RESUMO
Eukaryote nuclear genomes predominantly replicate through multiple replication origins. The number of replication origins activated per chromosome during the S-phase duration may vary according to many factors, but the predominant one is replication stress. Several studies have applied different approaches to estimate the number and map the positions of the replication origins in various organisms. However, without a parameter to restrict the minimum of necessary origins, less sensitive techniques may suggest conflicting results. The estimation of the minimum number of replication origins (MO) per chromosome is an innovative method that allows the establishment of a threshold, which serves as a parameter for genomic approaches that map origins. For this, the MO can be easily obtained through a formula that requires as parameters: chromosome size, S-phase duration, and replication rate. The chromosome size for any organism can be acquired in genomic databanks (such as NCBI), the S-phase duration can be estimated by monitoring DNA replication, and the replication rate is obtained through the DNA combing approach. The estimation of MO is a simple, quick, and easy method that provides a new methodological framework to assist studies of mapping replication origins in any organism.
RESUMO
Eukaryote nuclear genomes predominantly replicate through multiple replication origins. The number of replication origins activated per chromosome during the S-phase duration may vary according to many factors, but the predominant one is replication stress. Several studies have applied different approaches to estimate the number and map the positions of the replication origins in various organisms. However, without a parameter to restrict the minimum of necessary origins, less sensitive techniques may suggest conflicting results. The estimation of the minimum number of replication origins (MO) per chromosome is an innovative method that allows the establishment of a threshold, which serves as a parameter for genomic approaches that map origins. For this, the MO can be easily obtained through a formula that requires as parameters: chromosome size, S-phase duration, and replication rate. The chromosome size for any organism can be acquired in genomic databanks (such as NCBI), the S-phase duration can be estimated by monitoring DNA replication, and the replication rate is obtained through the DNA combing approach. The estimation of MO is a simple, quick, and easy method that provides a new methodological framework to assist studies of mapping replication origins in any organism.
RESUMO
Pseudomonas syringae pv. phaseolicola (P.s. phaseolicola) is one of about 45 recognized pathovars within the P. syringae group and is the causal agent of halo-blight disease of beans. DNA from this bacterium digested to completion with two different restriction enzymes, PacI and PmeI, yielded 15 and 16 fragments, respectively. These were separated using PFGE and sized by comparison to known molecular mass markers. The P.s. phaseolicola chromosome was determined to be approximately 5.64 Mb in size. To link the different fragments obtained into a circular chromosome map for both enzymes, 150 random Tn5 mutants of P.s. phaseolicola were used as a source of DNA and the identification of the band carrying the transposon 'tag' in each mutant was done after PFGE and Southern hybridization of a complete chromosomal digestion using a Tn5 probe. Partial digestions of DNA from different Tn5 mutants 'tagging' specific bands were then generated and the complete and partial products of the digestion separated by PFGE and identified with a Tn5 probe. By calculating the size of the partial products, it was then possible to link different bands into a physical map. This is the first report on the construction of a physical map of a member of the P. syringae group and should be invaluable for molecular genetic analysis in this species and in evolutionary or taxonomic studies when compared to similar data obtained for any of the other recognized pathovars.