FULL-malaria: a database for a full-length enriched cDNA library from human malaria parasite, Plasmodium falciparum

Abstract

FULL-malaria is a database for a full-length-enriched cDNA library from the human malaria parasite Plasmodium falciparum (http://133.11.149.55/). Because of its medical importance, this organism is the first target for genome sequencing of a eukaryotic pathogen; the sequences of two of its 14 chromosomes have already been determined. However, for the full exploitation of this rapidly accumulating information, correct identification of the genes and study of their expression are essential. Using the oligo-capping method, we have produced a full-length-enriched cDNA library from erythrocytic stage parasites and performed one-pass reading. The database consists of nucleotide sequences of 2490 random clones that include 390 (16%) known malaria genes according to BLASTN analysis of the nr-nt database in GenBank; these represent 98 genes, and the clones for 48 of these genes contain the complete protein-coding sequence (49%). On the other hand, comparisons with the complete chromosome 2 sequence revealed that 35 of 210 predicted genes are expressed, and in addition led to detection of three new gene candidates that were not previously known. In total, 19 of these 38 clones (50%) were full-length. From these obser­vations, it is expected that the database contains ∼1000 genes, including 500 full-length clones. It should be an invaluable resource for the development of vaccines and novel drugs.

INTRODUCTION

Malaria is the most devastating parasitic disease in the world; it kills two million people every year. Thus, the causative agent, Plasmodium falciparum, has been the first target for genome sequencing of a eukaryote pathogen. Its 30 Mb genome with an unusually high AT content (80%) is divided into 14 chromosomes, for two of which the complete nucleotide sequence has already been deciphered (1,2) and the rest will be determined within a few years.

This parasite has a quite complex life cycle: a sexual stage in mosquitoes and an asexual stage in humans, with proliferation first in hepatocytes and then in erythrocytes. Analysis of gene expression at different stages is critical in understanding how this organism has adapted to two hosts.

Recently we have developed the ‘oligo-capping’ method to produce full-length enriched cDNA libraries (3,4). Briefly, it first replaces the cap structure at the 5′-end of eukaryotic mRNA with a synthetic RNA oligo primer, then uses an oligo–dT primer for production of cDNA with reverse transcriptase. After amplification by PCR using 5′- and 3′-end primers, cDNAs are digested with restriction enzymes and ligated to a vector in a directional manner. Though a huge clone has thereby been cloned from human tissue (4), large transcripts may be under-represented in the library, and 3′-end truncated clones may be included.

As our first attempt to produce a full-length cDNA library from P.falciparum, we focused on the erythrocytic stage parasites, which cause lethal symptoms. Plasmodium falciparum 3D7 strain, which was used for the genome sequencing project, was obtained from Dr Walliker, propagated in vitro as described previously (5) and used for the production of the library.

DATABASE DESCRIPTION

One-pass readings of the 5′-end nucleotide sequences of 3500 randomly picked clones were determined and analyzed using BLASTN. Two thousand four hundred and ninety reads constitute the first version FULL-malaria database. As shown in Table 1, it is possible to perform the BLAST search of the database with nucleotide sequences or by gene names. The whole sequence in the database can be retrieved using ftp. Full-length clone name lists will expand as the genome sequencing proceeds.

Table 1. Contents of the database.

About this database

Retrieve our sequence dataa

BLAST search of this databaseb

In silico mapping to finished chromosome sequencesc

List of full cDNA clonesd

Statistics of this databasee

Related sitesf

Acknowledgments

Who we are …

^aYou can display clone sequences in the database by annotation category, or search clones by BLAST definition. It is also possible to download the whole sequence data.

^bYou can search the database with a nucleotide sequence using BLASTN.

^cGraphical display of expression mapping to finished chromosome sequences.

^dList of the 5′-ends of full-length cDNA clones.

^eStatistics of database; clone numbers by definition and chromosome localization and full-ratio.

^fLink to related sites.

The summary of the characterization of the database is shown in Table 2. Three hundred and ninety sequences (16%) correspond to defined P.falciparum genes in GenBank. They represented 98 different genes, and complete protein coding sequences are included for 48 of these genes (49%). Comparison with the complete chromosome 2 sequence revealed that 143 clones are located on this chromosome. Expression of 38 genes among these 143 clones was confirmed, for 19 (50%) of which the cDNA clones are full-length. The most important finding was the identification of three new gene candidates that had been missed by previous computational predictions using Glimmer M. Recent disputes about the difficulty of gene prediction in the malaria genome underscore the importance of comparisons of genomic DNA with cDNA (6,7).

Table 2. Results of BLASTN of FULL-malaria.

Genea	390	15.7%
Genomea	565	22.7%
ESTb	65	2.6%
GSTc	47	1.9%
rRNAa	63	2.5%
Mitochondriaa	2	0.1%
No hit	1324	53.2%
Human	5	0.2%
Vector	30	1.2%
Total	2490	100.0%

^aBLASTN search of nr-nt database of GenBank showed homology with genes or genome of P.falciparum (E value <e^–20).

^bEST reported in (8).

^cGST reported in (9).

Because chromosome 2 contains one-thirtieth of the total genome, it is expected that the FULL-malaria database contains ∼1000 genes and 500 full clones. Further sequencing will reveal more full-length cDNA clones, which should help with the development of new vaccines and reagents to this disease.

As the oligo-capping method is applicable for most eukaryotic pathogens, similar approaches will provide information needed about and resources to combat various diseases.

DDBJ/EMBL/GenBank accession nos AU086071–AU088560