Brazilian Genome Sequencing Projects: State of the Art

Recent Patents on DNA & Gene Sequences 2008, 2, 111-132 111

1872-2156/08 $100.00+.00 © 2008 Bentham Science Publishers Ltd.

Brazilian Genome Sequencing Projects: State of the Art

Eduardo Righi Capanema Xavier1,2,

*, Beatriz Parreiral Xavier Capanema

3, Jerônimo Conceição

Ruiz2, Guilherme Oliveira

2, Roberto Meyer

4, Vivian D’Afonseca

1, Anderson Miyoshi

1 and

Vasco

Azevedo1

1Departamento de Biologia Geral do Instituto de Ciências Biológicas da Universidade Federal de Minas Gerais.

Avenida Antônio Carlos, 6627. Campus Pampulha. Belo Horizonte, Minas Gerais. CEP 31.270-010, Brasil. 2Centro de

Pesquisas René Rachou. Fundação Oswaldo Cruz - FIOCRUZ. Ministério da Saúde. Avenida Augusto de Lima, 1715.

Barro Preto. Belo Horizonte, Minas Gerais. Brasil. CEP 30.190-002, 3Faculdade de Direito da Universidade Federal

de Minas Gerais. Avenida João Pinheiro, 100. Centro. Belo Horizonte, Minas Gerais. CEP 30.130-180, Brasil. 4Laboratório de Imunologia, Instituto de Ciências da Saúde. Universidade, Federal da Bahia. Avenida Reitor Miguel

Calmon, S/N-Vale do Canela. Salvador, Bahia. CEP 40140-100, Brasil.

Received: February 2, 2008; Accepted: April 2, 2008; Revised: April 14, 2008

Abstract: This review covers all Brazilian Genome, EST and Metagenome Projects, Sequencing Networks’ history and

structure, and patents related to Brazilian Genome Projects, beginning with the first genome sequenced in this country, i.e.

the 9a5c strain of Xylella fastidiosa CVC, up till the recently sequenced 1002 strain of Corynebacterium

pseudotuberculosis, which was done with a mixed strategy that included both traditional Sanger methodology and Avant

Garde 454 Life Sciences pyrosequencing technology. Almost 90% of all genomic research that has been done in Latin

America is a product of Brazil’s effort to support and stimulate OMICs in our country. Consequently, we gave special

attention to patents registered by Brazilian genome networks and/or Brazilian scientists involved in genomics,

transcriptomics, proteomics, EST, and metagenome projects, as well as in the development of bioinformatics software and

techniques.

Keywords: Genome, EST, metagenome, sequencing projects, Brazil, Xylella fastidiosa, Corynebacterium pseudotuberculosis, sanger, pyrosequencing, 454, OMICs, bioinformatics, patents.

INTRODUCTION

Those who potentially will benefit from DNA patenting around the globe should keep their sights turned towards Brazil. More than 85% of all sequencing projects in Latin America have been conducted in our country [1]. Along with coffee, soybean, banana, eucalyptus and sugar cane [2-4], Brazil’s key crop for renewable energy, many other orga-nisms have been and are being researched and sequenced throughout the country [5, 6]. We have focused our attention on genome projects executed in Brazil as well as EST and metagenome sequencing projects, such as the Coryne-bacterium pseudotuberculosis Genome Project, Brazil’s first genome sequencing project, carried out with 454 Life Sciences pyrosequencing technology [J.C. Ruiz, G. Oliveira - personal communication], along with the traditional Sanger method [7-10], conducted by the Minas Gerais Genome Network. We also made an effort to include in each section all available information on patents directly or indirectly related to these genome projects. It is likely that this type of systems biology research (omics) will reach a vertex in the next years due to the delivery of new sequencing techniques [7, 8, 9, 11, 12] that both drastically reduce sequencing costs and are able, in some specific cases, to provide up to a 100-fold increase in throughput over current Sanger sequencing

*Address correspondence to this author at the Departamento de Biologia

Geral do Instituto de Ciências Biológicas da Universidade Federal de Minas Gerais. Avenida Antônio Carlos, 6627. Campus Pampulha. Belo Horizonte,

Minas Gerais. CEP 31.270-010, Brasil; Tel/Fax: + 55 31 3409-2610; E-mail: [email protected]

technology [7, 12, 13]. Moreover, sequencing will become increasingly more viable for those interested in participating, thereby widening access to the global benefits provided by omics research.

A breakthrough for Brazilian molecular biologists and the first bioinformaticians, the Xylella fastidiosa genome was resolved in a coordinated effort involving various university laboratories [14]. It was the first phytopathogen to have its entire genome sequenced, and it provided a tremendous stimulus for the development of sequencing technology in Brazil [5, 6, 15]. It resulted in the creation of new genome networks [6], as well as relevant know-how, software and technical training and information. This first genome project involved a tremendous coordinated effort; consequently, some believed that Brazil could become a Panama Canal for scientists interested in real science and not in corporate profits. This is because science in Brazil was built and is sustained by federal (mostly) and state universities and research centers, all supported by public funds. Clearly, while this cooperative effort avoids the costs of competitive duplication, the possibility of profit is not ignored, since patenting is necessarily done in cooperation with the government whenever the research is government sponsored in federal and state universities and government-supported research centers [6]. That said; there is less incentive for entities such as corporations, joint-ventures and foreign parties to share intellectual property rights with these federal and state universities. Consequently, the commercial side of this process has not thrived and the patenting has not progressed much. However, this non-commercial orientation

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112 Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 Capanema Xavier et al.

has actually helped keep scientists interested in participating and cooperating. This is a paradox that works in Brazil for one specific reason: the strength and consistency of the sequencing networks concentrated in the south-east region of the country [5]. Fig. (1) illustrates this scenario. Brazil is a strong participant globally in genome sequencing projects Fig. (2).

BACKGROUND

The first organism to have its genome sequenced in Brazil was Xylella fastidiosa (species name reflecting its complex nutritional requirements condition); it is the cau-

sative agent of citrus variegated chlorosis (CVC), a disease that affects orange trees, grapevine, citrus and coffee, and it was the first phytopathogen worldwide to have its genomic sequence fully read. This sequencing was done in a cooperative effort by the Xylella fastidiosa consortium of the Organization for Nucleotide Sequencing and Analysis (ONSA) [16, 17]. This bacterium is a parasite of the host xylem; pathogenicity is a consequence of the activity of toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions media-ted by a range of proteins [18]. According to Doddapaneni et al. [19], there are currently four sequenced strains (9a5c, Dixon, Ann1 and Temecula-1). NCBI’s TaxBrowser

1 repor-

1 http://www.ncbi.nlm.nih.gov/sites/entrez?db=taxonomy

*Brazilian states by regions: North Region: AC - Acre; AM - Amazonas; RR - Roraima; RO - Rondônia; PA - Pará; AP - Amapá; TO -

Tocantins; Northeast Region: MA - Maranhão; PI - Piauí; CE - Ceará; RN - Rio Grande do Norte; PB - Paraíba; PE - Pernambuco; AL -

Alagoas; SE - Sergipe; BA - Bahia; Center-West Region: GO - Goiás; MT - Mato Grosso; MS - Mato Grosso do Sul; DF - Distrito Federal;

Southeast Region: SP - São Paulo; RJ - Rio de Janeiro; MG - Minas Gerais; ES - Espírito Santo; South Region: PR - Paraná; SC - Santa

Catarina; RS - Rio Grande do Sul.

** Abbreviations: CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico (National Council of Scientific and

Technological Development); FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo (Foundation for Research Support of the

São Paulo State); FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais (Foundation for Research Support of the Minas

Gerais State); FAPERJ - Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (Foundation for Research Support of the Rio de

Janeiro State);

Find figures at http://www.lgcm.icb.ufmg.br/publications.

Fig. (1). Map showing genome/EST/metagenome projects in Latin America with the working networks and the sequenced organisms.

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Brazilian Genome Projects Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 113

ted in November 2007 that two strains, 9a5c and Temecula1, both sequenced by the São Paulo State Consortium, had complete sequence status, whereas the Dixon and Ann1 strains were in the draft assembly stage [20]. Sequencing of Xyllela fastidiosa strains M12 and M23 is in progress and is being done by the DOE Joint Genome Institute. The State of São Paulo Research Foundation - FAPESP et al. has issued a patent [21] concerning the isolation and characterization of an operon of Xylella fastidiosa; this was published Novem-ber 15, 2001. It “relates to the isolation and characterization of an operon (…) referred to as the XfGUM operon, which contains nine genes and is involved in the production of a compound with xanthan-gum-like properties. The expression products of the operon and genes are also a part of the invention, as are various uses of these”. Moreover, the entire genome of X. fastidiosa was patented, with various uses described. This patent number WO02082432 [22], claims a “computer readable medium having recorded thereon a nucleic acid molecule from X. fastidiosa genome” that bears the X. fastidiosa genome sequence and allows therefore a determination as to whether a plant, a plant part, or a collection of plants are infested with X. fastidiosa or if any given organism is potentially pathogenic to plants by measuring homology against the X. fastidiosa genome, which is an interesting bioinformatics-generated offshoot of this patent. Another patent related to the X. fastidiosa genome was issued for Kirkpatrick and Guilhabert (2006) [23], who demonstrated an engineering resistance to Pierce’s Disease. A protein called HecA-like hemagglutinin and its gene are described; according to the inventors, this protein

constitutes a “molecular glue” that attaches to the bacteria surface, causing cells to aggregate. This mechanism is involved in bacteria-plant pathogenicity and invasion pro-cesses and could be used as a factor to slow down infection if transgenic plants expressing HecA-like hemagglutinin were to be engineered with this technology. It involves gene sequences, i. e. the HecA-like hemagglutinin sequence corresponding to the active bacterial gene, the physiological traits of this gene’s expression and orthologous-induced expression. The latest patent related to X. fastidiosa that we could find is entitled “Biocontrol of X. fastidiosa” [24]. It does not claim any sequences; rather it concerns specific strains, providing possible mechanisms or strategies for preventing disease in affected plants. The proposed biocon-trol could be attained by inoculating plants with the patented X. fastidiosa “benign” strain, so that bacteria populations can reach equilibrium, preventing harmful strains from causing damage.

Since the classic Xylella publication in Nature [14] in 1997, new genome, EST and metagenome projects (“soil microbial communities from contaminated sediments” developed by the Federal University of Santa Catarina - UFSC) have grown, and a number of genome networks have emerged following the ONSA example. Among these, the ONSA consortium along with the Minas Gerais Genome Network, a network managed by the René Rachou Research Center of the Oswaldo Cruz Foundation (FIOCRUZ), have conducted the Schistosoma mansoni Genome/EST Project, which was initiated in April 2001 [25-27]. Almost 180,000 EST clones were sequenced, providing 68,664,542

Source: GOLD - Genomes OnLine Database [1].

http://www.lgcu.icb.ufung.br/publications.

Fig. (2). Percentage of genome sequencing projects by country from January 2005 until September 2007, including complete, draft

assemblies and ongoing efforts, according to the GOLD Server in December 2007.

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114 Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 Capanema Xavier et al.

nucleotide sequences, of which 17,998,756 are non-redundant, according to the ONSA WebSite

2 [28-30]. This

flatworm, which belongs to the Trematoda class [31], along with two other Schistosoma species, Schistosoma japonicum and Schistosoma haematobium, infects more than 200 million people in over 70 countries [29, 32-35]. Verjovsky-Almeida [36] states in the Schistosoma Genome Project web site

3 of the University of São Paulo (USP) that “no patent

will be sought for sequence or clone information. It is expected that patents might result from future work with the clones, possibly for the protection of inventions and uses of such clones or sequences for diagnostic procedures, vac-cines, etc.” [37, 38]. Nevertheless, a patent with publication number WO05023979, registered in 2005, bearing the project title “Patente para o uso de genes encontrados no Projeto Genoma do Schistosoma como diagnóstico, alvo de desenvolvimento de vacina ou de drogas”

4 was deposited in

the WIPO database with the title “Isolated S. mansoni Nucleic Acid Molecules and Uses thereof” [39]; it was registered, with FAPESP as first applicant and Sérgio Verjovski-Almeida as first inventor. This patent claims “an isolated nucleic acid molecule which encodes an S. mansoni protein, or a portion thereof which is at least 20 amino acids in length”. Also, the Oswaldo Cruz Foundation (FIOCRUZ) developed a kit for schistosomiasis diagnosis through PCR. This invention was patented [40], with publication number WO01075148. Also related to the Schistosoma mansoni research developed in Brazil, the Federal University of Minas Gerais, along with Dr. Sérgio Costa Oliveira has a patent (WO07118292) that involves the production of recombinant proteins by means of genetic engineering techniques (recombinant DNA) - ad hoc membrane protein Sm29 - and an immunoenzymatic assay to detect IgG antibodies against the Sm29 protein [41]. Similarly, many other S. mansoni patents involve proteins and their encoding regions for vaccination uses [42-47] and for antibody production [48]. Glutathione-s-transferase Sm26 of S. mansoni, the DNA encoding 28 kDa glutathione-s-trans-ferase of Schistosoma mansoni and the protein product are described. There are also patents for antibody-producing clones that react with antigens produced by S. mansoni for diagnostic purposes and for pharmaceutical preparations [48]. An especially interesting patent involving Schistosoma describes a method of transgene expression and secretion in this trematode via viral or non-viral vectors in order to turn the parasite into a drug delivery organism [49]. It is a method to produce Schistosoma worms that would serve as intermediate vectors that could express gene products into the patient’s bloodstream and could also be used for mass production, quality control, termination of therapy and dose titration [50]. It is a gene therapy method that would substitute the patient's own cells to produce the desired gene product, e. g. insulin for diabetic patients or erythropoietin for patients with anemia caused, for instance, by chronic kidney disease [51] or by solid organ transplantation [52, 53].

2 http://verjo18.iq.usp.br/schisto/

3 http://verjo18.iq.usp.br/schisto/About/info.html

4 A translation to the patent’s project title: “Patent to the use of genes found in the

Schistosoma Genome Project as diagnostic, development of vaccine targets or drugs”.

Considerable effort was made to put together a large national genome network. This resulted in the creation of the Brazilian National Genome Project Consortium, which brought together 25 laboratories, a bioinformatics center and three coordination laboratories. The first project carried out by the Brazilian National Genome Project Consortium, published in 2003, was the Chromobacterium violaceum Genome Project [54-56]. Chromobacterium violaceum is a Gram-negative free-living bacteria that inhabits soil and water in tropical areas [57, 37]; it infects mammals, inclu-ding human beings, [58] often causing death by septicemia [59]. The Brazilian National Council for Scientific and Technological Development (CNPq) et al. issued in 2004 a patent [60] entitled “Gene-Coding Polynucleotides of the Chromosome of the Bacterium Chromobacterium violaceum, Expression and Activity of these Polynucleotides and their Applications”, which constitutes more than a whole-genome patent in its claiming extensions. It claims for polynuc-leotides or groups that are identical or have a similarity of at least 70% to those identified with SEQ ID numbers 1-29, along with fragments thereof coding for the oxidation-reduction of arsenic, cyanide, cyanate and other heavy me-tals, synthesis of hydrocyanic acid and biocide compounds, chitinases and chitin dextrinases, hemolysins, polyhydroxy-alkanoates, violacein, and polypeptides inducible by herbicides and that can be preferentially isolated from the chromosome of the ATCC 12472 strain of C. violaceum. The patent also claims complementary polynucleotides of those cited or that include at least 15 successive nucleotide bases of the polynucleotide sequences from those cited. It constitutes a massive patent effort to obtain control of the sequenced polynucleotides and forthcoming sequences of any C. violaceum strain or other organisms bearing such sequences that could eventually be sequenced. A German organization has patented a method for biosynthesis of the natural blue-violet colorants violacein and desoxyviolacein that is based on the use of C. violaceum pigments [61]. Another patent related to this organism makes use of C. violaceum as well as Chlorella vulgaris to recover and concentrate gold utilizing the microorganism’s capa-bility of producing cyanide ion, forming a cyanide ion-con-taining culture with gold ore. It is therefore a methodological patent [62].

The second project that the Brazilian National Genome Project Consortium worked on was a Mycoplasma sequence project, which comprised: (i) Mycoplasma hyopneumoniae pathogenic 7448 strain; (ii) non-pathogenic Mycoplasma hyopneumoniae strain J; and (iii) Mycoplasma synoviae, an avian pathogen. According to Vasconcelos et al. (2005) [63], Mycoplasmas are a group of more than 180 species with no cell wall that parasitize various organisms, including plants, humans and other animals. Due to their nutritional requirements, they typically exhibit host tissue specificities. This is a feature that is likely related to the fact that this group has undergone considerable genome reduction during its evolution, probably as a consequence of taking advantage of complementary host metabolism mechanisms. However, according to the authors [63], bottlenecks and asexual reproduction have been responsible for the accumulation of deleterious mutations in these organisms, resulting in

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Table 1. Brazilian Genome, EST and Metagenome Projects, with All Related Patents

Organism or Subject Genome Network or

Responsible Center Status Type of Project Related Patent Numbers

Anopheles darlingi CNPqa, LNCCa Incomplete Genome and EST WO07073591*

WO06029605

WO04075912

WO05103243

WO04034783

WO04029088

WO03087322

WO03079796

WO03020913

WO03013238

WO01000667

WO94006295

Bos indicus ESALQa/ USPa Incomplete EST WO98050401

Bradyrhizobium japonicum

SEMIA 5079

EMBRAPAa Incomplete Genome WO07094885

WO02008386

WO01038492

WO00004778

WO87007910

Cancer Genome Project Ludwig Institute for Cancer Research

- SP

Incomplete EST WO04031229

Chromobacterium violaceum

ATCC 12472

Brazilian National Genome Project

Consortium, CNPq, MCTa

Complete Genome WO04056960

WO02050299

WO91008316

Coffea arabica UNICAMPa, FAPESPa

EMBRAPA/Café, EMBRAPA/Café,

Brazilian Consorcium for the

Research and Development of Coffee

Complete Genome -

Corynebacterium pseudotuberculosis

1002

FIOCRUZa/CPqRRa/ UFMGa/

FAPEMIGa

Incomplete Genome WO90011351

WO07074991

WO06008098

WO06008097

WO04050694

WO03046123

WO02051231

WO02022799

WO01066573

WO01049854

WO01049854

WO01002583

WO01002583

WO95019442

WO03040290

WO03040180

WO02100530

WO01085967

WO01000843

WO01000843

WO01000844

WO01000842

WO01000805

WO01000804

WO01000802

WO03040181

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116 Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 Capanema Xavier et al.

(Table 1) Contd....

Organism or Subject Genome Network or

Responsible Center Status Type of Project Related Patent Numbers

Eimeria acervulina

USP Incomplete EST WO06113594

WO04052393

WO04002527

WO03004684

WO03004683

WO99050387

Eimeria maxima

USP Incomplete EST WO06113594

WO04052393

WO04002527

WO03054157

WO03004684

WO03004683

WO99050387

Eimeria tenella

USP Incomplete EST WO06113594

WO05070180

WO05010040

WO05005472

WO04052393

WO04002527

WO03004684

WO99050387

Eucalyptus grandis

FAPESP Incomplete EST WO06052554

WO05032241

WO04048595

WO98013503

WO97030162

Gluconacetobacter diazotrophicus

PAL5

UFRJa, LNCC/MCT, EMBRAPA,

UENFa, UERJa, FAPERJa, CNPq,

MCT

Incomplete Genome WO02045513

Herbaspirillum seropedicae

Z67

GenoPar Consortiuma Incomplete Genome WO02045513

WO07100162

Leifsonia xyli xyli

CTCB07

FAPESP/ UNICAMP Complete Genome WO07026860

Leishmania chagasi ProGeNea, MCT, BNBa, CNPq Incomplete EST WO96033414

Leptospira interrogans Copenhageni

Fiocruz L1-130

FAPESP, CNPq, USP, UNICAMP Complete Genome WO06002631

Litopenaeus Vannamei UFSCara, CNPq Incomplete EST WO00034476

Moniliophthora (Crinipellis)

perniciosa

Singer

UNICAMP, SEAGRIa Incomplete Genome -

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(Table 1) Contd....

Organism or Subject Genome Network or

Responsible Center Status Type of Project Related Patent Numbers

Mycobacterium bovis BCG

Moreau RDJ

FAPa, FENAMa

Incomplete Genome WO07130846

WO07091881

WO07014885

WO07010413

WO06128390

WO06102767

WO06060663

WO05111205

WO05077411

WO05005639

WO05004911

WO04083459

WO04055214

WO04011436

WO03093307

WO03089462

WO03070981

WO03053459

WO03053459

WO03038402

WO03011336

WO02050262

WO99045119

WO99002670

WO98032862

Mycoplasma hyopneumoniae

7448

LNCC, UFRGSa Complete Genome WO07116032

WO07103042

WO06056841

WO05112995

WO05060328

WO05009462

WO04058142

WO04003161

WO03004051

WO03003941

WO02049666

WO02010343

WO00031115

WO99026664

WO96028472

WO95009870

WO93016726

WO92018161

WO92003157

WO91018627

WO86000019

Mycoplasma hyopneumoniae J

LNCC, UFRGS Complete Genome WO07116032

WO07103042

WO06056841

WO05112995

WO05060328

WO05009462

WO04058142

WO04003161

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(Table 1) Contd....

Organism or Subject Genome Network or

Responsible Center Status Type of Project Related Patent Numbers

WO03004051

WO03003941

WO02049666

WO02010343

WO00031115

WO99026664

WO96028472

WO95009870

WO93016726

WO92018161

WO92003157

WO91018627

WO86000019

Mycoplasma synoviae

53

LNCC, UFRGS Complete Genome -

Paracoccidioides brasiliensis

Pb01 ATCC-MYA-826

UnBa, UFGa, MCT, CNPq Incomplete Differential Gene

Expression

WO98055649

Pichia (Hansenula) angusta

(polymorpha)

UNICAMP Incomplete Genome WO03052063

Rhizobium tropici

PRF 81

EMBRAPA Incomplete Genome WO07137075

WO07107000

WO07058086

WO07006318

WO06134623

WO06090913

WO05115412

WO05054462

WO05019439

WO04005517

WO03089640

WO03020014

WO02015703

WO02015702

WO02013614

WO01060159

WO00044221

WO99033966

WO99013855

WO98012206

WO98002560

WO95023156

WO95017806

WO94025568

WO94019924

Saccharum sp. UNICAMP, FAPESP Incomplete EST WO07106966

WO98043631

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(Table 1) Contd....

Organism or Subject Genome Network or

Responsible Center Status Type of Project Related Patent Numbers

Schistosoma mansoni ONSAa, FAPESP Complete EST WO05023979

WO01075148

WO07118292

WO06088951

WO04043998

WO03002597

WO02085930

WO00017654

WO97033613

WO97033610

WO91012327

WO91009621

WO90002563

WO83001837

Soil

microbial communities

from contaminated sediments

UFSCa, CNPq Incomplete Metagenome WO05090595

WO04018673

WO03054177

Trypanosoma rangeli

SC-58, Choachi

UFSC, CNPq Incomplete EST -

Xanthomonas axonopodis pv.

aurantifolii B

11122

FAPESP, USP Incomplete Genome WO03001911

Xanthomonas axonopodis pv.

aurantifolii C

10535

FAPESP, USP Incomplete Genome WO03001911

Xanthomonas axonopodis pv. citri

XV101, 306

FAPESP, USP, UNICAMP Complete Genome WO03001911

Xanthomonas campestris campestris

ATCC 33913

FAPESP, USP Complete Genome WO06003989

WO05054470

WO02072795

WO02044352

WO02012293

WO01007635

WO00078967

WO00020616

WO98041612

WO98012301

WO98002545

WO96036765

WO93021307

WO93017096

WO93017096

WO90015143

WO88001172

WO87005937

WO87000006

WO86001830

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(Table 1) Contd....

Organism or Subject Genome Network or

Responsible Center Status Type of Project Related Patent Numbers

Xanthomonas smithii citri FAPESP, USP, UNICAMP Incomplete Genome -

Xylella fastidiosa

Pierce's Disease Strain

UNICAMP, FAPESP, USDAa Incomplete Genome WO06069160

WO05023005

WO05023005

WO02082432

WO01085905

Xylella fastidiosa CVC

8.1.b clone 9.a.5.c

ONSA, FAPESP Complete Genome WO02082432

WO01085905

Xylella fastidiosa-grape

Temecula1

FAPESP, USDA, AEGa, Brazilian

National Genome Project Consortium

Complete Genome WO02082432

WO01085905

Zea mays FAPEMIG, UFMG Incomplete Differential Gene

Expression

WO06084868

WO04063379

WO02006322

WO01083790

WO00053732

* Brazilian Group or Researcher’s patents set in boldface; a Abbreviations used herein: ESALQ - Escola Superior de Agricultura “Luiz de Queiroz”; USP - Universidade de São Paulo;

EMBRAPA - Empresa Brasileira de Pesquisa Agropecuária; CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico; MCT - Ministério da Ciência e Tecnologia;

UNICAMP - Universidade de Campinas; FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo; FIOCRUZ - Fundação Oswaldo Cruz; CPqRR - Centro de Pesquisas

René Rachou; UFMG - Universidade Federal de Minas Gerais; FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais; UFRJ - Universidade Federal do Rio de

Janeiro; LNCC - Laboratório Nacional de Computação Científica; UENF - Universidade Estadual do Norte Fluminense; UERJ - Universidade do Estado do Rio de Janeiro; FAPERJ -

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro; ONSA - Organization for Nucleotide Sequencing and Analysis; GenoPar - Programa Genoma do Estado do Paraná;

ProGeNe - Programa de Genética e Farmacogenética do Instituto de Psiquiatria do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo; UFSCar -

Universidade Federal de São Carlos; SEAGRI - Secretaria da Agricultura, Irrigação e Reforma Agrária do Estado da Bahia; FAP - Fundação Ataulpho de Paiva; FENAM - Fundação

Nacional dos Médicos; UFRGS - Universidade Federal do Rio Grande do Sul; UnB - Universidade de Brasília; UFG - Universidade Federal de Goiás; UFSC - Universidade Federal

de Santa Catarina; USDA - United States Department of Agriculture; AEG - Agronomical and Environmental Genomes.

Source: GOLD - Genomes OnLine Database. http://www.genomesonline.org [1].

minimal genomes that maintain basic core functions and adaptations required for their survival through specific hosting and intrinsic parasitism. Mycoplasma hyopneu-moniae is the causative agent of enzootic pneumonia in pigs, whereas M. synoviae is responsible for respiratory tract disease and synovitis in chickens and turkeys. Gene sequen-ces of M. hyopneumoniae have been patented by the National Council for Scientific and Technological Develop-ment (CNPq) et al., referring to the polypeptides encoded by these genes and the uses thereof, including the protection induced by the immune response animals produce against these polypeptides [64]. Antigenic potential is therefore one of the aims of these patents. They also specify intellectual property of similar polynucleotides that have at least 70% identity, along with nucleotide sequences coding for polypeptides by amino acid sequences and fragments. These correspond to pathogenic strain 7448 of M. hyopneumoniae. Methods for serological diagnosis of M. hyopneumoniae infection in mammals and a cultivation process that is preferential for this organism were also included. A SEQ ID Nr. 1 was sought, together with the DNA coding sequence for the protein described, its regulator sequence, the vector used to express the claimed protein, the host cell (E. coli) that was used to express the protein, the immunogenic

composition that the claimed protein bears as well as the immunogenic composition of a fragment of the claimed protein, the method used to induce the immune response in the animal with the claimed protein, a method to detect antibodies produced by the administration of the protein (diagnostic method) and the amount of protein that should be used. A large number of patents related to M. hyopneu-moniae describe vaccination methods, including a vaccination kit with an immunogenic dose of live attenuated Porcine Reproductive Respiratory Syndrome (PRRS) virus, which involves a novel method of vaccination of a pig by administering a whole or partial cell, a modified live preparation, a subunit, a nucleic acid or DNA vaccine of M. hyopneumoniae to a weaned piglet; a M. hyopneumoniae multi-strain vaccine in the form of whole or partial cell bacteria or in the form of a subunit vaccine; a mixed vaccine for preventing porcine respiratory disease, comprising inacti-vated Haemophilus parasuis S4 and S5, M. hyopneumoniae and PRRS virus to prevent swine enzootic pneumonia, PRRS, porcine respiratory disease complex (PRDC) and post-weaning multisystemic wasting syndrome (PMWS); an oil-in-water emulsion of bacterial or viral suspensions that acts as a vehicle for the delivery of a pharmaceutical com-position, ad hoc inactivated M. hyopneumoniae bacterium,

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Brazilian Genome Projects Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 121

an inactivated d porcine circovirus type 2 (PCV-2) virus or combinations thereof; another combined vaccine with M. hyopneumoniae with at least one viral antigen comprising an adjuvant mixture of acrylic acid polymer, a mixture of a metabolizable oil, such as one or more unsaturated terpene hydrocarbons, and a polyoxyethylene-polyoxypropylene block copolymer; M. hyopneumoniae polypeptides and nuc-leic acids, as well as nucleic acid expression vectors con-taining nucleic acids for treating swine to prevent enzootic pneumonia; another vaccine with whole or partial cell inactivated or modified live preparations, a subunit vaccine, or a nucleic acid or DNA vaccine that is synthesized or recombinantly produced; a M. hyopneumoniae bacterin vaccine composition that provides immunity against infec-tion after a single administration; a temperature-sensitive vaccine; methods of identifying gene sequences of potential vaccine antigens and polypeptides encoded by the gene sequences as well as the use of such sequences to induce a protective immune response in animals with the use of a marker (preferably polyHis tag) co-expressed with a protein translated from the nucleotide sequence; a M. hyopneu-moniae protein prepared by recombinant DNA or synthetic means, its DNA coding sequence, the expression vector and methods of treating swine to prevent enzootic pneumonia using vaccines as well as diagnostic tests based on the protein or antibodies produced against the protein [65]; a putative protective antigen against Mycoplasma spp. prepared by a method that involves applying a Mycoplasma with an antibody probe, producing a biological sample taken a short time after an immune animal has been challenged with Mycoplasma; a method for making an inactivated vaccine of M. hyopneumoniae by inactivating the bacteria with thimerosal and mixing it with aluminum hydroxide adjuvant and DEAE dextran; a process of inactivation of organisms of the genera Mycoplasma and Acholeplasma, comprised of treating them with ethyleneimine or a suitable derivative thereof that is particularly applicable to the production of a vaccine against M. hyopneumoniae; a bac-terin comprised of a sufficient amount of a virulent M. hyopneumoniae isolate that has been inactivated with binary ethyleneimine to immunize a pig, along with a suitable physiologically acceptable carrier; vaccine components and compositions useful for vaccinating pigs against M. hyopneumoniae and secondary pathogenic infections; and a vaccine intended to protect against diseases caused by micro-organisms that have a membrane as the most external envelope, such as M. hyopneumoniae, which contains, as the active principle, plasmic membranes of the microorganisms that cause these diseases [66-84, respectively]. Finally, a patent was applied for that involves adhesins, a feature that Mycoplasma exhibit [85]. This patent describes the invention as the identification, purification and characterization of M. hyopneumoniae adhesins with an affinity chromatography procedure, which incorporates a novel M. hyopneumoniae receptor analogue for the attachment and/or removal of the adhesins from a mycoplasmal preparation. It also describes an in vitro microtiter plate adherence assay for the charac-terization of M. hyopneumoniae and adhesins from that microorganism. Monoclonal antibodies against the adhesins are also included.

The Brazilian National Genome Project Consortium, through its management of the National Laboratory for Scientific Computing (LNCC), has started to sequence the Anopheles darlingi genome and has undertaken the challenge to develop the corresponding EST project simultaneously [86-88]. Anopheles darlingi is a mosquito vector of malaria, a disease affecting more than 300 million people yearly according to the WHO and other sources [89-94]. This disease is caused by the protozoan Plasmodium. In Brazil, especially in Amazonia, this disease has increased because of uncoordinated land settlement [95-98]. According to the LNCC Anopheles darlingi Genome/EST Project Web Site

5,

studies performed in the Rio Negro region have shown that about 95% of the anophelines in contact with man are A. darlingi species; it also states that the project’s main goal is to compare the A. darlingi genome with that of A. gambiae [99-101]. Malaria causes the individual to experience fever and chills at differing regular time intervals, depending on the type of Plasmodium the individual is infected with [89, 102, 103]. If not treated appropriately, malaria can cause severe complications and death. Prophylaxis can be achieved by the use of bed netting and insecticides to avoid mosquitoes, along with antimalarial drugs [89-91,104]. There are also preventive drugs that can be taken, for instance, by travelers who go to malaria-risk areas [90, 91]. Plasmodium falciparum is the agent of the most severe form of malaria in humans [91,96,105]. It has a complex life cycle; during asexual stages, the protozoan multiplies within red blood cells, causing them to literally explode or break apart at the end of the schizogony manifestation. This pheno-menon occurs cyclically and is responsible for the inter-mittent pathognomonic characteristic of Plasmodium infec-tion [106-108]. While in the invertebrate host, Plasmodium can also produce via asexual reproduction infective stages known as sporozoites that accumulate in the mosquito’s salivary glands and can initiate vertebrate infection [91,92, 109]. Patents related to Anopheles have been concentrated on drugs able to control the spread of Plasmodium within the mosquito population and on techniques to map and diminish mosquito population growth. Molecules have also been registered, such as proteins, with their coding DNA seq-uences and organic compounds that could trigger immune responses in the anophelines in order to make them refrac-tory to the Plasmodium invasion [110, 111]. A patent applied for by the European Molecular Biology Laboratory (EMBL) describes a procedure that attempts to prevent the trans-mission of malaria through the mosquito by modulating the vector’s innate immune responses to the Plasmodium parasite by focusing on gene families that encode pedtidoglycan recognition proteins (PGRPs), leucine-rich repeat proteins (LRRPs) and C type lectins (CTLs) using pattern recognition receptors (PRRS) [112]. Similarly, another patent focuses on the use of thioester-containing proteins that are part of the immune system of the mosquito A. gambiae, using them in mosquitoes of the genus Ano-pheles in order to make them refractory towards the malaria parasite [112]. Two carboxy-peptidase B enzymes from A. gambiae and homologs thereof have been patented by the Pasteur Institute in France. The carboxypeptidases would be used in the production of vaccines; the patent includes the

5 http://www.darlingi.lncc.br/index.htm

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122 Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 Capanema Xavier et al.

antibodies produced in this manner along with the methods of using them. They are aimed at blocking Plasmodium development in the inver-tebrate host. Another patent developed by the Pasteur Institute isolates nucleic acid molecules that encode multiple drug resistance proteins from Drosophila melanogaster or A. gambiae (dMRP or gMRP vectors - as cited in the patent registry) and describes how host cells can be transformed with vectors containing these nucleic acids [113]. Patent WO03087322 specifies equipment and methods for the production of strains of haematophagous insects with desired properties, such as hypoallergenicity or hyperinfectivity, along with methods of producing a parasite strain able to withstand cryopreservation at temperatures close to freezing, equipment and methods for the injection of an attenuated parasite vaccine and for the production of parasites and haematophagous insects that are free from contamination by unwanted biological agents, as well as equipment and procedures for the aseptic rearing of parasites with complex life cycles to avoid contamination [114]. Patent WO03020913 describes an invention related to A. gambiae odor receptor genes as well as methods for identifying odor receptor genes; it describes nucleotide sequences of A. gambiae odor receptor genes and amino acid sequences of their encoded proteins (including peptides or polypeptides), with their related products and methods. The inventors state that these sequences may be operatively linked to promoter sequences and transformed into host cells. They describe methods of production, i.e. recombination methods, for antibodies to an odor receptor protein, along with derivatives and analogs. Methods for identifying molecules to bind or modulate the activity of these odor receptor genes are also provided [115]. The government of the United States of America, represented by the Secretary of the Department of Health and Human Services is an applicant for a patent claiming the DNA and amino acid sequences of a novel anti-thrombin peptide, named anophelin, which was isolated from the salivary glands of the mosquito Anopheles albimanus.

Anti-thrombotic therapeutic applications of anophelin are also disclosed [116]. A patent presented by A.E. Eiras [117] along with the Federal University of Minas Gerais describes a trap and methods to capture mosquitoes of the species Aedes aegypti, Aedes albopictus, Anopheles sp. and Culex sp. in order to monitor, detect and control them. The trap is a dark container with at least one opening, with a totally or partially sticky inner surface [118-120].

NETWORK STRUCTURE

Brazil is currently the Latin American leader of the genomics field Fig. (1); the nearly 40 genome/EST/ metagenome projects around the country follow the operational design that is typical of our multi-institution government funded omics projects [121]. Since 1997, with the foundation of the ONSA Network [16], most of the sequencing projects that have been carried out in the various states that support these programs (São Paulo, Minas Gerais, Rio de Janeiro, Santa Catarina, Rio Grande do Sul, Bahia and Santa Catarina) have followed this design [122]. As A.J.G. Simpson [5] states, these “contributions have been achieved exclusively through the use of large networks, a strategy that has proved to be particularly appropriate for our own, relatively small scientific community in a developing country”.

A genome network is an association of university laboratories, federal and state research centers and colla-borators willing to share both time and resources on a given sequencing project. These groups include laboratory leaders and co-workers who divide the sequencing work, with overall financing by government research and development funding agencies. These research support agencies finance such projects (i) directly, by granting funds to keep the sequencing process running, buying reagents, dyes and cloning kits and (ii) indirectly, by maintenance of the lab facilities, including sequencing machines, staff payrolls and general lab equipment. Consequently, these funding agencies

Abbreviations: JGI - Joint Genome Institute (DOE/USA); TIGR - The Institute for Genomic Research; JCVI - J. Craig Venter Institute;

WashU - Washington University; Sanger - Wellcome Trust Sanger Institute; BROAD - Broad Institute (MIT/Harvard); GEN

(GENOSCOPE) - Centre National de Séquençage (France); FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo; CNPq -

Conselho Nacional de Desenvolvimento Científico e Tecnológico; FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais;

FAPERJ - Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro. Source: GOLD - Genomes OnLine Database [1].

Find figures at http://www.lgcm.icb.ufmg.br/publications.

Fig. (3). Foundations that fund genome projects in Brazil and in the world showing main institutes and groups responsible for these genome

projects.

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Brazilian Genome Projects Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 123

are applicants for all the patents that are products of this genome research see Table (2).

ORGANIZATION FOR NUCLEOTIDE SEQUENCING

AND ANALYSIS (ONSA)

Financed by the Foundation for the Support of Research in the State of São Paulo

6 (FAPESP), the Organization for

6 A “Fundação de Amparo” could be translated as “Foundation for Research Support”.

For instance, the “Fundação de Amparo à Pesquisa do Estado de São Paulo” could be

translated as “The São Paulo State Foundation for Research Support” or simply “The

São Paulo State Research Foundation”. Thus, the Foundations for Research Support are

organized by state but are sustained by federal funds.

Nucleotide Sequencing and Analysis (ONSA) is a pioneer sequencing network in Brazil [134]. It was responsible for carrying out the full sequencing of the phytopathogen Xylella fastidiosa CVC 9a5c. It basically involves two major univer-sities in Brazil, the University of São Paulo (Universidade de São Paulo - USP) and the University of Campinas (Univer-sidade de Campinas - UNICAMP). Other major genome projects, including Saccharum sp. (sugar cane), Coffea arabica (tetraploid coffee species) and Schistosoma mansoni, are being conducted by ONSA through partnerships with other networks and collaborators.

Table 2. Funding Agencies and Managers

Group Location (City, State) Genome/EST/Metagenome Projects Web Sources

Funding Agency

CNPq/MCT

Brasília, Distrito

Federal (DF)

Anopheles darlingi, Chromobacterium violaceum, Mycoplasma

hyopneumoniae, Mycoplasma synoviae, Rhizobium tropici,

Paracoccidioides brasiliensis, Crinipellis perniciosa, Leishmania

chagasi, Gluconacetobacter diazotrophicus, Leptospira interrogans,

Litopenaeus vannamei, Herbaspirillum seropedicae, Soil microbial

communities from contamined sediments

http://www.cnpq.br

FAPESP

São Paulo, São Paulo

(SP)

Bos indicus, Coffea arabica, Mycobacterium bovis, Human Cancer

Genome, Leifsonia xyli xyli, Saccharum sp., Schistosoma mansoni,

Xanthomonas axonopodis, Xanthomonas campestris, Xanthomonas

smithii, Xylella fastidiosa

http://www.fapesp.org

FAPEMIG Belo Horizonte, Minas

Gerais (MG)

Schistosoma mansoni, Corynebacterium pseudotuberculosis, Zea mays http://www.fapemig.br

FAPERJ Rio de Janeiro, Rio de

Janeiro (RJ)

Gluconacetobacter diazotrophicus http://www.faperj.br/

Manager Group

LNCC

Petrópolis, Rio de

Janeiro (RJ)

Anopheles darlingi, Bradyrhizobium japonicum, Chromobacterium

violaceum, Mycoplasma hyopneumoniae, Mycoplasma synoviae

http://www.lncc.br/frame.html

CPqRR/FIOCRUZ Belo Horizonte, Minas

Gerais

Schistosoma mansoni, Corynebacterium pseudotuberculosis, Zea mays http://www.cpqrr.fiocruz.br

EMBRAPA Brasília, Distrito

Federal (DF)

Bradyrhizobium japonicum, Coffea arabica, Zea mays http://www.embrapa.br/

USP São Paulo, São Paulo

(SP)

Xylella fastidiosa, Xanthomonas axonopodis, Xanthomonas

campestris,Xanthomonas smithii

http://www2.usp.br/portugues

/index.usp

UNICAMP Campinas, São Paulo

(SP)

Xylella fastidiosa, Xanthomonas axonopodis, Xanthomonas campestris,

Xanthomonas smithii

http://www.unicamp.br/

UFMG Belo Horizonte, Minas

Gerais (MG)

Corynebacterium pseudotuberculosis, Schistosoma mansoni, Zea mays http://www.ufmg.br

UnB Brasília, Distrito

Federal (DF)

Paracoccidioides brasiliensis http://www.unb.br/

UFBA Salvador, Bahia (BA) Crinipellis perniciosa http://www.portal.ufba.br/

Abbreviations: CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico; MCT - Ministério da Ciência e Tecnologia; FAPESP - Fundação de Amparo à Pesquisa do

Estado de São Paulo; FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais; FAPERJ - Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro; LNCC -

Laboratório Nacional de Computação Científica; CPqRR - Centro de Pesquisas René Rachou; FIOCRUZ - Fundação Oswaldo Cruz; EMBRAPA - Empresa Brasileira de Pesquisa

Agropecuária; USP - Universidade de São Paulo; UNICAMP - Universidade Estadual de Campinas; UnB - Universidade de Brasília; UFBA - Universidade Federal da Bahia.

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124 Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 Capanema Xavier et al.

BRAZILIAN NATIONAL GENOME PROJECT CONSORTIUM

The Brazilian National Genome Project Consortium includes 25 laboratories, a bioinfomatics center and three coordinating laboratories distributed throughout much of the country. It is likely the largest genome network currently active in Brazil and was responsible for the Chromo-bacterium violaceum Genome Project, which started in 2001 and was published in 2003 [54]; it also has taken on the Mycoplasma Genome Project, which comprises Mycoplasma hyopneumoniae strains 7448 and J and Mycoplasma synoviae [63], and runs the Anopheles darlingi Genome/EST Project.

MINAS GERAIS GENOME NETWORK (RGMG)

Supported by the State of Minas Gerais Research Foundation (FAPEMIG), the RGMG includes collaborating laboratories of the Federal University of Ouro Preto (UFOP), EMBRAPA (Empresa Brasileira de Pesquisa Agropecuária - Brazilian Agricultural Research Corporation) Milho e Sorgo (Maize and Sorghum), the Federal University of Minas Gerais (UFMG), the Federal University of Viçosa (UFV), the Federal University of Lavras (UFLA) and the Federal University of Uberlândia (UFU). This network is managed by the Oswaldo Cruz Foundation (FIOCRUZ), through one of its research centers in Belo Horizonte, the capital of Minas Gerais state, the René Rachou Research Center (CPqRR).

The Minas Gerais Genome Network has organized two genome projects to date; these are the Schistosoma mansoni Genome Project

7 and the Corynebacterium pseudotuber-

culosis Genome Project [123-125]. The latter is the first genome project in Brazil that has used both Sanger traditional sequencing technology and a pyrosequencing technology developed by Roche Applied Sciences, 454 Life Sciences pyrosequencer [7,126,127]. Corynebacterium pseudotuberculosis is a gram-positive actinobacteria that causes a disease that affects small ruminants, which is com-monly known as caseous lymphadenitis (CLA) [128]. This disease is hard to eradicate due to the limited effectiveness of available drug therapy [123]. Actinobacteria is a group that includes many organisms that have already been sequenced [129, 130]. Genera of considerable medical, veterinary and biotechnological interest, such as Mycobacterium, Nocardia and Rhodococcus, are also part of this suprageneric taxon [131, 132]. One of the most important toxins of C. pseudo-tuberculosis involved in caseous lymphadenitis infection is phospholipase D (PLD) toxin, which was patented in 1990 by the Commonwealth Serum Laboratories Commission [133]. A vaccine composed of a toxoided pure PLD toxin was developed for use against CLA in sheep, based on nucleotide and amino acid sequences of the toxin; these are disclosed in the patent, together with the cloning and expression of the PLD toxin gene in E. coli and coryneform bacteria. Another patent registered in 2007 [134] describes an inducible expression vector that can be used against

7 The Schistosoma mansoni Genome Project carried out by the Minas Gerais Genome

Network is working along with the EST Project managed by the ONSA Network. The

results have been delivered together and most of the work has been done as a form of

partnership, including microarray analysis carried out at the Federal University of

Minas Gerais - UFMG.

Coryneform bacteria. It describes a system for inducible expression of a foreign gene in Corynebacteria, which is triggered by a temperature increase; an operator is sequen-tially introduced into a promoter system derived from Corynebacterium ammoniagenes, effectively expressing GFP in the bacteria. Two patents [135, 136], describe these methods: “nucleic acid sequences for regulating gene transcription and expression, said novel promoters and expression units, methods for modifying or inducing the transcription rate and/or expression rate of genes, expression cassettes containing said expression units, genetically modified microorganisms with a modified or induced transcription rate and/or expression rate and methods” for producing biosynthetic products by cultivating the gene-tically modified microorganisms to produce two different products, P1-34 expression units and PEF-TS expression units. Given the biotechnological expression members that the actinobacteria group has been known for, some patents relate specifically to the biotechnological products from this group, such as patent WO04050694 [137, 138], which spe-cifies methods for the biosynthesis of methionine, including nucleic acid molecules from C. glutamicum that are involved in this process. Also, following this biotechnological line in genome projects, Basf Aktiengesellschaft et al. (2003) [139] filed a patent involving novel nucleic acid molecules for the construction of genetically improved microorganisms and methods for the production of fine chemicals, in particular amino acids. This line of research is also responsible for a number of patents [140-157]. An interesting feature that Actinobacteria exhibit as a group is their capacity to produce regulatory proteins that act epigenetically along the bac-teria’s genome, as claimed by patent number WO03040181 [158], entitled “Genes from Corynebacterium glutamicum Coding for Regulatory Proteins”.

The RGMG network is also involved with a project to detect genes expressed in corn under drought conditions, in a partnership with the EMBRAPA Maize & Sorghum center. This project seeks to understand the molecular basis of water stress tolerance in plants by identifying differentially expressed genes in roots of maize lines that have different levels of drought tolerance [159]. Corn is a very important agricultural product; therefore many patents have been deposited claiming a number of Zea mays gene regions, DNA sequences and genetic engineering methods [160]. For instance, patent WO06084868 presents an invention for the expression of cassettes comprising at least one transcription regulating nucleotide sequence obtained from the group of genes of monocotyledonous plants consisting of coffeoyl-CoA-O-methyltransferase genes, C8,7-sterol isomerase genes and chloroplast protein genes, which are mainly transcription regulating sequences obtained from Z. mays and Oryza sativa; these sequences are mainly involved in root/kernel-preferential, leaf/endosperm-preferential, root/silk/kernel-preferential and constitutive expression. Another patent includes methods for modulating develop-ment and/or developmental pathways that comprise nucleo-tide sequences (SEQ ID Nr. 1, 3 and 5) and amino acid sequences (SEQ ID Nr. 2, 4 and 6) for maize REVOLUTA. These sequences are involved in controlling or modulating cell division, differentiation and development of the plant’s meristem. Similarly, polycomb genes MEZ1 and MEZ2 from

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maize have been patented by the Wisconsin Alumni Research Foundation et al. [161] and Monsanto Technology Llc. [162], describing promoters from male reproductive tissues isolated from Z. mays that can be used “in plants to regulate transcription of target genes including genes for control of fertility, insect or pathogen tolerance, herbicide tolerance or any gene of interest”. There is also a patent involving transgenic features from Z. mays [163], providing nucleic acids encoding polypeptides of DNA methyltrans-ferase that can be used to stabilize transgene expression in transgenic plants, to alter the yield or biochemical qualities of plants by silencing targeted genes in plants in vivo.

THE NATIONAL LABORATORY FOR SCIENTIFIC COMPUTING - LABORATÓRIO NACIONAL DE COMPUTAÇÃO CIENTÍFICA (LNCC)

The National Laboratory for Scientific Computing (LNCC) is a major reference laboratory for both sequencing projects and bioinformatics. It is located in the city of Petrópolis, in the state of Rio de Janeiro; among other acti-vities, the LNCC coordinates a number of sequencing projects. It is responsible for the delivery of Chromo-bacterium violaceum, Mycoplasma hyopneumoniae strains 7448 and J, and Mycoplasma synoviae complete genome sequences [63, 164]. The LNCC also runs the Anopheles darlingi Genome/EST Project, which is currently active, managing the Brazilian National Genome Project Con-sortium’s projects and delivering the genomes [165].

The LNCC has made a great effort to develop and imp-rove software that has been used to assemble and annotate genome projects throughout Brazil. The software, baptized SABIA [166], which stands for “System for Automated Bacterial Integrated Annotation”, consists of a web-based platform used for scaffolding and clustering sequences as well as performing general assemblies and running anno-tations. Most of the work delivered so far by the networks has been done using SABIA technology. Although initially designed for the processing of prokaryote data, the SABIA software now also distributes a module for eukaryotic genomes [167-169].

THE BRAZILIAN CENTRAL REGION CONSOR-TIUM

The Brazilian Central Region Consortium has started to map the funcional and differential genomes of the yeast and mycelium forms of Paracoccidioides brasiliensis [170- 173]. This dimorphic fungus causes paracoccidioido-mycosis (PCM), which is especially severe for children. This infection can be acquired by humans who inhale spores or propagules from mycelium form found in natural habitats; it reaches the lungs and then becomes infective [174]. The P. brasiliensis Genome Project is integrated with the Brazilian National Genome Project Consortium; it includes 13 university institutions and is managed by the Universidade de Brasília (UnB). Mapping is based on ESTs for detecting genes in the yeast and mycelium forms, as well as genes that are differentially expressed during fungus cell transition. According to the genome network’s website

8, the strategies

8 https://dna.biomol.unb.br/Pb-eng/

proposed in the project include sequencing of ESTs from the mycelium and yeast forms and identification of stage-specific differential cDNAs (ESTs) from one of the two cellular forms of P. brasiliensis by using (i) electronic subtraction, (ii) DDRT-PCR and (iii) microarray chips. Bayer Corporation et al. has issued a patent describing nucleic acid probes and primers from hybridization assays for detecting two fungi (P. brasiliensis and Pneumocystis carinii) that cause disease in humans and animals [84]. These probes can detect rRNA, rDNA or polymerase chain reaction products from most fungi in clinical, environmental or food samples.

GENOME PROGRAM FROM THE PARANÁ STATE - GENOPAR

This network is responsible for the sequencing of a nitrogen-fixing bacterium that associates with gramineae, called Herbaspirillum seropedicae [175, 176]. It regulates nitrogen fixation by a transcriptional activator named NifA protein, which has not yet been patented [177]. Nevertheless, two patents were found in the WIPO database [178] concerning this organism, both claiming the whole organism as the patent’s main issue or invention. These include patent number WO07100162 which discloses a bacterium belong-ing to the genus Azospirillum or Herbaspirillum that can live symbiotically in a plant to impart resistance against disease caused by pathogenic molds, bacteria or viruses; WO02045513 describes biological inoculants for enhancing the growth of plants, including bacterial strains of Herbaspirillum seropedicae 2A, Pantoea agglomerans P101, Pantoea agglomerans P102, Klebsiella pneumoniae 342, Klebsiella pneumoniae zmvsy, Herbaspirillum serope-dicae Z152 and Gluconacetobacter diazotrophicus PA15.

GENOMICS IN A NEW ERA

The genome projects are reaching a new level of technology [179-182]. This is a consequence of new sequencing technologies, including the 454 Life Sciences pyrosequencing method, Solexa-Lynx, Helicos Bioscience Corp. [7,126,127,183-186], and others. The first successful whole-genome shotgun sequencing project performed with-out using Sanger-based sequencing was completed in 2005 [126,127,187] using the 454 Life Sciences platform. Sanger technology is costly, time consuming and labor intensive [186,188]. Methods and equipment for rapid, efficient and inexpensive delivery of DNA sequence information are knocking at the scientific community’s door. A great deal of work has been done during the past 12 years since the complete sequencing of Haemophilus influenzae [189-193] by the Institute for Genomic Research (TIGR) in the United States. Currently, strategies for genotyping and re-sequencing are changing, permitting a broader overview into DNA’s most important features; costs are also being drastically reduced by novel detection methods, miniatu-rization of instrumentation, microfluidic separation and by an increase in the number of assays per run [7,183,185, 186,194-196].

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YET, THERE IS STILL A LACK OF FUNCTIONAL GENOME PROJECTS

Although many sequencing projects are being delivered and large collections of expressed sequences have been obtained, there is an important gap in functional genome projects since many genes have been annotated so far as hypothetical, possible and putative genes. “Functional geno-mics, defined as applying global experimental approaches to assess gene function, by using the information and reagents provided by structural genomics (i.e. mapping and sequen-cing)” seems therefore to be the challenge for omics groups nowadays [197]. Given the current explosion of computer tools for comparative analysis of genomic sequences, comparisons of genomic sequences from different organisms being a central focus of contemporary genome analysis, functional genomics has become imperative for relevant progress [198, 199]. Consequently, structural approaches need to be targeted; genome projects produce an enormous amount of sequence data that needs to be annotated in terms of molecular structure and biological function. The objective therefore would be to determine protein structures efficiently and to “exploit the solved structures for the assignment of biological function to hypothetical proteins” [200-204].

CURRENT & FUTURE DEVELOPMENTS

It is amazing how far genomics has gone. However, some estimates of the number of species on planet Earth reach up to a number of 50 million species. That means that only 4-20% of species have been described (rounding to an estimate of about 10 million species) [201]. Based on these pro-jections, we have completely sequenced only 0.00136-0.00684% of the organisms inhabiting this planet [205].

By the time the Human Genome Project was completed, numerous respected scientists and philosophers had agreed that an antique and somewhat old fashioned branch of biology called taxonomy would inexorably suffer a rebirth process, which would be stimulated by the flood of mole-cular data that genome projects would soon deliver. That actually did not happen; at least, it has not till now. However, ask any colleague if they know an official taxo-nomist, PhD in taxonomy or really dedicated specialist, that is, someone whose job it is to analyze, deploy and curate databases who has no specific interest in a given organism orgroup that he might be working on. Taxonomists have not been reborn as prophesized; but they actually do exist and the field itself has suffered a rebirth process to some extent. These putative professionals have mingled with molecular

Table 3. Patents Claimed by Brazilian Groups and/or Brazilian Researchers Related to Genome/EST/Metagenome Projects

Related

Organism

Publication Number and

International Application

Number

Ref. No. and Title Inventors Publication

Date

Support or

Financial

Group(s)/Applicant

Chromobacterium

violaceum

WO04056960

PCT/BR2003/000207

[60] Gene-Coding Polynucleotides

of the Chromosome of the

Bacterium Chromobacterium

violaceum, Expression and Activity

of these Polynucleotides and their

Applications

De Vasconcelos,

A.T.R., Simpson,

A.J.G., Abreu,

H.N.S., et al.

08/07/2004 CNPq/ Yes

Mycoplasma

hyopneumoniae

WO05060328

PCT/BR2004/000257

[64] Encoding Nucleotides of

Genes from the Chromosome of the

Bacterium Mycoplasma

hyopneumoniae, Expression and

Activity of the Polynucleotides and

their Applications

De Vasconcelos,

A.T.R., Camargo,

A.A., Carraro, D.M.,

et al.

07/07/2005 CNPq/ Yes

Schistosoma

mansoni

WO05023979

PCT/BR2004/000170

[39] Isolated S. mansoni Nucleic

Acid Molecules and Uses thereof

Verjovski-Almeida,

S., Leite, L.C.C.,

Farias, L.P., et al.

17/03/2005 FAPESP/ Yes

Schistosoma sp.

WO01075148

PCT/BR2001/000035

[40] Method and Kit for the

Detection of Schistosomiasis

through the Polymerase Chain

Reaction

Teles Rabello, A.L.,

Dias Neto, E.,

Pontes, L.A.

11/10/2001 FIOCRUZ/ Yes

Xylella fastidiosa WO01085905

PCT/IB2001/001004

[21] Isolated Gum Operon from

Xyllela fastidiosa, isolated Nucleic

Acid Molecules therefrom, and uses

thereof

Arruda, P., Da Silva,

F.R., Kemper, E.L.,

et al.

15/11/2001 FAPESP/ Yes

Xylella fastidiosa

WO02082432

PCT/IB2001/001618

[22] Isolated Genome of Xyllela

fastidiosa and uses thereof

Simpson, A.,

Reinach, F., Setubal,

J., et al.

17/10/2002 FAPESP/ Yes

Source: WIPO - World Intellectual Property Organization.

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Brazilian Genome Projects Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 127

biologists and computer scientists working directly with sequencing and bioinformatics. Every single molecular biologist and computer scientist working in the bioinfor-matics field has become a taxonomist by definition.

PATENTS AND THE WTO

Another phantom that the international community faces, and shall increasingly keep on facing, relates to the Brazilian patent-breaking policy [206, 207] based on WTO dispatches, which somehow are discrepant with the national health assistance program’s capacity to maintain drugs or drug combinations accessible. The Brazilian government compulsory licences the the drugs that it supplies gratis to

most of the affected population during epidemic/pandemic threats, e. g. the anti-HIV cocktail sold by Merck Sharp & Dohme and currently utilized by the Brazilian National AIDS Program [207, 208]. Therefore, it is reasonable to expect the same attitude when it comes to genomic discoveries, gene and/or techniques patents [209]. Brazilian legislation allows compulsory licensing, which is often misunderstood as patent infringement, in cases that involve public interest, health issues, nutrition, environmental defense and national technological or sociological develop-ment [210]. The authorization can be found in the “Brazilian Patent Law” (Lei de Patentes do Brasil - 9279/96. Artigo 71), by decrees 3201/99 and 4830/03; moreover, it is

* European Patent Office comprising France, Germany and the European Consortium as well as all other European countries. International

genomes could not be plotted for obvious reasons. Data for China’s 2006, 1990 and 1998 production was not retrievable at the source.

Source: WIPO - World Intellectual Property Organization.

Find figures at http://www.lgcm.icb.ufmg.br/publications.

Fig. (4). Total number of patents registered for the top 10 sequencing countries from 1985 to 2006.

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128 Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 Capanema Xavier et al.

sustained by the TRIPS Agreement and by the Doha Declaration [206]. Compulsory licensing allows the country to obtain drugs or other products from another supplier without being obliged to pay the fees charged by the original developers after quality standards are met by the new producers. For instance, Brazil buys some AIDS medicines from India at a much lower cost than charged by an English company (Merck Sharp & Dohme Efavirenz, which patented a non-nucleoside reverse transcriptase inhibitor used in the treatment of HIV infections).

ACKNOWLEDGEMENTS

It is our pleasure to thank all research centers, bioinfor-matics groups, financing foundations and laboratories directly involved in the sequencing projects carried out in Brazil. Special acknowledgements go to all of the sequen-cing staff at the Universidade Federal de Minas Gerais - UFMG and the Centro de Pesquisas René Rachou - CPqRR,

which constitute the core with which our group (Laboratório de Genética Celular e Molecular - LGCM, from the Instituto de Ciências Biológicas of the Universidade Federal de Minas Gerais) has done their work so far.

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