Graduate Thesis Or Dissertation
 

Determination of differential gene action in Bdellovibrio bacteriovorus using RNA-DNA hybridization techniques

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  • In an attempt to study the times in the lifecycle of Bdellovibrio bacteriovorus at which various genes are transcribed, the patterns of messenger ribonucleic acid (mRNA) and ribosomal ribonucleic acid (rRNA) syntheses were examined using two different types of hybridization systems. The hybridization system using membrane bound deoxyribonucleic acid (DNA) and saturating amounts of RNA, provides the fraction of DNA which is transcribing RNA at various time periods in the organisms lifecycle. The hybridization system using DNA in solution in excess of the RNA, provides the fractions of the the total RNA being transcribed at the various time periods in the organisms life cycle. Using pulse labeled RNA from hostdependent Bdellovibrio bacteriovorus 109(Davis), (HD 109D), incubated in a casamino acids medium, the fraction of DNA transcribing RNA at that time period in the lifecycle was found to be 4.3% of the total DNA, or 8.6% of the potential gene weight of the cell. Competition with unlabeled r-RNA purified from host independent Bdellovibrio bacteriovorus 109(Davis), (HI 109D), in late logarithmic growth showed that 48% of the transcribed DNA, or 2% of the total DNA, was producing 65% of the total labeled RNA as r-RNA. This amount of DNA was calculated to code for 13 ribosomal cistrons. Total RNA extracted from HD 109D grown through a complete developmental cycle in Spirillum serpens cell sonicate, and RNA extracted from HD 109D grown only to the mid-point in its life-cycle in S. serpens cell sonicate was also used as cold competitor. Both competed for 92% of the sites on the DNA available to the labeled HD 109D RNA and made up 81% of the total labeled RNA. Total RNA extracted from HI 109D in logarithmic growth also competed successfully for the DNA sites available to 81% of the labeled RNA. This indicates that 92% of the DNA transcribed during the labeling period is also transcribed during the time periods from which the competitor RNA was extracted. As r-RNA was demonstrated to comprise 48% of the transcribed DNA cistrons, this leaves 44% of the transcribed DNA producing 16% of the m-RNA during all the time periods tested, and 8% (that fraction which retained the labeled RNA after competition) producing 19% of the m-RNA unique to the labeling period.
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