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Experimental RNomics in Aquifex aeolicus: identification of small non-coding RNAs and the putative 6S RNA homolog.

Willkomm DK, Minnerup J, Hüttenhofer A, Hartmann RK - Nucleic Acids Res. (2005)

Bottom Line: The most abundant intergenic RNA of the library was identified as the 6S RNA homolog of A.aeolicus.We identifed novel 6S RNA candidates within the gamma-proteobacteria but were unable to identify reasonable 6S RNA candidates in other bacterial branches, utilizing mfold analyses of the region immediately upstream of ygfA combined with 6S RNA blastn searches.By RACE experiments, we mapped the major transcription initiation site of A.aeolicus 6S RNA primary transcripts, located within the pheT gene preceding ygfA, as well as three processing sites.

View Article: PubMed Central - PubMed

Affiliation: Philipps-Universität Marburg, Institut für Pharmazeutische Chemie Marbacher Weg 6, D-35037 Marburg, Germany.

ABSTRACT
By an experimental RNomics approach, we have generated a cDNA library from small RNAs expressed from the genome of the hyperthermophilic bacterium Aquifex aeolicus. The library included RNAs that were antisense to mRNAs and tRNAs as well as RNAs encoded in intergenic regions. Substantial steady-state levels in A.aeolicus cells were confirmed for several of the cloned RNAs by northern blot analysis. The most abundant intergenic RNA of the library was identified as the 6S RNA homolog of A.aeolicus. Although shorter in size (150 nt) than its gamma-proteobacterial homologs (approximately 185 nt), it is predicted to have the most stable structure among known 6S RNAs. As in the gamma-proteobacteria, the A.aeolicus 6S RNA gene (ssrS) is located immediately upstream of the ygfA gene encoding a widely conserved 5-formyltetrahydrofolate cyclo-ligase. We identifed novel 6S RNA candidates within the gamma-proteobacteria but were unable to identify reasonable 6S RNA candidates in other bacterial branches, utilizing mfold analyses of the region immediately upstream of ygfA combined with 6S RNA blastn searches. By RACE experiments, we mapped the major transcription initiation site of A.aeolicus 6S RNA primary transcripts, located within the pheT gene preceding ygfA, as well as three processing sites.

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Composition of the A.aeolicus cDNA library (clones identified by sequencing and/or dot blot hybridization). (A) Number of clones in the different categories. Unclear assignment: sequences with several or no matches to the A.aeolicus genome, mostly due to shortness of the respective sequence. (B) Antisense and intergenic sequences categorized in more detail by genetic context.
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fig1: Composition of the A.aeolicus cDNA library (clones identified by sequencing and/or dot blot hybridization). (A) Number of clones in the different categories. Unclear assignment: sequences with several or no matches to the A.aeolicus genome, mostly due to shortness of the respective sequence. (B) Antisense and intergenic sequences categorized in more detail by genetic context.

Mentions: Among the 1042 cDNA library clones, 994 yielded either dot blot signals and/or analyzable sequence information (Figure 1A): 80 out of the 994 clones represented either very short sequence stretches or non-Aquifex sequences, predominantly originating from the pSPORT 1 vector. Of the remaining 914 clones, the majority (68%) corresponded to fragments of ribosomal RNAs (5S, 16S and 23S rRNA). The most prevalent group of non-ribosomal RNAs then were intergenic RNAs, with 90 out of the 100 clones derived from the intergenic region between reading frames pheT and aq_1731. The group of intergenic RNAs was followed in number by tRNAs and their precursors (68), mRNAs (66), tmRNA (43) and, as the least abundant group of A.aeolicus RNAs within the cDNA library, 18 RNAs with antisense orientation to annotated reading frames. For a complete list of cDNA sequences, excluding rRNAs and tmRNA, see Table S1 of the Supplementary Material. Library members that represented intergenic and antisense RNAs were categorized according to their genetic context (Figure 1B), their genomic locations are illustrated in Figure 2A and B, and further details are listed in Table 1.


Experimental RNomics in Aquifex aeolicus: identification of small non-coding RNAs and the putative 6S RNA homolog.

Willkomm DK, Minnerup J, Hüttenhofer A, Hartmann RK - Nucleic Acids Res. (2005)

Composition of the A.aeolicus cDNA library (clones identified by sequencing and/or dot blot hybridization). (A) Number of clones in the different categories. Unclear assignment: sequences with several or no matches to the A.aeolicus genome, mostly due to shortness of the respective sequence. (B) Antisense and intergenic sequences categorized in more detail by genetic context.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1074721&req=5

fig1: Composition of the A.aeolicus cDNA library (clones identified by sequencing and/or dot blot hybridization). (A) Number of clones in the different categories. Unclear assignment: sequences with several or no matches to the A.aeolicus genome, mostly due to shortness of the respective sequence. (B) Antisense and intergenic sequences categorized in more detail by genetic context.
Mentions: Among the 1042 cDNA library clones, 994 yielded either dot blot signals and/or analyzable sequence information (Figure 1A): 80 out of the 994 clones represented either very short sequence stretches or non-Aquifex sequences, predominantly originating from the pSPORT 1 vector. Of the remaining 914 clones, the majority (68%) corresponded to fragments of ribosomal RNAs (5S, 16S and 23S rRNA). The most prevalent group of non-ribosomal RNAs then were intergenic RNAs, with 90 out of the 100 clones derived from the intergenic region between reading frames pheT and aq_1731. The group of intergenic RNAs was followed in number by tRNAs and their precursors (68), mRNAs (66), tmRNA (43) and, as the least abundant group of A.aeolicus RNAs within the cDNA library, 18 RNAs with antisense orientation to annotated reading frames. For a complete list of cDNA sequences, excluding rRNAs and tmRNA, see Table S1 of the Supplementary Material. Library members that represented intergenic and antisense RNAs were categorized according to their genetic context (Figure 1B), their genomic locations are illustrated in Figure 2A and B, and further details are listed in Table 1.

Bottom Line: The most abundant intergenic RNA of the library was identified as the 6S RNA homolog of A.aeolicus.We identifed novel 6S RNA candidates within the gamma-proteobacteria but were unable to identify reasonable 6S RNA candidates in other bacterial branches, utilizing mfold analyses of the region immediately upstream of ygfA combined with 6S RNA blastn searches.By RACE experiments, we mapped the major transcription initiation site of A.aeolicus 6S RNA primary transcripts, located within the pheT gene preceding ygfA, as well as three processing sites.

View Article: PubMed Central - PubMed

Affiliation: Philipps-Universität Marburg, Institut für Pharmazeutische Chemie Marbacher Weg 6, D-35037 Marburg, Germany.

ABSTRACT
By an experimental RNomics approach, we have generated a cDNA library from small RNAs expressed from the genome of the hyperthermophilic bacterium Aquifex aeolicus. The library included RNAs that were antisense to mRNAs and tRNAs as well as RNAs encoded in intergenic regions. Substantial steady-state levels in A.aeolicus cells were confirmed for several of the cloned RNAs by northern blot analysis. The most abundant intergenic RNA of the library was identified as the 6S RNA homolog of A.aeolicus. Although shorter in size (150 nt) than its gamma-proteobacterial homologs (approximately 185 nt), it is predicted to have the most stable structure among known 6S RNAs. As in the gamma-proteobacteria, the A.aeolicus 6S RNA gene (ssrS) is located immediately upstream of the ygfA gene encoding a widely conserved 5-formyltetrahydrofolate cyclo-ligase. We identifed novel 6S RNA candidates within the gamma-proteobacteria but were unable to identify reasonable 6S RNA candidates in other bacterial branches, utilizing mfold analyses of the region immediately upstream of ygfA combined with 6S RNA blastn searches. By RACE experiments, we mapped the major transcription initiation site of A.aeolicus 6S RNA primary transcripts, located within the pheT gene preceding ygfA, as well as three processing sites.

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