Researchers from the ONSA (Organization for the Sequencing and Analysis of Nucleotides) have identified 200 new genes associated with stages in the life of theSchistosoma mansoni worm, which causes schistosomiasis, a disease that is typical of poor countries and affect 200 million people in the world and, in Brazil alone, some 10 million inhabitants. With the conclusions that are popping up from the analysis of the genome of the parasite, also studied by research groups in Minas Gerais,new prospects are being opened up for fighting the disease. Today, it is treated with medicines that reduce the number of parasites in the blood, but that do not prevent reinfection – and there is evidence that the parasites are becoming resistant to the drugs that are in use.
The findings about Schistosoma are happening at a particularly fertile moment in the Genome – FAPESP Program, kicked off in 1997 with the mapping of the Xylella fastidiosa bacterium, one of the pests of orange groves. Last month, the genome of another bacterium was concluded, Leifsonia xyli subspxyli, which attacks sugarcane (Saccharum officinarum) and reduces by up to 27% the biomass usable for the production of sugar and alcohol. Leifsonia is the first entirely brazilian project in the ambit of a program that is the offspring of Genome-FAPESP, called Agronomic and Environmental Genomes (AEG), created in 2000, after the sequencing of a variety of Xylella that attacks the vines, in conjunction with the United States Department of Agriculture.
There are important results coming also from the sequencing of the chloroplast – a cellular structure – of sugarcane, which shows an impressive similarity with corn (Zea mays); and sequencing is practically finished of a seaweed, Gracilaria tenuistipitata, which produces agar, a gel that has a wide industrial application. The chloroplast has its own genome, which can be manipulated to create safer genetically modified plants.
As information accrues, it is possible to find out, at a pace that is always speeding up, the function of each gene of the organisms studied, as well as the most vulnerable points of the disease causing ones, whether in human beings or in plants, from which routes can be drawn up for combating them. This is the main objective of the work withSchistosoma, a worm that is between 6 and 10 millimeters in length and 0.5 millimeters in diameter, which reaches human beings through the Biomphalaria glabrata snail. The teams from São Paulo and Minas that are working to check their advance are reinforcing the international projects to identify and analyze the genomes of agents of typical diseases of underdeveloped countries, such protozoa like Plasmodium falciparum, which causes malaria,Trypanosoma cruzi, responsible for Chagas’s disease, and Leishmania, for leishmaniasis.
In little more than a year, a group from the ONSA network coordinated by Sergio Verjovski-Almeida, from USP’s Institute of Chemistry – in collaboration with other laboratories from USP itself, the University of Campinas (Unicamp), and from the Butantan and Adolfo Lutz institutes – produced 130,000 sequences of expressed (active) regions of genes – the so-called ESTs, or expressed sequence tags, which are the fragments of DNA (deoxyribonucleic acid), with the information for the production of proteins. These sequences of genes reflect the various stages of the life of the Schistosoma – adult, egg, miracidium, germball (the stage that lives inside the snail), cercaria and schistosomulum. But they represent just a part of the genetic baggage: while the genome of the majority of parasites has from 10 to 30 million base pairs (chemical units of the DNA), it is estimated that this worm has 300 million pairs – and an as yet undetermined number of genes.
The researchers from São Paulo have identified complete sequences of 200 new genes to which some function has been attributed, as they show some similarity with those of other organisms, and another 1,500 are almost complete. They also found about 20,000 totally new fragments, without any similarity to those from other organisms. Amongst the genes that attract interest are those connected with the production of proteins on the surface of the worm. The team from USP obtained the complete sequence of one of them, which is apparently connected with how the worm escapes from the human organism’s defenses – it may therefore be a way towards the production of a vaccine. A medicine based on a drug called praziquantel is used against this disease, which has a social impact second only to malaria. Applied on a large scale in Senegal, it generated strains of resistant parasites, characterized in October 2001 in the Annals of Tropical Medicine and Parasitology.
“The World Health Organization recommends looking for a vaccine, even though it may take a few years”, says Verjovski, “because the medicine works only after a person has become infected, but it does not interrupt the cycle of the disease”. On sunny days, the worm leaves the snails and goes to the water of lakes with stagnant waters or little currents. It gets into human beings through the skin, reaches the blood circulation and installs itself in the liver. There, it is transformed into an adult and reproduces itself. The eggs cause the destruction of the liver, which prevents the circulation of the blood and makes the belly swell – which is when the ‘water belly’ is characterized, the popular name given to it. The eggs reach the intestines, and, eliminated with the feces, fall into the water where the snails circulate – and the cycle starts again.
In Minas, a group coordinated by Guilherme Oliveira, from the René Rachou Research Center of the Oswaldo Cruz Foundation (Fiocruz), concluded in 2001 the sequencing of 16,000 ESTs. Another project is now at the stage of being implanted, carried out by Fiocruz and by universities from Minas Gerais, with funding from the State of Minas Gerais Research Support Foundation (Fapemig) and of the National Council for Scientific and Technological Development (CNPq). The researchers’ wish is to reach over 50,000 sequences. Also making headway are the comparative analyses of the genetic load of the organisms. These comparisons are giving rise to traces of the evolutionary processes that have taken place over millions of years, which define both the similarities and the differences between the lineages of living beings. The groups from São Paulo discovered that, curiously, the Schistosoma is genetically more similar to the fruit fly (Drosophila melanogaster) than to the other worm whose genome has already been sequenced, Caenorhabditis elegans.
This surprising information is coming to light with the comparisons established using Leifsonia xyli subsp.xyli, which causes annual loses in the order of R$ 50 million, in just one of the varieties of sugarcane that are susceptible to it. “Leifsonia is very similar to Streptomyces and Mycobacterium, two genera that house species that are pathogenic for animals”, comments Luís Eduardo Aranha Camargo, a researcher from the Luiz de Queiroz College of Agriculture (Esalq) and one of the coordinators of the sequencing, co-financed by the Cooperative of the Producers of Sugarcane, Sugar and Alcohol of the State of São Paulo (Copersucar).
The close similarity of the Leifsonia genome – with 2,584,462 base pairs and around 2,600 genes, similar in size toXylella ‘s – with Streptomyces may have an immediate commercial value: Streptomyces is regarded as a biofactory of antibiotics, many of which are also found inLeifsonia. This explains the interest in patenting genes that are very similar to those that produce the two antibiotics, streptomycin and nisin. There is also the hope of arriving at new paradigms of plant/pathogen interaction, based on the analysis of the Leifsonia genome. “Perhaps this bacterium starts the process of infestation in sugarcane using a secreting system (through which the pathogenic bacterium exports its toxins to the outside the cell), similar to Mycobacterium, which causes tuberculosis and leprosy”, Camargo ponders.
The comparative analyses are revealing subtle genetic distinctions between bacteria of the same species, but with different victims. his is the case of the two varieties of Xylella fastidiosa, the one that attacks the orange tree, the subject of a pioneering work concluded in 2000, and the one that attacks grapevines – or Xylella PD, so called for causing Pierce’s Disease, in the region of wines in California, United States, and causes losses estimated at US$ 40 million a year. Concluded in the middle of last year, the sequencing of Xylella PD establishes some interesting parallels with the strain of the bacterium that infects the orange plantations in the interior of the state of São Paulo, causing Citrus Variegated Chlorosis (CVC), a pest also known in Brazil as the yellowing disease – responsible for the eradication of 10 million orange trees in 2001 and for losses estimated at R$ 650 million by the Fund for Citrus Plant Protection [RJS1] of São Paulo (Fundecitrus), which co-funded the sequencing.
The researchers identified a sequence of 70,000 base pairs found only in the citrus Xylella. Part of this sequence is also in Xanthomonas citri, which causes citrus canker, but was not detected in the grape Xylella, nor inXanthomonas campestris, which attacks cabbages. The strain of Xylella that attacks grapes is, by the way, smaller than the one that causes the yellowing disease: with some 2.5 million base pairs, it has 200,000 base pairs less. “This distinction may be a consequence of the evolutionary history of the bacterium, the result of the interaction between the bacterium and the plant, and between the plant and the environment”, is the deduction made by Marie-Anne Van Sluys, from USP’s Institute of Biosciences and one of the coordinators of the sequencing of the grape Xylella.
In the agriculture, prospects of work are opening up with the projects for sequencing the chloroplast. Located in the cytoplasm of plant cells, this structure contains a mini-genome: it also contains genetic information, like the chromosomes of the nucleus of the cell. It may be possible to create genetically modified plants by just altering the chloroplast, and in a way that is safer for the environment, since over 95% of plant species have no chloroplasts in their grains of pollen, where the male sexual cells are to be found. Modifications made to this mini-genome would not, then, be transmitted to other plants via pollen.
Helaine Carrer, from Esalq, is coordinating one of the pioneering works in the use of this method in Brazil: it is the sugarcane chloroplast genome project, with 141,182 base pairs, under the Sugarcane Genome project. Helaine is now able to say that the genome of the sugarcane chloroplast has greater similarity with the genome of the corn chloroplast than with that of other Gramineae, like rice (Oryza sativa) and wheat (Triticum vulgare). “The gene content observed in the sugarcane chloroplast is identical to corn chloroplast, including the positional arrangement of the genes”, she says. Intergenic regions exclusive to corn have proved to be present in sugarcane, with over 98% of similarity. “This is the greatest similarity between chloroplasts so far described, up to this moment”, she emphasizes.
The secret of this incredible similarity may lie in the mechanism of photosynthesis developed in the course of the plant’s evolutionary process. Both sugarcane and corn are plants with C4 photosynthesis (that is, the first product formed in the process of photosynthesis has 4 carbon atoms), while rice and wheat are C3 plants. “While the nuclear genomes of sugarcane and corn show many differences due to the evolutionary processes of each species, the chloroplast genomes have proved to be practically identical”, explains Helaine. The discovery has an application: any difference in the chloroplast may serve as a marker in research into genetic improvement.
Mariana Cabral de Oliveira, from USP’s Institute of Biosciences, is doing similar workwith Gracilaria tenuistipitata, a seaweed of Asian origins that produces agar, a gel used in the food, pharmaceutical and cosmetic industries. The economic importance of this organism was one of the reasons that elected it to be the subject of study, besides the fact that this species is easy to cultivate in the laboratory, and frequently used for studies in the physiology of photosynthesizing organisms. “It is almost a model organism”, says Mariana. The sequencing of the seaweed’s chloroplast, with its 183,883 base pairs, which began in October 2000, has revealed a group of genes related to photosynthesis, which could be manipulated to expand the seaweed’s productivity.
Mariana is also dedicated to the EST sequencing of Gracilaria, in collaboration with researchers from USP and from Sweden. 3,000 ESTs have now been sequenced – a small part of the organism, which has from 24 to 32 chromosomes, according to the species. The size that the nuclear genome of this seaweed can reach is not known, but species of seaweed with genomes larger than the human genome, which has 3 billion base pairs, are now known. Prospecting for genes promises other surprises.
Sugarcane well understood
The genome of the varieties of sugarcane most cultivated in Brazil has never been analyzed with so many approaches as in the 37 scientific articles that make up the most recent issue of the Brazilian magazine, Genetics and Molecular Biology, of the Brazilian Genetics Society. It was dedicated to the results of the Sugarcane Genome project, or Sucest, financed by FAPESP and Copersucar.
The articles – produced by about 200 researchers from 39 teams from the ONSA network – discuss varied aspects of the project and of the more than 43,000 clusters, regions of the sugarcane genome that may have one or more genes, identified by Sucest. “These works bring important information, not only for an understanding of the biology of the sugarcane, but also of other cultivated gramineae with a high commercial value, such as corn, rice and sorghum”, says Paulo Arruda, from the State University of Campinas (Unicamp), the coordinator of the project.
Some works are opening up prospects in the direction of a possible genetic manipulation, with the intention of improving the yield of sugarcane crops, increasing its resistance to diseases, or reducing the costs of production. The team led by Adriana Hemerly, of the Federal University of Rio de Janeiro (UFRJ), has identified a group of genes that seems to be associated with the symbiotic (beneficial) relationship between the two nitrogen fixing bacteria, Gluconacetobacter diazotrophicus and Herbaspirillum rubrisubalbicans and sugarcane. When they install themselves in the inside of the plant, they fix the nitrogen, which naturally reduces the need for using nitrogenous fertilizer. “We want to perfect this relationship of symbiosis”, says she.
The first results are heartening. After inoculating the two species of bacteria into the sugarcane, Adriana and her colleagues (from Embrapa Agrobiology, in Rio de Janeiro, and Unicamp as well) measured the expression (the level of use) of the genes of the plant in several situations. They saw that 274 genes from the Sucest database expressed themselves only in plants with G. diazotrophicus , 198 were used only when the sugarcane hosted H. rubrisubalbicans , and 62 only showed themselves in activity only when this plant showed the two bacteria simultaneously. “Before Sucest, we knew only four or five genes related to symbiosis.”
Along another line of work, a team coordinated by Marcelo Menossi, from Unicamp, identified 43 genes that may be related to the plant’s tolerance of aluminum, the excess of which reduces its productivity. “These genes may perhaps make it possible to control the plant’s resistance to aluminum”, says Menossi. Another article from Genetics of importance for biotechnology deals with the identification of SNPs, a kind of mutation, in the genome of sugarcane, a highly complex plant in whose DNA there may be up to 10 copies of the same gene. SNP stands for single nucleotide polymorphism, the chemical unit that forms the genetic sequence. The term indicates, then, mutations in pieces of genes that are caused by just one nucleotide being changed.
The majority of these mutations tends to be harmless, but others may affect the plant’s appearance or alter its behavior. If they are well understood and mapped, SNPs can be used as molecular markers. In a work with as yet preliminary results, researchers from Unicamp gave a small proof that the data from Sucest is useful for setting up a possible map of sugarcane SNPs: they analyzed two genes that codify the alcohol dehydrogenase enzyme, already known from other species, in which they identified 40 SNPs. “It is very difficult to differentiate the alleles (copies) of a sugarcane gene”, says Laurent Grivet, a researcher with Cirad, a French institute specialized in tropical agriculture, who works at Unicamp today. “I think that, with sugarcane, it is not going to be possible to relate the SNPs with their phenotype (the plant’s appearance), but we must use these polymorphs as molecular markers”.
1. Xylella fastidiosa PD Genome; Modality Agronomic and Environmental Genome Subprogram; Coordinators Marie-Anne Van Sluys and Mariana Cabral de Oliveira – USP; Investment US$ 925,181.84
2. Leifsonia xyli Genome; Modality Agronomic and Environmental Genome Subprogram; Coordinators Luís Eduardo Aranha Camargo and Cláudia Barros Monteiro Vitorello – USP; Investment US$ 1,150,841,.4
3. Schistosoma mansoni Genome; Modality Genoma – FAPESP Program; Coordinator Sérgio Verjovski-Almeida – USP; Investment US$ 1,244,697.50
4. Gracilaria tenuistipitata Genome; Modality Regular research benefit line; Coordinator Mariana Cabral de Oliveira – USP; Investment R$ 68,000.00 and US$ 34,000.00
5. Sugarcane Chloroplast Genome; Modality SUCEST – Sugarcane EST Sequencing Project; Coordinator Helaine Carrer – USP; Investment R$ 49,954.45 and US$ 207,341.15