Antigenic variation of a cysteine-rich protein in Giardia lamblia.

The WB isolate of Giardia lamblia expresses a cysteine-rich 170-kD surface antigen (CRP170) that undergoes antigenic variation. An (6E7), cytotoxic for isolates expressing CRP170, was used in another study to select antigenic variants from clones of the WB isolate of Giardia. CRP170 was replaced by surface-labeled bands ranging in size from approximately 50 to 170 kD. In this study, mAb 6E7 was used to isolate a 1-kb portion of the CRP170 gene (M2-1) from a lambda gt 11 expression library. The M2-1 clone hybridized to a 5.4-kb transcript from isolates expressing CRP170 but did not hybridize to RNA from antigenic variants. Evidence was found for frequent rearrangements at the CRP170 gene locus. DNA sequencing of the M2-1 clone revealed the presence of long tandem repeats. The putative amino acid sequence of M2-1 reveals a 12% cysteine content, and CRP170 is readily labeled in vivo with cysteine.

Evolutionary relatedness of Plasmodium species as determined by the structure of DNA.

Malaria parasites can be grouped evolutionarily by analysis of DNA composition and genome arrangement. Those that vary widely with regard to host range, morphology, and biological characteristics fit into only a small number of distinctive groups. The DNA of the human parasite Plasmodium falciparum fits into a group that includes rodent and avian malarias and is unlike the DNA of other primate malaria parasites. The DNA of Plasmodium vivax, which is also a human parasite, fits into a distinctly different group that includes Plasmodium cynomolgi, a parasite of monkeys. The evolutionary lines suggested here appear to be consistent with similarities seen among malaria parasites with regard to gene sequence.

Mung bean nuclease cleaves Plasmodium genomic DNA at sites before and after genes.

Mung bean nuclease was found to cut the genomic DNA of the malaria parasite Plasmodium at positions before and after genes but not within gene-coding regions. This cleavage, which had nearly the preciseness of a restriction nuclease, required controlled conditions in the presence of formamide. Southern blot analysis showed that the coding areas for Plasmodium actin, circumsporozoite protein, histidine-rich protein, ribosomal RNA's, and tubulin are each cleaved from genomic DNA to yield a single major band on an agarose gel. DNA sequence data on several clones of mung bean nuclease cleavage products containing the gene for the circumsporozoite protein of Plasmodium falciparum confirmed that cleavage sites are before and after genes. Recognition and cleavage of DNA did not seem to be related to any primary sequence but may be related to structural features of the DNA duplex that demarcate genes. Mung bean nuclease-cleaved DNA could be inserted directly into a lambda expression vector, yielding a representative but small gene bank of intact gene fragments.

Structurally distinct, stage-specific ribosomes occur in Plasmodium.

Two structurally distinct nuclear genes code for cytoplasmic small subunit ribosomal RNA's in the parasite Plasmodium berghei. Stable transcripts from one of the ribosomal RNA genes are found almost exclusively in those stages of the life cycle that develop in the mosquito. When the parasite infects the mammalian host, transcripts from the second gene become the predominant small subunit ribosomal RNA species.

Sequence of the immunodominant epitope for the surface protein on sporozoites of Plasmodium vivax.

Plasmodium vivax is one of the four malaria parasites that cause disease in humans. The structure of the immunodominant repeating peptide of the circumsporozoite (CS) protein of P. vivax was determined. A fragment of P. vivax DNA that encodes this tandemly repeating epitope was isolated by use of an oligonucleotide probe whose sequence is thought to be conserved in CS protein genes. DNA sequence analysis of the P. vivax clone indicates that the CS repeat is nine amino acids in length (Gly-Asp-Arg-Ala-Asp-Gly-Gln-Pro-Ala). The structure of the repeating region was confirmed with synthetic peptides and monoclonal antibodies directed against P. vivax sporozoites. This information should allow synthesis of a vaccine for P. vivax that is similar to the one being tested for P. falciparum.

Genetic analysis of the human malaria parasite Plasmodium falciparum.

Malaria parasites are haploid for most of their life cycle, with zygote formation and meiosis occurring during the mosquito phase of development. The parasites can be analyzed genetically by transmitting mixtures of cloned parasites through mosquitoes to permit cross-fertilization of gametes to occur. A cross was made between two clones of Plasmodium falciparum differing in enzymes, drug sensitivity, antigens, and chromosome patterns. Parasites showing recombination between the parent clone markers were detected at a high frequency. Novel forms of certain chromosomes, detected by pulsed-field gradient gel electrophoresis, were produced readily, showing that extensive rearrangements occur in the parasite genome after cross-fertilization. Since patients are frequently infected with mixtures of genetically distinct parasites, mosquito transmission is likely to provide the principal mechanisms for generating parasites with novel genotypes.

Structure of the gene encoding the immunodominant surface antigen on the sporozoite of the human malaria parasite Plasmodium falciparum.

The gene for the circumsporozoite (CS) protein of Plasmodium falciparum has been cloned and its nucleotide sequence determined. The gene encodes a protein of 412 amino acids as deduced from the nucleotide sequence. The protein contains 41 tandem repeats of a tetrapeptide, 37 of which are Asn-Ala-Asn-Pro and four of which are Asn-Val-Asp-Pro. Monoclonal antibodies against the CS protein of Plasmodium falciparum were inhibited from binding to the protein by synthetic peptides of the repeat sequence. The CS protein of Plasmodium falciparum and the CS protein of a simian malaria parasite, Plasmodium knowlesi, have two regions of homology, one of which is present on either side of the repeat. One region contains 12 of 13 identical amino acids. Within the nucleotide sequence of this region, 25 of 27 nucleotides are conserved. The conservation of these regions in parasites widely separated in evolution suggests that they may have a function such as binding to liver cells and may represent an invariant target for immunity.

Three segments from the monkey genome that hybridize to simian virus 40 have common structural elements.

Three cloned segments that hybridize to a region of simian virus 40 (SV40) deoxyribonucleic acid including the origin of replication have been isolated from a monkey genomic library. The primary structure of one segment was previously reported (T. McCutchan and M. Singer, Proc. Natl. Acad. Sci. U.S.A. 78:95-99, 1981). We report here the sequences of the other two segments and a comparison of all three. The SV 40-hybridizing region in each segment is limited to several hundred base pairs. All of the segments contain multiple and disconnected sequences homologous to the region of SV40 directly surrounding the viral replication origin. The number and arrangement of the homologous sequences is different in the three segments. However, the segments have the following features in common: (i) each contains multiple copies of the sequence GGGCGGPuPu, which also appears six times near the origin of SV40; (ii) each contains several strong homologies to the central dyad symmetry of SV40; (iii) each contains a long internal repeat, as does the origin region of SV40. The three SV40-hybridizing segments are members of a larger family of genomic sequences that hybridize well to each other, but not necessarily to SV40.

A novel class of supercoil-independent nuclease hypersensitive site is comprised of alternative DNA structures that flank eukaryotic genes.

The cell makes a fundamental distinction between genes and non-gene sequences, which mechanistically underlies the process of gene regulation. Here, we describe the properties of a novel class of genetic sites that reproducibly flank and delineate the coding regions of the eukaryotic genes tested. Defined in vitro reaction conditions that include altered solvation and elevated temperature rendered the sites hypersensitive to nuclease cleavage. Consequently, the complete coding regions of the Drosophila genes tested were quantitatively excised from genomic DNA or genomic clones by this treatment. Identical reaction products were generated from linear or supercoiled DNA substrates. Chemical modification and fine-structure analysis of several cleavage sites flanking Drosophila genes showed that the cleavage sites were stable nucleic acid structures that contained specific arrangements of paired and unpaired nucleotides. The locations and properties of the cleavage sites did not correspond to previously known nuclease hypersensitive sites nor to known alternative DNA structures. Thus, they appear to represent a new class of genetic site. In a deletion analysis, the minimal sequence information necessary to direct in vitro nuclease cleavage 3' to the Drosophila GART gene co-localized with the signal required for termination of transcription in vivo. The data suggest that a novel class of DNA site with distinct structural properties encodes biological information by marking the boundaries of at least some gene expression units in organisms as diverse as Plasmodium and Drosophila.

The ribosomal DNA loci in Plasmodium falciparum accumulate mutations independently.

Homogeneity of rDNA sequence within a cell is maintained by mechanisms working at the DNA level. The imperative to maintain homogeneity is thought to result from pressure to maintain the sequence of the rRNA transcript. We have investigated the extent of sequence variation within and between members of a species that is unable to utilize some standard mechanisms of rDNA sequence correction. We have compared the sequence of the internal transcribed spacer (ITS1) located between the 18 S rRNA and 5.8 S rRNA genes of five different loci of a single Plasmodium falciparum genotype. The ITS1 sequences are identical at 80 to 91% of the positions among the three asexually expressed genes (A-types) and 75% between the two genes expressed during sporogony (S-types), with only 42 to 57% identity between the types. This is rather startling in that the differences described here for a single genome are greater than those normally seen when comparing rDNA units from distantly related organisms. We observe an apparent conservation of secondary structure within ITS1 sequences from the different transcription units, which would reflect a level of selection at the rRNA but the organism seems to be quite tolerant of primary sequence variation. Investigation of the mature coding region within the 18 S rRNA genes did not reveal sequence variation within A- and S-types from a single genotype. However, comparison of the 18 S rRNA coding region from 17 geographically distinct strains reveals up to 10% sequence variation within a 400 nucleotide region. Hence homogeneity of rRNA units within a species does not seem to be an imperative driven totally by selection at the RNA level. The extraordinary maintenance of homogeneity within rDNA units normally seen within a species appears to have significance beyond those that can be ascribed to the events involved in processing, assembly and function of the ribosome.

Regulation and trafficking of three distinct 18 S ribosomal RNAs during development of the malaria parasite.

The human malaria parasite Plasmodium vivax has been shown to regulate the transcription of two distinct 18 RNAs during development. Here we show a third and distinctive type of ribosome that is present shortly after zygote formation, a transcriptional pattern of ribosome types that relates closely to the developmental state of the parasite and a phenomenon that separates ribosomal types at a critical phase of maturation. The A-type ribosome is predominantly found in infected erythrocytes of the vertebrate and the mosquito blood meal. Transcripts from the A gene are replaced by transcripts from another locus, the O gene, shortly after fertilization and increase in number as the parasite develops on the mosquito midgut. Transcripts from another locus, the S gene, begins as the oocyst form of the parasite matures. RNA transcripts from the S gene are preferentially included in sporozoites that bud off from the oocyst and migrate to the salivary gland while the O gene transcripts are left within the oocyst. Although all three genes are typically eukaryotic in structure, the O gene transcript, described here, varies from the other two in core regions of the rRNA that are involved in mRNA decoding and translational termination. We now can correlate developmental progression of the parasite with changes in regions of rRNA sequence that are broadly conserved, where sequence alterations have been related to function in other systems and whose effects can be studied outside of Plasmodium. This should allow assessment of the role of translational control in parasite development.

Sequence and structure of a Plasmodium falciparum telomere.

We have isolated a 3 kb cloned DNA fragment which originated from a telomere of the human malaria parasite Plasmodium falciparum. The complete nucleotide sequence of the clone is presented. The clone is composed of several distinct structural regions which vary in their sequence complexity. Using oligonucleotide probes for the different structural regions, we analyzed the genetic conservation of sequence organization near telomeres in various strains of P. falciparum. Our results suggest that rapid sequence variability is generated in the vicinity of chromosome ends.

Identification of 5 S and 5.8 S ribosomal RNA molecules and their genes in Plasmodium berghei.

The ribosomes of Plasmodium berghei contain two small RNA molecules approximately 150 and 120 bases long. These correspond in size to 5.8 and 5 S RNA molecules found in the cytoplasmic ribosomes of eukaryotic cells. Segments homologous to the 5.8 S RNA are present in each of the four ribosomal RNA gene units. Restriction fragments which span the region between the coding areas of the two major ribosomal RNA molecules hybridize to 5.8 S RNA, indicating that the gene is located between the small and large ribosomal RNA genes. The gene(s) for the 5 S RNA is not associated with any of the four ribosomal RNA gene units.

Isolation of a gene segment expressed by mature schizonts of Plasmodium knowlesi.

Fragments of Plasmodium knowlesi DNA, generated by mung bean nuclease digestion, were ligated into the lambda gt11 vector. This expression library was immunoscreened with a serum which inhibits invasion of erythrocytes by merozoites in vitro and whose primary specificity is directed against a Mr 140 000 merozoite surface antigen. One of the isolated clones contained a 125 base pair insert which hybridized to a 1.8 kilobase species of schizont RNA, indicating that this insert is part of a gene expressed during schizogony.

Cloning and characterization of a ribosomal RNA gene from Plasmodium berghei.

The ribosomal RNA (rRNA) of Plasmodium berghei strain NYU2 was characterized with respect to size and used to identify molecular clones of ribosomal RNA genes (rDNA) from this organism. The intact large rRNA species (Mr 1.40 X 10(6] is intermediate in size between prokaryotic and eukaryotic large rRNAs. It is specifically cleaved in vivo near the 5'-end yielding one large component (Mr 1.10 X 10(6] and one small component (Mr 0.30 X 10(6]. The small rRNA species (Mr 0.75 X 10(6] is larger than in higher eukaryotic organisms, but is similar in size to some other protozoa. Using 5'-end labeled rRNA as a probe, clones of rDNA were selected from genomic DNA libraries prepared in lambda phage Charon 4A and plasmid pBR322. The clones prepared in lambda were prone to recombination. The 14.7 kilobase (kb) insert in phage lambda PbR27 contains the complete coding sequences for both the small and larger rRNA species. A complete set of small plasmid clones were isolated which hybridize to the different segments of the rRNA transcription unit. A restriction map of the genes cloned in lambda PbR27 with the approximate position of the coding region is presented. The polarity of transcription of the rDNA was determined by hybridizing 3'-OH end labeled rRNA to Southern blots of genomic and cloned rDNA. The results indicate that a transcription unit of P. berghei rDNA is arranged as in other organisms with the small rRNA at the 5' end and the large rRNA at the 3' end.

The structure of the large subunit rRNA expressed in blood stages of Plasmodium falciparum.

Here we present the sequence of the large subunit (LSU) rRNA expressed in blood-stage forms (and therefore A-type) of the malaria parasite, Plasmodium falciparum, from two different isolates. We determined the genomic sequence of a rRNA unit of the CAMP parasite strain from within the internal transcribed spacer 1 (ITS1) through the 5.8S rRNA gene, the ITS2 and the entire large subunit rRNA gene. We have also determined the corresponding sequence of the gene of the FVO strain by sequencing cDNA clones derived from blood-stage asexual parasites. Differences between the two were due to scattered point mutations in expansion segments of the mature rRNA regions. In addition to the point mutations, the rRNA genes from the two strains could be distinguished by the presence of a more complex polymorphism near the 3' end of the molecule. The most complex polymorphic form was localized on a single chromosome and found in only a subset of geographically distinct isolates. The sequences of the 5.8S rRNA unit and the LSU rRNA unit reported here can be logically assembled into a complete secondary structure which conforms to the standard structure conserved in all eukaryotic ribosomes. The construction of a model of secondary structure for the LSU rRNA has allowed the identification of phylogenetically conserved sequences involved in drug interaction with the ribosome, as well as those sequences involved in tertiary interactions within the rRNA itself.

The genome of Schistosoma mansoni: isolation of DNA, its size, bases and repetitive sequences.

DNA has been prepared from adults and cercariae of Schistosoma mansoni utilizing a technique that involves centrifugation through cesium chloride. The DNA isolated from S. mansoni adults and that isolated from cercariae were found to be indistinguishable in all analyses. No modified bases were detected by chromatography or comparative endonuclease restriction. Cot analysis demonstrated that the haploid genome of S. mansoni is 0.26 pg (2.7 X 10(8) base pairs) and that the genome contains both moderately and highly repeated components. Some of the repetitive fraction of DNA consists of tandemly repeated ribosomal genes of which there are 500-1000 copies per genome (1.8-3.6% of the total DNA). Four other non-ribosomal repetitive sequences (comprising at least a further 2.0% of the total DNA) have been isolated from a DNA clone bank and their arrangement within the S. mansoni genome investigated by restriction and Southern blot analysis. These cloned segments of DNA appear in many different locations within the genome and thus are reminiscent of the interspersed DNA sequences described in higher eukaryotic organisms.

Comparison of the primary structure of the 25 kDa ookinete surface antigens of Plasmodium falciparum and Plasmodium gallinaceum reveal six conserved regions.

The gene encoding the 25 kDa ookinete surface antigen (Pgs25) of Plasmodium gallinaceum has been cloned using an oligonucleotide probe directed against one of the EGF-like domains of the P. falciparum 25 kDa ookinete surface antigen (Pfs25). The Pgs25 gene codes for a polypeptide of 215 amino acids, two amino residues less than Pfs25. The deduced amino acid sequence contains a putative signal sequence at the amino-terminus, four tandemly repeated EGF-like domains, and a hydrophobic region at the carboxyl-terminus. By comparing Pgs25 with Pfs25, six conserved regions, consisting of six or more amino acid residues, have been identified. Most of the conserved regions are outside EGF-like core consensus sequences. The most striking conservation is the spacing of the cysteines.

Variation among circumsporozoite protein genes from rodent malarias.

We investigated the effect of long term passage of parasites in naive animals on the circumsporozoite protein (CSP) gene of Plasmodium yoelii. The CSP gene sequence was determined from a non-lethal cloned line of P. yoelii and compared to the CSP gene sequence from a lethal strain of P. yoelii. The two parasite lines were originally derived from the same isolate, but were separated 17 years ago followed by continued passage. The sequence of the CSP gene and its surroundings from the non-lethal line remains identical to that from the lethal isolate except for a deletion within the repeated central domain. This result contrasts with the results obtained by sequencing a number of clones from different geographical field isolates of Plasmodium falciparum where there appears to be rapid accumulation of sense mutations within putative functional domains. These observations are consistent with the suggestion that strong biological pressure in a field environment results in selection of parasite types on the basis of different CSP gene sequences.

Ribosomal RNA-based diagnosis of Plasmodium falciparum malaria.

We have identified useful target sites for the diagnosis of malaria infections by oligonucleotide hybridization on the small subunit RNA of Plasmodium falciparum. Acetic acid works as effectively as formaldehyde or methyl mercuric hydroxide in procedures designed to apply RNA to filters. We have confirmed the findings of others that the stability of ribosomal RNA suffices for its use as a target for diagnosis. We have achieved a detection level of at least 0.00046% parasitemia and suggest that detection of a single parasite is well within reach of this technology.