Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2001 Nov 15;29(22):4674-83.
doi: 10.1093/nar/29.22.4674.

The Abundance of Sterile Transcripts in Giardia Lamblia

Affiliations
Free PMC article

The Abundance of Sterile Transcripts in Giardia Lamblia

H G Elmendorf et al. Nucleic Acids Res. .
Free PMC article

Abstract

The protozoan parasite Giardia lamblia synthesizes a diverse and surprisingly abundant array of sterile transcripts unable to code for proteins. Random sampling of cDNAs from two evolutionarily divergent Giardia strains indicates that approximately 20% of cDNAs in the libraries represent polyadenylated sterile transcripts. RNase protection analysis and northern blot hybridization of three sterile transcript loci demonstrated that both the sterile transcript and a complementary mRNA were made in each case, further categorizing these sterile transcripts as antisense transcripts. Investigation of the genomic loci for these same three sterile antisense transcripts showed typical transcription units for the sense transcripts, but still failed to reveal a usable open reading frame for the sterile antisense transcripts. 5'-RACE mapped the transcription start site for one of the sterile antisense transcripts to an AT-rich region, as is typical for GIARDIA: It is unclear whether these sterile transcripts represent errors in transcription or whether they have regulatory functions within the cell, although preliminary investigations failed to reveal evidence for a role in developmental gene regulation. In either case, the presence of such a large pool of sterile antisense transcripts is dramatic evidence of the unusual molecular machinery of the early diverging protist G.lamblia.

Figures

Figure 1
Figure 1
Sterile antisense cDNAs in G.lamblia. Individual plaques were picked from cDNA libraries from two strains of Giardia, WB and GS. The cDNAs were amplified by PCR and fully sequenced on one strand. Open reading frames were analyzed in all six reading frames using DNA Strider 1.2. The sequences have been translated in all six reading frames, with F and R indicating forward and reverse strands, respectively. Half lines indicate ATG codons and full lines indicate stop codons. Possible ORFs longer than 300 nt are indicated by hatched boxes and labeled F# and R#. Arrows above and below the plots indicate the location of consensus polyadenylation signal sequences. (A) Sterile antisense cDNAs from the WB library. (B) Sterile antisense cDNAs from the GS library.
Figure 1
Figure 1
Sterile antisense cDNAs in G.lamblia. Individual plaques were picked from cDNA libraries from two strains of Giardia, WB and GS. The cDNAs were amplified by PCR and fully sequenced on one strand. Open reading frames were analyzed in all six reading frames using DNA Strider 1.2. The sequences have been translated in all six reading frames, with F and R indicating forward and reverse strands, respectively. Half lines indicate ATG codons and full lines indicate stop codons. Possible ORFs longer than 300 nt are indicated by hatched boxes and labeled F# and R#. Arrows above and below the plots indicate the location of consensus polyadenylation signal sequences. (A) Sterile antisense cDNAs from the WB library. (B) Sterile antisense cDNAs from the GS library.
Figure 2
Figure 2
Transcriptional maps of loci expressing sense and antisense RNAs. Genomic clones for the three cDNAs sequenced in Figure 1 were isolated from a Giardia strain WB genomic DNA library (44) and the loci sequenced. The sequences corresponding to the original cDNAs are indicated by long gray arrows preceded by open boxes. The transcription initiation sites were mapped by 5′-RACE and are indicated by the end of the black boxes (sense transcripts) or open boxes (antisense transcripts). Polyadenylation signals are indicated by open arrowheads. All other open reading frames greater than 100 amino acids and beginning with a methionine are indicated by short gray arrows and are labeled ‘ORF’s. These open reading frames have no similarity to known proteins and are labeled simply ORF. (A) Cysteine protease. (B) NOR. (C) ATP-dependent RNA helicase.
Figure 3
Figure 3
Sequence of the genomic locus for NOR. The sequence is shown as a single strand encoding the ‘forward’ direction; hence the original mRNA (the sterile, antisense transcript) would appear on this strand. The sequence complementary to the NOR gene is underlined. The AT-rich sequences surrounding the transcription start sites and the polyadenylation signals for both the sense and antisense transcripts are boxed and are as follows: transcription start site for sense transcript (AAAAAATG at 2040, start codon underlined); polyadenylation signal for sense transcript (AGTAAA at 1545, stop codon underlined); transcription start site for antisense transcript (AAAA at 510); polyadenylation signal for antisense transcript (TGTAAA at 2610).
Figure 4
Figure 4
RNase protection analysis of Giardia RNA. Lane M shows a 100 nt RNA ladder. The cDNAs analyzed in Figure 1 were used to generate 32P-labeled riboprobes. Riboprobes complementary to the antisense (A) or sense (S) strands of the cysteine protease, NOR or ATP-dependent RNA helicase loci were added to yeast RNA and left undigested as an indication of riboprobe size (-). To detect the presence of the antisense and sense transcripts, the riboprobes were added to yeast (Y) RNA or G.lamblia (G) polyadenylated trophozoite RNA and digested with a 1:100 dilution of RNase A + RNase T1, except for the NOR probe complementary to the sense strand, for which a 1:1000 dilution was used. Results are not quantitative since titrations of RNA were not used.
Figure 5
Figure 5
Northern blot analysis of sense and antisense transcripts. Oligonucleotides complementary to the cysteine protease antisense (A) or sense (S) transcripts or to the NOR antisense or sense transcripts were end-labeled with [γ-32P]ATP to equivalent specific activities and hybridized to either WB trophozoite (0 h) or encysting cell (24 h) polyadenylated RNA.

Similar articles

See all similar articles

Cited by 18 articles

See all "Cited by" articles

MeSH terms

Associated data

Feedback