Experiencias que ayudan a formar el vínculo del Apego

To obtain a method of preparation of streptococcal plasmid DNA which was quick, with as few steps in the procedure as possible, was reproducible and suitable for all the strains under study, preliminary experiments were carried out usihg several well documented methods.

On two separate occasions, attempts were made to prepare plasmid DNA from strains DS5, K87, JH2-2 and transconjugant derivatives of matings between DS5 and JH2-2 by the Vapnek method. The samples thus obtained were

run on horizontal 0.8% (w/v) agarose gels and the DNA visualised in ultra­ violet light after staining with ethidium bromide. All samples had an identical diffuse hand of DNA which co-migrated with that obtained from the plasmid free strain JH2-2 and was, therefore, most likely chromosomal DNA.

The lysis of cells directly in the slots of a vertical gel as described by Eckhardt was tested as a rapid method of plasmid isolation which should have minimised nuclease digestion and the possibilities of mechanical

shearing of large plasmids. The strains used in the first experiment were

DS5, JH2-2 and two tetracycline resistant transconjugants from a mating of these strains, and the procedure was repeated using strains DS5, JH2-2, K55 and K60. On both gels, the position of chromosomal DNA was taken as

those bands co-migrating with that from the plasmid free JH2-2. Such a

band was seen in DS5 and transconjugants on the first gel but only in

JH2-2 on the second. On the first gel, a faint band which had migrated

faster than chromosomal DNA was seen in all samples except JH2-2 and it was assumed that this was the tetracycline resistance plasmid pAMal but other hands representing pAMyl or pAM&l were not seen in the DS5 sample. The second gel gave a more promising result for DS5 with pAMal clearly visible and a faint band migrating more slowly than chromosomal DNA,

Assuming that under the conditions used lysis of cells, was obtained it is possible that no plasmid DNA was seen in K55 and K60 because the concen­

tration of cells was too high resulting in a reduced yield of plasmid

(Eckhardt, 1978) although a band of chromosomal DNA would have been expected in these circumstances. Alternatively, if the cell concentration was too low and low copy number plasmids present, the concentration of DNA released would probably have been insufficient to visualise after staining.

The mutanolysin lysis method was used to isolate plasmid DNA from strains DS5, K88 and K87 in a series of experiments. As stated previously, density gradient centrifugation steps were omitted and replaced by phenol

and chloroform extractions. This lysis method must have released a large

amount of soluble protein as, on addition of phenol for the first extraction, the samples became completely white due to precipitated protein. A further phenol extraction followed by two chloroform extractions were necessary to produce a clear aqueous phase from which DNA was precipitated with ethanol. Figure 4 is a representation of the band patterns in 0.6% (w/v) agarose obtained from the above strains and the plasmid free JH2-1 on four separate occasions. As the running conditions were slightly different on each occasion, band sizes were calculated from the standard DNA samples on the respective gels and re-drawn in the appropriate position. The pattpm of bands obtained from DS5 was similar in both experiments with bands at

positions expected for ccc DNA of pAMal (9.1kb) pAMBl (26.5kb) and possibly

one or more of the pAMyl plasmids (54kb). The other bands in the samples

were either chromosomal DNA or other forms of these plasmids. The pro­

cedure was less successful with strain K88 - apparently only chromosomal DNA was isolated in the first experiment. However three bands, one

probably chromosomal, were isolated when the procedure was repeated, The most striking feature of the K87 samples was the large amount of chromosomal

mutanolysin method after electrophoresis in 0.6% agarose gels.

Numbers in parenthesis indicate the experiment number. Arrow

indicates chromosomal DNA and show the positions of pAMy,

pAM3l and pAMal of DS5 respectively.

lO 00 cathode origin 55 45 35 30 25 20 a - a anode

figure does not show was the distortion of the four small bands of K87

which was a characteristic of these samples and possibly caused by incomplete deproteinisation or overloading due to the high proportion of chromosomal DNA.

The method was successful in isolating several plasmid species from the strains tested but there was also a large amount of chromosomal DNA in the sançles which could conceal the presence of co-migrating plasmid DNA. Another disadvantage of the method was the inefficient separation of cccDNA

from open circular or linear forms of plasmids, exemplified in DS5 samples where there were several bands in addition to those expected.

The method recommended by Crosa and Falkow (1981) for isolation of large plasmids was tested twice using all the parental antibiotic resistant strains and the plasmid free strain JH2-1 and the appearance of the samples from the first experiment after electrophoresis in a 0.6% (w/v) agarose gel

is shown in Figure 5. Two DS5 samples, one of which was prepared from

double the cell density, were included in the preparation and only the sample from the higher DS5 cell density showed any bands, one of which was probably

chromosomal DNA. The probable positions of pAMy and pAMal are marked but

there was no DNA visible at the position expected for pAMal or pANgl. Samples prepared from K55, K60 and K87 all had the same band pattern which differed markedly from that obtained from K87 using the mutanolysin procedure. Only chromosomal DNA was isolated from K88, SB94, SB69 and JH2-1 while no DNA was visible in the K46 sample. In general, the DNA bands obtained were very faint. At the second attempt to isolate plasmids using this method, no DNA was seen in any of the samples with the. exception of K87 which had one hand of approximately 26kb. The most probable reasons for

the. failure, of this method were either not enough starting material i.e. insufficient cells to yield detectable DNA at the end of the procedure, or incomplete lysis of cells in the absence of lysozyme treatment leading to a

Figure 5. Schematic representation of DNA samples prepared by the Crosa and Falkow method after electrophoresis in a 0.6% agarose gel.

Arrow indicates the position of chromosomal DNA and y, a the probable positions of DS5 plasmids pAMy and pAMal respectively.

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low DNA yield.

Large scale preparations from 1000ml culture which included a caesium chloride-ethidium bromide density gradient centrifugation were carried out once for strains DS5, K87, K88, SB94, twice for strains K55, SB69 and K46 and three times for K60. After centrifugation, all gradients except those of K60, SB69 and one of K46 had two and sometimes three bands visible with

ultraviolet light. The uppermost band contained mainly chromosomal DNA

and the lowest band the ccc plasmid DNA. Gradients from K60 preparations did not have any visible DNA bands while SB69 and one of the K46 preparations had one faintly fluorescent band at a density equivalent to that of the

chromosomal DNA in the other preparations. Since samples were run on

different agarose gels with slightly altered running conditions, the sizes of the bands obtained have been estimated from standard size DNA samples on the individual gels and re-drawn in Figure 6 at the appropriate positions. Samples DS5, K55, K87, SB94 and SB69 all had bands of approximately 21kb which may have been contaminating chromosomal DNA. As the method is designed to separate the linear chromosomal DNA from ccc plasmids, however, it is possible, especially in samples K55 and K87 where the bands were clear and sharp, that these bands represent plasmids. In addition to this band, the DS5 sample contained bands migrating to positions expected for pAMy,

pAMgl and pAMal with a further,, band of 5.5kb. This latter could have been