Determination of microbial genome sizes by two-dimensional denaturing gradient gel electrophoresis

Abstract

In two-dimensional denaturing gradient gel electrophoresis, DNA is digested with a restriction endonuclease and the resulting DNA fragments are separated as a function of size by conventional agarose gel electrophoresis. Following this first dimension electrophoresis, the fragment distribution is placed at the top of a denaturing gradient slab gel and electrophoresis is carried out parallel to the gradient direction. This second dimension separation is a complex function of the base sequence of each fragment. Analysis of the DNA fragment distribution as a function of fragment size allows the DNA size to be calculated. This method has been applied to calculate three microbial genome sizes: Mycoplasma capricolum, 724 kb; Acholeplasma laidlawii, 1646 kb; and Hemophilus influenzae, 1833 kb.

Cloning of the completeMycoplasma pneumoniaegenome

“…mycoides, Y strain. The discrepancies have not yet been resolved, though recently the validity of PFGE for AT-rich DNA sequences (as most of the Mollicutes genomes are) has been questioned by Maniloff and Poddar (20,21). In case of M. pneumoniae our size determination agrees fairly well with the value of 735 kbp derived from renaturation kinetics.…”

Organization of the bacterial chromosome.

“…Some examples of early estimates of genome sizes are as follows: Azotobacter chroococcum, 1,940 kb (291); Desulfovibrio gigas, 1,630 kb (274); Desulfovibrio vulgaris, 1,720 kb (274); E. coli, 3,520 kb (274); and Myxococcus xanthus, 5,690 kb (384). Recent values (which still must be regarded as approximations) in closer agreement with results of PFGE are as follows: Mycoplasma capricolum, 742 kb; Acholeplasma laidlawii, 1,646 kb; Haemophilus influenzae, 1,833 kb; and E. coli, 4,399 kb (272). In comparison, the currently accepted value for the size of the E. coli chromosome is 4.7 kb (56, 324), and for M. xanthus the value is 9.45 kb (51).…”

Restriction Fragment Fingerprint and Genome Sizes ofStaphylococcusSpecies Using Pulsed-Field Gel Electrophoresis and Infrequent Cleaving Enzymes

“…However, a large number of chromosomal fragments generated by these enzymes produce complicated DNA banding patterns that are difficult to resolve and compare. High-resolution restriction fragment fingerprints of bacterial chromosomes can be produced with frequent-cutting restriction enzymes using two-dimensional electrophoresis (Poddar and Maniloff, 1986;Poddar and Maniloff, 1989). However, multiple strains cannot be compared in one such experiment.…”