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Cloning fibrillarin and snoRNA genes from Sulfolobus acidocaldarius

University of British Columbia

Recently, a Sulfolobus acidocaldarius RNA-protein complex with rRNA precursor 5' leader processing activity was purified and U3 snoRNA was found to be an essential component (Potter et al., 1995). Since fibrillarin is a universal snoRNA binding protein and has important functions in ribosome synthesis, this processing complex was suspected to contain fibrillarin. Fibrillarin gene of M. voltae was used as a heterologous probe to attempt to isolate Sulfolobus fibrillarin gene. By southern hybridizations, a 1.7 kb cross-hybridizing S. acidocaldarius DNA fragment was identified and subsequently isolated. Database searches indicated that it was not the fibrillarin gene. Some homology was found between this DNA fragment and the methanogen fibrillarin gene probe which may account for the southern signal. Since this 1.7 kb DNA fragment contains a 1.1 kb open reading frame, its sequence was deposited into genbank. PCR reactions using degenerate primers were also conducted to clone this gene. While PC R products at the expected lengths were obtained using M. voltae DNA as templates, negative results were obtained using S. acidocaldarius genomic DNA. Because the degenerate primer sequences were based on universally conserved fibrillarin sequences, the negative PCR result suggested that Sulfolobus may not have this gene. In order to detect the Sulfolobus fibrillarin protein directly, the methanogen fibrillarin protein was overexpressed in E. coli and used for raising polyclonal antibodies. In a western blot, this antibody can detect fibrillarin in methanogen cell lysates, but no protein the size of fibrillarin can be detected in Sulfolobus extracts. This antibody can also crossreact with a protein in a yeast cell lysate whose size is similar to that anticipated for yeast fibrilllarin (NOP1). Taken together, these observations suggest that Sulfolobus lacks a close fibrillarin homologue. Since fibrillarin antibodies can coimmunoprecipitate the snoRNAs associated with fibrillarin, they are a powerful tool in studying eukaryotic snoRNAs. Experiments have been initiated to use the methanogen fibrillarin antibody for immunoprecipitations of potential snoRNP s from the methanogen cell extract. Reverse transcription coupled PCR was used to clone other snoRNAs in addition to U3 that are possibly present in the Sulfolobus purified extract. Four cloned cDNA s were obtained but sequence analysis did not reveal homology to any known eukaryotic snoRNA. They are possibly RNA degradation products. Interestingly, one of the clones shares an eight nucleotide sequence that is identical to the S. acidocaldarius U3 box C sequence, a sequence present in most of the snoRNAs.

Thesis/Dissertation

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2009-02-16

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http://hdl.handle.net/2429/4629

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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