GENOME ORGANIZATION
IN Paracoccidioides brasiliensis: ANALYSIS OF TEN LARGE CLONED SEGMENTS
Marina P. Nobrega; Simone C. B.
Bandeira;Flavia V. Morai; Amanda B.Ferrari; Carolina P. Barco; Andréa G.
Csoknyai; Carla S. Dias; Luciano A S.Bernades; Francisco G. Nóbrega
Laboratório de Genética Molecular e Genomas,
Instituto de Pesquisa e Desenvolvimento, Universidade do Vale do Paraíba, S.J.
Campos, SP (e-mail:mnobrega@univap.br)
Paracoccidioides brasiliensis (PB) is a thermodimorphic fungus
that causes a deep systemic infection in humans and is endemic in South
America. There is already a good deal of information about expressed genes
(ESTs) for this organism. To further characterize the nuclear genome of this
pathogen we constructed genomic libraries in the yeast shuttle plasmids
YEp351/352 with clones ranging from 5 to 20 Kbp and also a fosmid library
(Epicentre). We started from total DNA highly purified by CsCl isopynic
centrifugation. The nDNAs fragments obtained is of high molecular size, around
50Kbp and was sheared accordingly to obtain the size ranges appropriate for the
different libraries. We obtained about 1.300 clones with the plasmid vectors.
Insert size was estimated in a set of 18 clones and ranged from 5.6 to 21 Kbp.
Ten clones were completely sequenced by subcloning and transposon island
insertion and a number of novel genes detected. In particular we found a NRPSs
(non ribosomal peptide synthetase) that is probably associated with the
synthesis of a low molecular weight iron chelator siderophore which plays an
important role in parasite survival and infection of the host. We annotated
also the following putative genes: DER-1 prot. 28,5Kb; transmembrane 9
super family protein; miosin; choline dehydrogenase oxiredutase;
chaperonine (domain TCP1/cpn60-family); Ser/Thr protein kinase;
2-hydroxiacid dehydrogenase NAD binding domain; AAA-superfamily of
ATPases associated with a wide variety of cellular activities. We succeeded in
obtaining about 2,800 clones in the pCC1FOS™
vector with long inserts (36 to 45 kb) and are in the process of
developing methods to obtain DNA sequence 5´ and 3´ tags from the clones and
preparing membrane arrays to be screened by specific gene probes for gene
discovery and further understanding of the gene organization in PB.
Financial
support: FAPESP and CNPq
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