One of the postdocs suggested we work our way through the old H. influenzae competence literature, so we've been meeting more-or-less weekly to do that. We pick a time interval (e.g. 1975-79) and decide which papers look like they deserve serious attention.
Last time we considered two papers from Jane Setlow's lab, one reporting that DNA of competent cells contains single-stranded regions, and one analyzing single-stranded regions that appear in the DNA such cells take up. This reminded me of a more recent paper from David McCarthy (1987), and of some experiments I did. Here I'll psot about the competent-cell DNA issue. Later I'll post about the strandedness of incoming DNA,a nd about the weird history and behaviour of rec-2 mutants.
The Setlow research was done in the mid-1970s. This was before agarose gels came into general use, and they analyzed the sizes of DNA fragments using sedimentation in sucrose density gradients (big fragments are rapidly pushed to the bottom while small fragments move only partway down the gradient). In the first paper they used gradients containing NaOH to separate the strands of the DNA, and pulse-chase labeling with 3H to identify newly synthesized segments. They found that newly synthesized DNA from competent cells contained many more short single-stranded segments than DNA from log-phase cells. They also used columns of BND-cellulose, which DNA with single-stranded regions should stick to. More competent-cell DNA stuck than log-phase cell DNA. The confirmed that this DNA was enriched for single-stranded regions by digesting it with the nuclease SI, which preferentially cuts single-stranded segments. And they used CsCl 'isopycnic' density gradients to confirm that the strands were indeed newly synthesized.
But there are good reasons why sucrose gradients were discarded once agarose gels became available. These experiments have very poor resolution and lots of artefacts, and it's hard for me to understand what they showed. The very existence of newly synthesized strands in competent-cell DNA may be an artefact...
The McCarthy paper used a different technique, electron microscopy, to directly compare the structures of DNA from log-phase and competent cells. They found DNA from competent cells to contain more single-stranded regions and single-stranded tails. They also used cross-linking of DNA to prevent branch migration. So their results confirmed Setlow's interpretation of the sucrose gradient results.
Although we might expect cells to develop some aberrant DNA structures after having been abruptly transferred from a rich replication-supporting medium to a starvation medium lacking DNA precursors, the presence of gaps and tails is a bit surprising. At that time I was a post-doc in Ham Smith's lab and had been doing a lot of work with pulsed-field agarose gels that nicely resolved very large fragments of chromosomal DNA. So I devised some experiments to look for evidence of these gaps and tails.
The first experiments used a nuclease from mung beans. This is like S1 nuclease in preferentially cutting single-stranded DNA, but is less likely to also cut double-stranded DNA. These experiments showed two things. First, DNA from competent cells is no more sensitive to mung bean nuclease than is DNA from log phase cells. Second, DNA from competent cells is much more sensitive than log-phase DNA to being heated in the presence of the buffer used for the nuclease digestions. This buffer contains zinc and is at pH 5; I had been heating the DNA preps before loading them into the gels because the DNA had been prepared from cells embedded in low-melting-point agarose to protect it from shearing. The DNAs were all fine if I had simply put slices of this DNA-in-agarose into the wells of the pulsed-field gels, but if I had instead melted the slices at 65C and pipetted them into the wells the competent-cell DNA bands looked very faint. This effect was independent of any muclease treatment.
My second experiments used a DNA polymerase to find out whether DNAs from log-phase and competent cells had different numbers of gaps and tails. The 'Klenow' fragment of DNA polymerase can replicate single-stranded DNA in vitro, provided that adjacent double-stranded DNA has a 3' end that can serve as a primer. So chromosomal DNA with a single-stranded gap is a perfect template. I incubated my log-phase and competent cell DNAs with Klenow polymerase and precursor nucleotides (including 32P-dGTP), and ran them in a pulsed-field gel. The two DNAs incorporated the same amount of radioactivity and gave the same patterns in the gel. As a control I used DNA from cells that had been treated with chloramphenicol - this protein-synthesis inhibitor lets cells complete any DNA replication they have initiated but blocks initiation of new rounds of replication. This DNA incorporated only about 1/3 as much radioactivity.
So neither experiment provided any support for the presence of frequent single-strand gaps or tails in the DNA of competent cells. The sensitivity to the nuclease buffer did indicate there there is something funny about this DNA. I was left wondering whether the DNA might have incorporated ribonucleotides or other non-standard subunits. Because ribonucleotides are sensitive to alkali, segments containing them would become gaps if the DNA was denatured with NaOH. But this wouldn't explain McCarthy's results.
The other weird thing about both McCarthy's and Setlow's results was that they found DNA from 'competent' cells of the non-transformable mutant rec-2 to be like DNA from log phase cells. I'll do a separate post about this.
Bedazzled by alternate truth
4 hours ago in The Phytophactor