Physical mapping across the dihydrofolate reductase-thymidylate synthase chromosome of Leishmania major.
We have used a chromosome-specific approach to generate a 300 kb long 'contig' across Leishmania major 500 kb chromosome. Clones from a 13-hit genomic library served as templates to generate end-specific probes that were used in hybridization to a high density array of the library. The 'contig' generated contained 12 markers uniformly spaced. Three restriction endonucleases were mapped within the map extending its resolution. Map extension indicated a peculiar feature of sequence organization in subtelomeric regions where chromosome-specificity of mapping is lost. End-probes generated from clones mapping to the extremes of a 300 kb 'contig' rescued a high percentage of 2 types of clones from the genomic library, 1 of which showed positive hybridization to the hexameric telomere repeat. Fine mapping at these regions revealed that these 2 clones contained elements common to all chromosomes of the parasite. The physical map generated constitutes ready-to-use data for the study of many aspects of genome organization. Being cloned in a shuttle vector, the genomic sequences reordered in the map can be used to generate genetic information by transfection into the parasite.
Tosi
L R
LR
Departamento de Bioquimica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brasil.
Casagrande
L
L
Beverley
S M
SM
Cruz
A K
AK
D000818:Animals; D002874:Chromosome Mapping; D018320:Leishmania major / Q000201:enzymology* / Q000235:genetics; D009097:Multienzyme Complexes / Q000235:genetics*; D015800:Protozoan Proteins / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D013940:Thymidylate Synthase / Q000235:genetics*
D009097:Multienzyme Complexes; D015800:Protozoan Proteins; C056930:thymidylate synthase-dihydrofolate reductase; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase