

RNA from intrascapular fat was chosen due to the high fraction of brown adipose tissue, whereas ventral fat consists of white adipose tissue only. Both were labelled with very low intensity by all the probes and did not show any clear preference among the isoforms.

Since it is unclear whether σ1A, -B and -C preferentially associate with either γ1 orγ2 or with both, we were interested to know if the expression of γ-adaptin genes

Figure 3.5:Expression profile ofσ1A, -B and -C mRNA.Northern blot signals were quantified and normalised to the signal intensity of hprt.

Figure 3.6:Northern blot of γ1- and γ2-adaptin. The position of 18S (1.9 kb) and 28S rRNA (4.7 kb) is indicated.

3.1 The σ1-adaptin family would show a tissue-specific pattern. For this purpose, the tissue northern blots were decorated with cDNA probes comprising hinge and ear region of γ1 or γ2 (fig.3.6).

For both genes, mRNA species of two different sizes were detected in most tissues, both longer than 4.7 kb forγ1 and one longer, one shorter than 1.9 kb for γ2. Both γ1 and γ2 were expressed in all tissues examined, in contrast to σ1 isoforms. The total level of γ-adaptin RNA appeared comparable between the different tissues.

But interestingly, the signal for γ2 was much stronger than that for γ1 in skeletal muscle RNA, which may indicate a closer relationship ofσ1B and γ2 in this tissue.