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Computational modeling of the Plasmodium falciparum interactome reveals protein function on a genome-wide scale.

Many thousands of proteins encoded by the genome of Plasmodium falciparum, the causal organism of the deadliest form of human malaria, are of unknown function. It is of utmost importance that these proteins be characterized if we are to develop combative strategies against malaria based on the biology of the parasite. In an attempt to infer protein function on a genome-wide scale, we computationally modeled the P. falciparum interactome, elucidating local and global functional relationships between gene products. The resulting interaction network, reconstructed by integrating in silico and experimental functional genomics data within a Bayesian framework, covers approximately 68% of the parasite genome and provides functional inferences for more than 2000 uncharacterized proteins, based on their associations. Network reconstruction involved the use of a novel strategy, where we incorporated continuously updated, uniform reference priors in our Bayesian model. This method for generating interaction maps is thus also well suited for application to other genomes, where pre-existing interactome knowledge is sparse. Additionally, we superimposed this map on genomes of three apicomplexan pathogens--Plasmodium yoelii, Toxoplasma gondii, and Cryptosporidium parvum--describing relationships between these organisms based on retained functional linkages. This comparison provided a glimpse of the highly evolved nature of P. falciparum; for instance, a deficit of nearly 26% in terms of predicted interactions is observed against P. yoelii, because of missing ortholog partners in pairs of functionally linked proteins.

Animals↗

Arginine biosynthesis in Campylobacter jejuni TGH9011: determination of the argCOBD cluster.

Arginine biosynthetic genes from Campylobacter jejuni TGH9011 were cloned by functional complementation of the respective Escherichia coli arginine biosynthetic mutants. Complementation of argA, argB, argC, argD, argE, argF, and argH auxotrophs was accomplished using a pBR322-based C. jejuni TGH9011 plasmid library. By cross-complementation analyses, the first four steps of arginine biosynthesis were shown to be closely linked on the genome. Two additional clones complementing the first (ArgA) and fifth (ArgE) steps in arginine biosynthesis were obtained. Neither recombinant showed linkage to the arg cluster, to each other, nor to other arginine biosynthetic functions by cross-complementation. Genes argF and argH were not linked to other arginine biosynthetic genes by cross-complementation analysis. Restriction enzyme patterns of recombinant plasmids fell into five groups. Group I contained the arg(ABCD) complementing locus. Group II and Group III were the two genetic loci corresponding to the argA and argE complementing genes. Group II contains the hipO gene encoding N-benzoylglycine-amino-acid amidohydrolase, also known as hippurate hydrolase. Group III contains the hipO homolog of C. jejuni. Group IV represents the argF gene. Group V is the argH gene. Functional complementation of mutations in the first four steps of the arginine biosynthetic pathway was obtained on recombinant plasmid pARGC2. The predicted order of gene complementation was argCargA(argBargD). The sequence of the insert in plasmid pARGC2 revealed direct homologs for argC, argB, and argD. However, sequence analysis of the gene complementing ArgA function in two separate E. coli argA mutants determined that the C. jejuni gene was not a canonical argA gene. The gene complementing the argA defect, which we call argO, showed limited homology to the streptothricin acetyltransferase gene (sat) of Escherichia coli. The flanking open reading frames in pARGC2 showed no homologies to arginine biosynthetic genes. The structure of the argCOBD gene arrangement is discussed with reference to the presence and location of other arginine biosynthetic genes on the genome of C. jejuni and other bacterial organisms.

Amino Acid Sequence↗

Polymerase chain reaction method for determining ratios of human immunodeficiency virus proviral DNA to cellular genomic DNA in brain tissues of HIV-infected patients.

A PCR method was developed to compare HIV-1 DNA loads in brain tissue samples. The method determines the ratio of the amplified product of an HIV DNA sequence to that of a host cellular DNA sequence using standard DNAs as reference. The standards include DNA from a line of human cells that harbor one HIV-1 provirus per cellular genome, and DNA from non-infected human cells. The standard DNAs were mixed in varying proportions and used to establish conditions of amplification under which the ratios of their PCR-amplified products corresponded with the ratios of the amounts of the DNAs themselves. The method was evaluated using known mixtures of the standard DNAs. Using the conditions thus obtained, ratios of HIV proviral DNA to cellular genomic DNA were obtained for tissue DNA samples taken from several different locations within the brain of two deceased HIV-infected patients. Results showed that HIV DNA was non-uniformly distributed within each brain (10-250 per 10(3) cellular genomes); the highest ratios were found in the hippocampus for each patient, independent of postmortem neuropathological findings. The criteria for quantitative PCR have general applicability to comparative studies of any proviral DNA loads in different tissue samples.

Acquired Immunodeficiency Syndrome↗

Biocontrol strain Pseudomonas sp. W34: specific detection and quantification in the rhizosphere of Cucumis sativus with a DNA probe and genotypic characterization by DNA fingerprinting.

A DNA probe specific for biocontrol strains of Pseudomonas was produced by screening randomly amplified polymorphic DNA (RAPD) PCR fragments. Specificity of the probe was assessed by dot blot and colony hybridization. It was used to specifically determine the population of these strains on roots of Cucumis sativus cv. Delikatess. Two polymorphic RAPD fragments of 750 bp, and 550 bp showed identical specificity. The biocontrol strain Pseudomonas sp. W34 was shown to be competitive in the rhizosphere of cucumber and to maintain a stable population for at least 10 days when inoculated on the seed. The phylogenetic relationships between the biocontrol and reference strains were analyzed at the strain level by means of RAPD and repetitive sequence-based PCR genomic fingerprinting (rep-PCR), and at higher taxonomic levels by means of amplified 16S ribosomal DNA restriction analysis ARDRA. It was shown that the antagonistic strains are closely related, forming a separate cluster from other non-antagonistic and reference Pseudomonas strains, their taxonomic placement remaining uncertain.

Cucumis sativus↗

Suppressive subtractive hybridization detects extensive genomic diversity in Thermotoga maritima.

Comparisons between genomes of closely related bacteria often show large variations in gene content, even between strains of the same species. Such studies have focused mainly on pathogens; here, we examined Thermotoga maritima, a free-living hyperthermophilic bacterium, by using suppressive subtractive hybridization. The genome sequence of T. maritima MSB8 is available, and DNA from this strain served as a reference to obtain strain-specific sequences from Thermotoga sp. strain RQ2, a very close relative (approximately 96% identity for orthologous protein-coding genes, 99.7% identity in the small-subunit rRNA sequence). Four hundred twenty-six RQ2 subtractive clones were sequenced. One hundred sixty-six had no DNA match in the MSB8 genome. These differential clones comprise, in sum, 48 kb of RQ2-specific DNA and match 72 genes in the GenBank database. From the number of identical clones, we estimated that RQ2 contains 350 to 400 genes not found in MSB8. Assuming a similar genome size, this corresponds to 20% of the RQ2 genome. A large proportion of the RQ2-specific genes were predicted to be involved in sugar transport and polysaccharide degradation, suggesting that polysaccharides are more important as nutrients for this strain than for MSB8. Several clones encode proteins involved in the production of surface polysaccharides. RQ2 encodes multiple subunits of a V-type ATPase, while MSB8 possesses only an F-type ATPase. Moreover, an RQ2-specific MutS homolog was found among the subtractive clones and appears to belong to a third novel archaeal type MutS lineage. Southern blot analyses showed that some of the RQ2 differential sequences are found in some other members of the order Thermotogales, but the distribution of these variable genes is patchy, suggesting frequent lateral gene transfer within the group.

Antigens, Bacterial↗

First chromosomal restriction map of Actinobacillus pleuropneumoniae and localization of putative virulence-associated genes.

Combined physical and genetic maps of the genomes of Actinobacillus pleuropneumoniae AP76 (serotype 7 clinical isolate) and of A. pleuropneumoniae ATCC 27088 (serotype 1 reference strain) were constructed by using the restriction endonucleases ApaI, AscI, NotI, and SalI. The chromosome sizes as determined by the addition of estimated fragment sizes were 2.4 Mbp, and both maps had a resolution of approximately 100 kbp. The linkages between the ApaI, AscI, NotI, and SalI fragments and their relative positions were determined by (i) fragment excision and redigestion and (ii) partial digests of defined fragments and Southern blot using end-standing probes. The single SalI site within the chromosome of strain A. pleuropneumoniae AP76 was defined as position 1 of the map; for the map of A. pleuropneumoniae ATCC 27088, the corresponding SalI site was chosen. Putative virulence-associated genes (apx, omlA, sodA, tbpBA, ureC, and a repeat element) and housekeeping genes (glyA, metJ, recA, and rhoAP) were positioned on the physical maps and located on the ApaI and NotI fragments of A. pleuropneumoniae serotype reference strains.

Actinobacillus pleuropneumoniae↗

Cytopathogenicity markers in the genome of Hungarian cytopathic isolates of bovine viral diarrhoea virus.

Since genetic recombination is a major factor in the evolution of the cytopathogenic (cp) bovine viral diarrhoea virus (BVDV) biotypes, in this study the cytopathogenicity markers were investigated in the genomes of two cp BVDV strains recently isolated from mucosal disease (MD) cases in Hungary. In the genome of strain H4956, a Jiv-like insertion was found similar to those described in reference strain NADL and in other BVDV 1, BVDV 2 and border disease virus (BDV) strains. The 133 amino acid Jiv-like sequence is inserted at nucleotide position 4984 (amino acid position 1533), 9 nucleotides upstream of that of strain NADL. The insertion showed 96% amino acid sequence identity with the cellular Jiv protein. In the genome of cp BVDV strain H115/PCR, an ubiquitin-containing duplication was found. The duplicated sequence started at nucleotide position 7978 (amino acid 2531) in the NS4B gene. The duplication contained a complete ubiquitin monomer of 76 amino acids and the complete NS3 gene starting at nucleotide position 5153 (amino acid 1589), which corresponds to the first N-terminal amino acid of NS3. The duplication was located further downstream of the known ubiquitin-containing genomic regions of cp BVDV strains, and it consisted of the shortest inserted nucleotide sequence. The insertions and duplication of strains H4956 and H115/PCR further confirmed that recombinations occurring at positions A and B are the most common mechanisms leading to the development of BVDV cytopathogenicity.

Amino Acid Sequence↗

Factors affecting codon usage in Yersinia pestis.

The complete genome of Yersinia pestis which was the causative agent of the systemic invasive infectious disease classically referred as plague, had been recently sequenced. In order to have a further insight into the synonymous codon usage evolution, factors shaping synonymous codon usage pattern of Yersinia pestis were analyzed in this paper. The coding sequences larger than or equal to 300 bp were used in codon usage analysis. Though "G"+"C" content in Y. pestis genome was slightly lower (47.64%), the highly expressed genes tended to use "C" or "G" at synonymous sites compared with lowly expressed genes. Conversely, lowly expressed genes tended to prefer "A" or "T" at synonymous positions. Gene expression level was strongly correlated with the first axis of the correspondence analysis (COA) (R=0.63, P<0.0001). By the analyses of the codon usage pattern of highly and lowly expressed genes, it was confirmed that gene expression level was partially responsible for the codon usage bias. GC-skew analysis showed that codon usage suffered replication-transcriptional selection. Codon adaptation index (CAI), frequency of "C"+"G" at the synonymous third position of codon (GC3s) and the effective number of codons (Nc) values showed some differences among different gene length groups. "G"+"C" content of genes was strongly correlated with the first axis of the COA (R=0.72, P<0.0001). It could be concluded that gene expressivity, replication-transcriptional selection, gene length and gene composition constraints were the main affecting factors of codon usage variation in Y. pestis.

Amino Acids↗

Detection of integration of plasmid DNA into host genomic DNA following intramuscular injection and electroporation.

Plasmid vectors have been widely used for DNA vaccines and gene therapy. Following intramuscular injection, the plasmid that persists is extrachromosomal and integration into host DNA, if it occurs at all, is negligible. However, new technologies for improving DNA delivery could increase the frequency of integration. In the present study, we tested the effect of electroporation on plasmid uptake and potential integration following intramuscular injection in mice, using a plasmid containing the mouse erythropoietin gene. Electroporation increased plasmid tissue levels by approximately six- to 34-fold. Using a quantitative gel-purification assay for integration, electroporation was found to markedly increase the level of plasmid associated with high-molecular-weight genomic DNA. To confirm integration and identify the insertion sites, we developed a new assay - referred to as repeat-anchored integration capture (RAIC) PCR - that is capable of detecting rare integration events in a complex mixture in vivo. Using this assay, we identified four independent integration events. Sequencing of the insertion sites suggested a random integration process, but with short segments of homology between the vector breakpoint and the insertion site in three of the four cases. This is the first definitive demonstration of integration of plasmid DNA into genomic DNA following injection in vivo.

Animals↗

The mouse genome: an overview.

A genetic map with one molecularly marked locus per cM will be available for the mouse in the near future. A map of this density should provide molecular reference points that connect genetic and physical maps, identify sites to initiate positional cloning studies for the molecular characterization of mutant loci, and define homologous regions of mouse and human genomes.

Animals↗

Evaluation of computational metabolic-pathway predictions for Helicobacter pylori.

MOTIVATION: We seek to determine the accuracy of computational methods for predicting metabolic pathways in sequenced genomes, and to understand the contributions of both the prediction algorithms, and the reference pathway databases used by those algorithms, to the prediction accuracy. RESULTS: The comparisons we performed were as follows. (1) We compared two predictions of the pathway complements of Helicobacter pylori that were computed by an early version of our pathway-prediction algorithm: prediction A used the EcoCyc E. coli pathway DB as the reference database (DB) for prediction, and prediction B used the MetaCyc pathway DB (a superset of EcoCyc) as the reference pathway DB. The MetaCyc-based prediction contained 75% more pathway predictions, but we believe a significant number of those predictions were false positives. (2) We compared two predictions of the pathway complement of H. pylori that used MetaCyc as the reference pathway DB, but that used different algorithms: the original PathoLogic algorithm, and an enhanced version of the algorithm designed to eliminate false-positive pathway predictions. The improved algorithm predicted 30\% fewer metabolic pathways than the original algorithm; all of the eliminated pathways are believed to be false-positive predictions. (3) We compared the 98 pathways predicted by the enhanced algorithm with the results of a manual analysis of the pathways of H. pylori. Results: 40 of the computationally predicted pathways were consistent with the manual analysis, 13 pathways are considered false-positive predictions, and four pathways had partially overlapping topologies. Twenty-six predicted pathways were not mentioned in the manual analysis; we believe these are correct predictions by PathoLogic that were not found by the manual analysis. Five pathways from the manual analysis were not found computationally. Agreement between the computational and manual predictions was good overall, with the computational analysis inferring many pathways that the manual analysis did not identify. Ultimately the manual analysis is also partially speculative, and therefore is not an absolute measure of correctness. The algorithm is designed to err on the side of more false positives to bring more potential pathways to the user's attention. The resulting H. pylori pathway DB is freely available at http://ecocyc.org:1555/HPY/organism-summary?object=HPY. AVAILABILITY: The Pathway Tools software is freely available to academic users, and is available to commercial users for a fee. Contact pkarp@ai.sri.com for information on obtaining the software.

Algorithms↗

Genome-wide mapping with biallelic markers in Arabidopsis thaliana.

Single-nucleotide polymorphisms, as well as small insertions and deletions (here referred to collectively as simple nucleotide polymorphisms, or SNPs), comprise the largest set of sequence variants in most organisms. Positional cloning based on SNPs may accelerate the identification of human disease traits and a range of biologically informative mutations. The recent application of high-density oligonucleotide arrays to allele identification has made it feasible to genotype thousands of biallelic SNPs in a single experiment. It has yet to be established, however, whether SNP detection using oligonucleotide arrays can be used to accelerate the mapping of traits in diploid genomes. The cruciferous weed Arabidopsis thaliana is an attractive model system for the construction and use of biallelic SNP maps. Although important biological processes ranging from fertilization and cell fate determination to disease resistance have been modelled in A. thaliana, identifying mutations in this organism has been impeded by the lack of a high-density genetic map consisting of easily genotyped DNA markers. We report here the construction of a biallelic genetic map in A. thaliana with a resolution of 3.5 cM and its use in mapping Eds16, a gene involved in the defence response to the fungal pathogen Erysiphe orontii. Mapping of this trait involved the high-throughput generation of meiotic maps of F2 individuals using high-density oligonucleotide probe array-based genotyping. We developed a software package called InterMap and used it to automatically delimit Eds16 to a 7-cM interval on chromosome 1. These results are the first demonstration of biallelic mapping in diploid genomes and establish means for generalizing SNP-based maps to virtually any genetic organism.

Arabidopsis↗

Challenges in human genetics in India in the new millennium.

This editorial provides information on the first draft of the human genome, and examines the impact this will have on the practice of medicine, with special reference to India. It examines what is currently possible in India, what we should do in the immediate future, and what is in store in the long run. The future is indeed bright and India needs to expand the genetic services and research facilities.

Forecasting↗

Analysis of the cytosolic proteome of Halobacterium salinarum and its implication for genome annotation.

The halophilic archaeon Halobacterium salinarum (strain R1, DSM 671) contains 2784 protein-coding genes as derived from the genome sequence. The cytosolic proteome containing 2042 proteins was separated by two-dimensional gel electrophoresis (2-DE) and systematically analyzed by a semi-automatic procedure. A reference map was established taking into account the narrow isoelectric point (pI) distribution of halophilic proteins between 3.5 and 5.5. Proteins were separated on overlapping gels covering the essential areas of pI and molecular weight. Every silver-stained spot was analyzed resulting in 661 identified proteins out of about 1800 different protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting (PMF). There were 94 proteins that were found in multiple spots, indicating post-translational modification. An additional 141 soluble proteins were identified on 2-D gels not corresponding to the reference map. Thus about 40% of the cytosolic proteome was identified. In addition to the 2784 protein-coding genes, the H. salinarum genome contains more than 6000 spurious open reading frames longer than 100 codons. Proteomic information permitted an improvement in genome annotation by validating and correcting gene assignments. The correlation between theoretical pI and gel position is exceedingly good and was used as a tool to improve start codon assignments. The fraction of identified chromosomal proteins was much higher than that of those encoded on the plasmids. In combination with analysis of the GC content this observation permitted an unambiguous identification of an episomal insert of 60 kbp ("AT-rich island") in the chromosome, as well as a 70 kbp region from the chromosome that has integrated into one of the megaplasmids and carries a series of essential genes. About 63% of the chromosomally encoded proteins larger than 25 kDa were identified, proving the efficacy of 2-DE MALDI-TOF MS PMF technology. The analysis of the integral membrane proteome by tandem mass spectrometric techniques added another 141 identified proteins not identified by the 2-DE approach (see following paper).

Bacterial Proteins↗

Genome organization and expression of the Penicillium stoloniferum virus S.

The complete sequences of two double-stranded RNAs (dsRNAs) (referred to S1 and S2) of Penicillium stoloniferum virus S (PsV-S) were established. The S1 dsRNA was 1,690 bp in length, and it contained a unique open reading frame (ORF) of 539 amino acids (molecular weight of 62 kDa, referred to P62). The S2 dsRNA was 1,523 bp in length, and also it contained one ORF of 434 amino acids (molecular weight of 47 kDa, referred to P47). Both S1 and S2 ORFs were identified only on the positive strand of each dsRNA segment. A sequence motif of (5'-CUG-3') was found at the 3'-termini of the positive strands of PsV-S1 and S2 dsRNAs. The predicted amino acid sequences of S1 dsRNA showed high sequence homology with the putative RNA-dependent RNA polymerases of RNA viruses. Near full-length and positive-sense single-stranded RNAs derived from the S1 and S2 dsRNAs were detected from the PsV-infected host cell. The expressed proteins of P62 and P47 showed a positive reaction against PsV-S antiserum in Western blot analysis. Phylogenetic analysis using the RDRP sequences and the capsid proteins of the various partitiviruses revealed that PsV-S is a definite member of the partitivirus, the family Partitiviridae, and especially clusters well along with D. destructiva virus 1 and 2.

Amino Acid Sequence↗

Reverse transcriptase: mediator of genomic plasticity.

Reverse transcription has been an important mediator of genomic change. This influence dates back more than three billion years, when the RNA genome was converted into the DNA genome. While the current cellular role(s) of reverse transcriptase are not yet completely understood, it has become clear over the last few years that this enzyme is still responsible for generating significant genomic change and that its activities are one of the driving forces of evolution. Reverse transcriptase generates, for example, extra gene copies (retrogenes), using as a template mature messenger RNAs. Such retrogenes do not always end up as nonfunctional pseudogenes but form, after reinsertion into the genome, new unions with resident promoter elements that may alter the gene's temporal and/or spatial expression levels. More frequently, reverse transcriptase produces copies of nonmessenger RNAs, such as small nuclear or cytoplasmic RNAs. Extremely high copy numbers can be generated by this process. The resulting reinserted DNA copies are therefore referred to as short interspersed repetitive elements (SINEs). SINEs have long been considered selfish DNA, littering the genome via exponential propagation but not contributing to the host's fitness. Many SINEs, however, can give rise to novel genes encoding small RNAs, and are the migrant carriers of numerous control elements and sequence motifs that can equip resident genes with novel regulatory elements [Brosius J. and Gould S.J., Proc Natl Acad Sci USA 89, 10706-10710, 1992]. Retrosequences, such as SINEs and portions of retroelements (e.g., long terminal repeats, LTRs), are capable of donating sequence motifs for nucleosome positioning, DNA methylation, transcriptional enhancers and silencers, poly(A) addition sequences, determinants of RNA stability or transport, splice sites, and even amino acid codons for incorporation into open reading frames as novel protein domains. Retroposition can therefore be considered as a major pacemaker for evolution (including speciation). Retroposons, with their unique properties and actions, form the molecular basis of important evolutionary concepts, such as exaptation [Gould S.J. and Vrba E., Paleobiology 8, 4-15, 1982] and punctuated equilibrium [Elredge N. and Gould S.J. in Schopf T.J.M. (ed). Models in Paleobiology. Freeman, Cooper, San Francisco, 1972, pp. 82-115].

Animals↗