Figure 1-Total RNA product after DNase treatment. 100 bp ladder (Lane 1) and DNase treated RNA extraction product (Lane 2). RNA isolation and DNase treatment was performed according to the Methods and Materials. RNA products were visualized by ethidium bromide staining and run on an agarose gel.
Figure 2-PCR amplification using various primer combinations. PCR amplification was performed as stated in the Methods and Materials. A 1.5 kb faint band was visualized (Lane 1) on the UV bed but cannot be seen in the picture. 100 bp ladder (Lane 9) and actin control (Lane 8). No products appeared in Lanes 2,3,4,5,6 and 7.
Figure 3-Nesting reaction using the 1.5 kb PCR product as a template for various primer combinations. Three bands of predicted size were visualized in this reaction. A 1.4 kb product (Lane 4), a 400 bp product (Lane 6), a 900 bp product (Lane 7). 100 bp ladder (Lane 1). No products appeared in Lanes 2,3, and 5.
Figure 4-Sequence alignment of the derived goldfish SRP54 sequence and the human SRP54 sequence from GenBank. Sequences were aligned using MacVector and the ClustalW Alignment Format.
RNA extraction was performed on the gills of goldfish. The product from the isolation yielded three distinct bands (data not shown). The first band visualized is DNA in the sample. The second band represents 28S RNA and the third band from the top is 18S RNA. Following DNase treatment the first band was eliminated (Fig 1, Lane 2) while the 28S and 18S bands remained. The DNase treated RNA was subjected to RT-PCR. The resulting cDNA was then PCR amplified using various combinations of forward and reverse primers. A faint band, representing the predicted 1.5 kb product, was detected in the reaction using SRP54-1 and SRP54-6 primers (Fig 2, Lane 1). This band does not show up in the picture but was visualized when the gel was placed on a UV bed. An actin positive control was used to verify the template had not degraded and produced a band. The actin control migrated correctly and is approximately 600 bp in length (Fig 2, Lane 8).
The next step taken was to run a nesting reaction, because of the low yield result from the initial PCR amplification. Primers that should sit on the inside of the 1.5 kb PCR product were used in a PCR amplification reaction. The PCR product itself was used as the template in the reaction. Several bands of predicted size were visualized from this PCR amplification (Fig 3). A PCR product approximately 1.4 kb in length was produced using primers SRP54-2 and SRP54-6 (Fig 3, Lane 4). Another PCR product approximately 400 bp in length was produced using primers SRP54-2 and SRP54-4 (Fig 3, Lane 6). Additionally, a third PCR product was produced approximately 900 bp in length, using primers SRP54-2 and SRP54-5 (Fig 3, Lane 7).
A sequencing reaction was then performed as described in the Methods and Materials. The resulting nucleotide sequences were aligned to the human SRP54 sequence from GenBank. The sequences aligned to the human SRP54 sequence at the predicted regions in relation to the primers used. Gaps in the sequence were filled by primer walking, using primers SRP54-7 and SRP54-8. By piecing together the resulting partial sequences, 1370 bps of the derived sequence aligned to the human SRP54 sequence with a 70% homology (Fig 4). The start codon for the human sequence is located at bp 228 while the stop codon is located at bp 1742.
There is a relatively high homology between the goldfish SRP54 sequence reported above and the published human sequence. There are still gaps in the beginning and the end of the goldfish sequence that need to be resolved but this should be possible in the future when the zebrafish project has been completed. The partial sequence of the zebrafish that was available for this experiment was only 600 bp and covered the beginning of the coding region. This made it difficult to develop primers that would span the entire coding region. With more time, the unknown bases in the sequencing reaction can be determined. Due to the presence of these unknown bases, translation of the amino acid sequence was not possible. With the amino acid sequence available it would be possible to determine if the goldfish SRP54 gene codes for the same methionine rich domain as its mammalian counterpart.
The author would like to express sincere thanks to Sabrina Volpi, Rocco Coli, and Ira Daly, without whose support this project could not have been completed. The author also thanks Dr. Berish Y. Rubin for his help with developing the idea for the project and allowing the use of his lab.
1. Lodish, H., A. Berk, S.L. Zipursky, P. Matsudaira, D. Baltimore, and J.E Darnell. 2000. Molecular Cell Biology 4th edition. W.H. Freeman and Co. New York, NY.
2. Rapiejko P.J. and R. Gilmore. 1994. Signal sequence recognition and targeting of ribosomes to the endoplasmic reticulum by the signal recognition particle do not
require GTP. Mol Biol Cell. 5: 887-897.
3. Bernstein, H.D., M.A. Poritz, K. Strub, P.J. Hoben, S. Brenner, and P. Walter. 1989. Model for signal sequence recognition from amino-acid sequence of 54K subunit of
signal recognition particle. Nature. 340: 482-486.
4. Gowda, K., S.D. Black, I. Moeller, Y. Sakaibara, M.C. Liu,and C. Zweib. 1998. Protein SRP54 of human signal recognition particle: cloning, expression, and comparative analysis of functional sites. Gene. 207: 197-207.
5. K. Gowda, K. Chittenden, and C. Zweib. 1997. Binding site of the M-domain of human protein SRP54 determined by systematic site-directed mutagenesis of signal recognition particle RNA. Nucleic Acid Res. 25: 388-394.
6. Romisch, K., J. Webb, J. Herz, S. Prehn, R. Frank, M. Vingron, and B. Dobberstein. 1989. Homology of 54K protein of signal-recognition particle, docking protein and
two E. coli proteins with putative GTP-binding domains. Nature. 340: 478-482.
7. Bacher, G., H. Lutcke, B. Jungnickel, T.A. Rapoport, and B. Dobberstein. 1996. Regulation by the ribosome of the GTPase of the signal-recognition particle during Protein targeting. Nature. 381: 248-251.
8. Rapiejko, P.J. and R. Gilmore. 1997. Empty site forms of the SRP54 and SRalpha GTPases mediate targeting of ribosome-nascent chain complexes to the endoplasmic recticulum. Cell. 89: 703-713.
9. Patel, S. and B. Austen. 1996. Sequence of the highly conserved gene encoding the human 54kDa subunit of signal recognition particle. 1996. 6: 167-170.
10. Trianedes, K., D.M. Findlay, T.J. Martin, M.T. Gillespie. 1995. Modulation of the signal recognition particle 54-kDa subunit (SRP54) in rat preosteoblasts by the extracellular matrix. J. Biol. Chem. 8: 20891-20894.
11. Adams, M.D. et al. 2000. The genome sequence of Drosophila melanogaster. Science. 287: 2185-2195.
12. No authors listed. 1998. Genome sequence of the nematode C. Elegans: a platform for investigating biology. The C. Elegans Sequencing Consortium. Science. 282: 2012-8.
13. Zopf, D., H.D. Bernstein, A.E. Johnson, and P. Walter. 1990. The methionine-rich domain of the 54 kd protein subunit of the signal recognition particle contains an
RNA binding site and can be cross-linked to a signal sequence. EMBO. 9: 4511-7.
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