Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2001 Oct;12(10):3114-25.
doi: 10.1091/mbc.12.10.3114.

Serial analysis of gene expression in Plasmodium falciparum reveals the global expression profile of erythrocytic stages and the presence of anti-sense transcripts in the malarial parasite

S Patankar  1 , A Munasinghe, A Shoaibi, L M Cummings, D F Wirth
Affiliations
Free PMC article

Serial analysis of gene expression in Plasmodium falciparum reveals the global expression profile of erythrocytic stages and the presence of anti-sense transcripts in the malarial parasite

S Patankar et al. Mol Biol Cell. 2001 Oct.
Free PMC article

Abstract

Serial analysis of gene expression (SAGE) was applied to the malarial parasite Plasmodium falciparum to characterize the comprehensive transcriptional profile of erythrocytic stages. A SAGE library of approximately 8335 tags representing 4866 different genes was generated from 3D7 strain parasites. Basic local alignment search tool analysis of high abundance SAGE tags revealed that a majority (88%) corresponded to 3D7 sequence, and despite the low complexity of the genome, 70% of these highly abundant tags matched unique loci. Characterization of these suggested the major metabolic pathways that are used by the organism under normal culture conditions. Furthermore several tags expressed at high abundance (30% of tags matching to unique loci of the 3D7 genome) were derived from previously uncharacterized open reading frames, demonstrating the use of SAGE in genome annotation. The open platform "profiling" nature of SAGE also lead to the important discovery of a novel transcriptional phenomenon in the malarial pathogen: a significant number of highly abundant tags that were derived from annotated genes (17%) corresponded to antisense transcripts. These SAGE data were validated by two independent means, strand specific reverse transcription-polymerase chain reaction and Northern analysis, where antisense messages were detected in both asexual and sexual stages. This finding has implications for transcriptional regulation of Plasmodium gene expression.

PubMed Disclaimer

Figures

Figure 1
Figure 1
BLASTx analysis of highly abundant SAGE tags. SAGE tags from the 3D7 library were analyzed with the use of the SAGE software (as described in MATERIALS AND METHODS). SAGE tags at an abundance level of five counts or greater were further analyzed as depicted in the flow chart. The percentage of single matches, no matches, and multiple matches to the databases are indicated. Numbers in brackets correspond to the proportion of tag counts in each group. Single matches were divided into tags that matched annotated and unannotated sequence reads. Tags in the latter group were characterized by BLASTx. Fourteen bp tags that failed to match either database were analyzed with the use of only the first 13 bp of the tag sequence.
Figure 2
Figure 2
SAGE 3D7 library analysis. (A) Cumulative total gene representation within the 3D7 SAGE library. Ascertained tags (from the 3D7 library) at increasing increments were plotted against the number of unique genes from which the tag subsets were derived. The solid line corresponds to all ascertained tags. The dotted line corresponds to the number of unique genes represented by tags at an abundance level of two counts or greater (some tags at an abundance level of one may be derived from sequencing errors). (B) SAGE abundance classes. SAGE tags (8335) are divided into abundance classes (percent abundance [first column] refers to the abundance of tags as a percentage of all 8335 tags in the SAGE library). The number of unique tags matching an entry in the P. falciparum National Center for Biotechnology Information database is listed per abundance class (last column), and the percentage of hits within the abundance class is given in brackets. ND, not determined.
Figure 3
Figure 3
Categories of highly expressed genes in the 3D7 control population. Highly abundant tags (tag counts of >0.05% of the total number of tags) were examined for their matches in P. falciparum databases (see analysis described in Figures 1 and 2) and categorized by putative functions. The number of genes in each category is depicted.
Figure 4
Figure 4
Validation of SAGE data by RT-PCR and Northern analysis (A) RT-PCR of genes represented in the 3D7 SAGE library. RT-PCR products generated by specific primers for pfg27/25 (lanes 1 and 2), rap-1 (lanes 3 and 4), msp-3 (lanes 5 and 6), and calmodulin (lanes 7 and 8) are shown. RT minus samples were also PCR amplified as negative controls (lanes 2, 4, 6, and 8). pBR322 MspI digest (lane M). (B) Summary of expression data. For Northern analysis 1 μg of mRNA from 3D7 cultures was gel electrophoresed, blotted onto a nylon membrane, and probed with gene specific 32P-labeled DNA probes. + indicates that a specific signal corresponding to each transcript was detected in the Northern analysis. Tag counts from the 3D7 library (8335 tags) for all four genes are listed. Quantitative Northern analysis was carried out for calmodulin and msp-3. Northern blots and gene-specific probes were prepared as described above. Known amounts of in vitro synthesized RNA fragments were run alongside the mRNA sample as markers for quantification. Signal intensities for each of the transcripts in the mRNA sample were converted to molar amounts by reference to those of the synthetic RNAs.
Figure 5
Figure 5
Validation of antisense SAGE data by strand-specific RT-PCR and strand-specific Northern analysis in asexual stage parasites. (A) Strand-specific RT-PCR analysis. First-strand cDNA from asexual stages was synthesized with a primer that specifically hybridizes to either antisense message (A) for calmodulin (lanes 1 and 2), rap-1 (lanes 5 and 6), msp-3 (lanes 9 and 10), and hsp-86 (lanes 13 and 14); or a primer that binds to sense message (S) for calmodulin (lanes 3 and 4), rap-1 (lanes 7 and 8), msp-3 (lanes 11 and 12), and hsp-86 (lanes 15 and 16). RT-PCR was performed on these cDNA samples with the use of the same primer pair for calmodulin (lanes 1–4), rap-1 (lanes 5–8), msp-3 (lanes 9–12), and hsp-86 (lanes 13–16). Products in lanes 1, 3, 5, 7, 9, 11, and 15 were cloned to confirm gene identity. + indicates that the gel product corresponded to the specific gene under investigation. In some cases, nonspecific PCR products were also generated, in addition to the specific product for the particular gene under study. RT minus samples were also PCR amplified as negative controls (lanes 2, 4, 6, 8,10, 12, 14, and 16). 100-bp ladder (NEB), lane M1; low DNA mass ladder (Invitrogen), lane M2. Genes for which antisense transcripts were found in the SAGE library are indicated by yes or no (last row). Products for msp-3 (lanes 9–12) were generated in a separate experiment. (B) Strand-specific Northern analysis. Synthetic sense (S) RNA fragments (lanes 1 and 4 for calmodulin; lanes 7 and 10 for msp-3), synthetic antisense (A) RNA fragments (lanes 2 and 5 for calmodulin; lanes 8 and 11 for msp-3), or 20 μg of total RNA from 3D7 cultures (lanes 3, 6, 9, and 12) were gel electrophoresed. Blots were transferred onto nitrocellulose membranes and probed with 32P-labeled sense (S) RNA probes (calmodulin, lanes 1–3; msp-3, lanes 7–9) or antisense (A) RNA probes (calmodulin, lanes 4–6; msp-3, lanes 10–12). Northern blots were exposed to x-ray film; optimal exposure times for signals from synthetic RNA fragments were 5–10 s, whereas those for total RNA were 5–10 min. Adobe Photoshop was used to make a composite image of these x-ray films.
Figure 6
Figure 6
Strand-specific RT-PCR analysis of pgs28 in sexual-stage parasites. First-strand cDNA from sexual stages was synthesized with a primer that specifically hybridizes to either antisense message (A) for pgs28 (lanes 1, 2, 5, 6, 9, and 10), or a primer that binds to sense message (S) for pgs28 (lanes 3, 4, 7, 8, 11, and 12). cDNA was synthesized from total RNA obtained from purified gametes and zygotes immediately (lanes 1–4), 24 h (lanes 5–8), and 48 h (lanes 9–12) after isolation. RT-PCR was performed on all samples with the use of the same primer pair. RT minus samples were also PCR amplified as negative controls (lanes 2, 4, 6, 8,10, and 12). pBR322 MspI digest (lane M).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Assaraf YG, Golenser J, Spira DT, Bachrach U. Polyamine levels and the activity of their biosynthetic enzymes in human erythrocytes infected with the malarial parasite Plasmodium falciparum. Biochem J. 1984;222:815–819. - PMC - PubMed
    1. Barnwell JW, Galinski MR. Invasion of vertebrate cells: erythrocytes. In: Sherman IW, editor. Malaria: Parasite Biology, Pathogenesis, and Protection. Washington, DC: ASM Press; 1998. pp. 93–120.
    1. Bowman S, Lawson D, Basham D, Brown D, Chillingworth T, Churcher CM, Craig A, Davies RM, Devlin K, Feltwell T, Gentles S, Gwilliam R, Hamlin N, Harris D, Holroyd S, Hornsby T, Horrocks P, Jagels K, Jassal B, Kyes S, McLean J, Moule S, Mungall K, Murphy L, Barrell BG, et al. The complete nucleotide sequence of chromosome 3 of Plasmodium falciparum. Nature. 1999;400:532–538. - PubMed
    1. Butler D. Funding assured for international malaria sequencing project. Nature. 1997;388:701. - PubMed
    1. Chen Q, Fernandez V, Sundstrom A, Schlichtherle M, Datta S, Hagblom P, Wahlgren M. Developmental selection of var gene expression in Plasmodium falciparum. Nature. 1998;394:392–395. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources