Matthew Meselson

Plasmid Screening at High Colony Density

A procedure is described for screening bacterial colonies containing recombinant plasmids by nucleic acid hybridization at high density, i.e., at 100 000 colonies per 150 mm diameter plate. Small colonies are established on nitrocellulose filters from which they can be faithfully replicated to additional filters. Chloramphenicol amplification may be carried out in situ before screening. The filters may be kept frozen for long-term storage of colonies which may be further replicated after thawing.

Massive Horizontal Gene Transfer in Bdelloid Rotifers

Horizontal gene transfer in metazoans has been documented in only a few species and is usually associated with endosymbiosis or parasitism. By contrast, in bdelloid rotifers we found many genes that appear to have originated in bacteria, fungi, and plants, concentrated in telomeric regions along with diverse mobile genetic elements. Bdelloid proximal gene-rich regions, however, appeared to lack foreign genes, thereby resembling those of model metazoan organisms. Some of the foreign genes were defective, whereas others were intact and transcribed; some of the latter contained functional spliceosomal introns. One such gene, apparently of bacterial origin, was overexpressed in Escherichia coli and yielded an active enzyme. The capture and functional assimilation of exogenous genes may represent an important force in bdelloid evolution.

Accumulation of a specific subset of D. melanogaster heat shock mRNAs in normal development without heat shock

During normal development in D. melanogaster, messenger RNAs for three of the seven heat shock proteins (hsp83, hsp28 and hsp26) accumulate in adult ovaries and are abundant in embryos until blastoderm. The three mRNAs appear to originate in nurse cells and subsequently pass, during stages 10-11, into the oocyte. Little if any of the four other heat shock mRNAs is present in unshocked ovaries or embryos at any time examined. Pre-blastoderm embryos fail to accumulate these heat shock mRNAs even if subjected to heat shock. The accumulation in normal oogenesis of mRNAs for only three of the seven heat shock proteins indicates the existence of differential, possibly multiple controls of heat shock gene expression, and suggests that heat shock proteins hsp83, hsp28 and hsp26 function in the oocyte or early embryo.

Density alterations associated with transducing ability in the bacteriophage lambda

The transducing variant of the bacteriophage λ is thought to arise by a double crossover in which a section of the normal chromosome is exchanged for a region of the bacterial chromosome containing galactose fermentation markers. If the exchange were unequal, the resulting transducing phages should have an altered DNA content which might be detected as a modification of the density of the phage particle. An examination of ten independently arising populations of transducing phages showed that each had a different but essentially uniform density. In contrast, the non transducing phage contained in these lysates possessed a constant density. The density of a transducing phage was stable to a cycle of lysogenization, prophage replication and multiplication following induction, as well as to genetic interactions with normal A and with the bacterial chromosome. Assuming the observed density changes to be due to changes in DNA content, it was calculated that the maximum change corresponded to 107 mol wt units, or 1·5 × 104 nucleotide pairs, and the smallest to 800 nucleotide pairs. It is proposed that the unequal exchanges giving rise to the density alterations are the result of poor homology and incomplete pairing between the Gal region of the bacterial chromosome and the region of λ which is deleted.

CONSERVATION OF RIBOSOMES DURING BACTERIAL GROWTH.

Two bands are observed when ribosomes from extracts of Escherichia coli are examined by density-gradient centrifugation in cesium chloride solution containing sufficient magnesium ions. The denser is called A and the less dense B. The B band diminishes and A augments as the concentration of magnesium ions is reduced below 0·04 M until at 0·002 M only A remains; below this concentration, particles in the A band are unstable and appear to release free RNA. Isolated 30 s and 50 s ribosomes form bands in the A region, but particles re-isolated from the A band sediment at 23 s and 42 s. The particles in the A band seem to be deficient in protein relative to normal ribosomes. The B band probably contains 70 s ribosomes, although this has not been definitely shown. An experiment was performed to learn whether parts of the 30 s and 50 s ribosomes undergo exchange or renewal during bacterial growth. A culture of E. coli uniformly labeled with heavy isotopes and phosphorus-32 was transferred to light non-radioactive medium in which growth was allowed to continue. At one and three generations after transfer, 30 s and 50 s ribosomes were isolated from samples of the culture and examined by density-gradient centrifugation. The dense 23 s and 42 s particles did not undergo any significant decrease in density as the bacteria multiplied in light medium, nor was phosphorus-32 seen to accumulate in light ribosomes much beyond a small amount found at the first generation. We conclude that the 23 s and 42 s nucleoprotein subunits of 30 s and 50 s ribosomes are conserved during bacterial growth.

Extreme resistance of bdelloid rotifers to ionizing radiation.

Rotifers of class Bdelloidea are common invertebrate animals with highly unusual characteristics, including apparently obligate asexuality, the ability to resume reproduction after desiccation at any life stage, and a paucity of transposable genetic elements of types not prone to horizontal transmission. We find that bdelloids are also extraordinarily resistant to ionizing radiation (IR). Reproduction of the bdelloids Adineta vaga and Philodina roseola is much more resistant to IR than that of Euchlanis dilatata, a rotifer belonging to the desiccation-intolerant and facultatively sexual class Monogononta, and all other animals for which we have found relevant data. By analogy with the desiccation- and radiation-resistant bacterium Deinococcus radiodurans, we suggest that the extraordinary radiation resistance of bdelloid rotifers is a consequence of their evolutionary adaptation to survive episodes of desiccation encountered in their characteristic habitats and that the damage incurred in such episodes includes DNA breakage that is repaired upon rehydration. Such breakage and repair may have maintained bdelloid chromosomes as colinear pairs and kept the load of transposable genetic elements low and may also have contributed to the success of bdelloid rotifers in avoiding the early extinction suffered by most asexuals.

On the Mechanism of Genetic Recombination between DNA Molecules

A two-factor cross was performed between bacteriophages labeled with heavy isotopes. Recombinants were found with chromosomes formed entirely or almost entirely of parental DNA. This and other features of the distribution of parental DNA among recombinant phages and among their descendants show that genetic recombination occurs by breakage and joining of double-stranded DNA molecules. Also, there is some indication that a small amount of DNA is removed and resynthesized in the formation of recombinant molecules.

Cyclic dissociation into stable subunits and re-formation of ribosomes during bacterial growth

Abstract Studies of the distribution of isotopic labels among ribosomes and ribosomal subunits following transfer of a growing bacterial culture from a heavy- to a light-isotopes medium show that: (1) During growth ribosomes frequently undergo subunit exchange, presumably by dissociation into their 30 s and 50 s subunits and re-formation from a pool of free subunits. (2) Bibosomal subunits are stable: they remain intact during growth and are continuously, recycled through ribosomes. On the basis of these results, it is suggested that ribosomes must dissociate into their subunits between successive rounds of translation.

14 – Plasmid Screening at High Colony Density

Publisher Summary This chapter discusses plasmid screening at high colony density. Bacterial plasmid vectors are widely employed in the isolation, amplification, mutagenesis, and analysis of DNA sequences. Maintaining colonies on filters also provides for long-term storage of large distributions. Colony hybridization can be performed under a wide variety of conditions. Chloramphenicol amplification technique has been employed in the isolation of both cDNA and genomic DNA sequences from a wide variety of organisms. No particular limitations because of fidelity of replication or frozen storage have been observed. This technique has been applied in probing colony arrays on nitrocellulose filters with radioactive antibodies to locate sequences through recognition of their gene products. This technique has also been applied to the construction and storage of cosmid banks and to the isolation of specific sequences from them.

Retroelements containing introns in diverse invertebrate taxa

We report that two structurally similar transposable elements containing reverse transcriptase (RT), Penelope in Drosophila virilis and Athena in bdelloid rotifers, have proliferated as copies containing introns. The ability of Penelope-like elements (PLEs) to retain introns, their separate phylogenetic placement and their peculiar structural features make them a novel class of eukaryotic retroelements.