A procedure for the isolation of deoxyribonucleic acid from micro-organisms

The 4,639,221-base pair sequence of Escherichia coli K-12 is presented. Of 4288 protein-coding genes annotated, 38 percent have no attributed function. Comparison with five other sequenced microbes reveals ubiquitous as well as narrowly distributed gene families; many families of similar genes within E. coli are also evident. The largest family of paralogous proteins contains 80 ABC transporters. The genome as a whole is strikingly organized with respect to the local direction of replication; guanines, oligonucleotides possibly related to replication and recombination, and most genes are so oriented. The genome also contains insertion sequence (IS) elements, phage remnants, and many other patches of unusual composition indicating genome plasticity through horizontal transfer.

/pdf/the-complete-genome-sequence-of-escherichia-coli-k-12-2lpi4d3nz4.pdf

The Complete Genome Sequence of Escherichia coli K-12

We describe a dominant behavioral marker, rol-6(su-1006), and an efficient microinjection procedure which facilitate the recovery of Caenorhabditis elegans transformants. We use these tools to study the mechanism of C.elegans DNA transformation. By injecting mixtures of genetically marked DNA molecules, we show that large extrachromosomal arrays assemble directly from the injected molecules and that homologous recombination drives array assembly. Appropriately placed double-strand breaks stimulated homologous recombination during array formation. Our data indicate that the size of the assembled transgenic structures determines whether or not they will be maintained extrachromosomally or lost. We show that low copy number extrachromosomal transformation can be achieved by adjusting the relative concentration of DNA molecules in the injection mixture. Integration of the injected DNA, though relatively rare, was reproducibly achieved when single-stranded oligonucleotide was co-injected with the double-stranded DNA.

/pdf/efficient-gene-transfer-in-c-elegans-extrachromosomal-rla2vqw8fr.pdf

Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences.

The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.

Firefly luciferase gene: structure and expression in mammalian cells.

https://www.cell.com/cell/pdf/0092-8674(87)90646-5.pdf

Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells.

https://www.cell.com/cell/pdf/0092-8674(83)90331-8.pdf

The double-strand-break repair model for recombination

Studies of bacterial transformation and bacteriaphage infection1–5 strongly indicate that deoxyribonucleic acid (DNA) can carry and transmit hereditary information and can direct its own replication. Hypotheses for the mechanism of DNA replication differ in the predictions they make concerning the distribution among progeny molecules of atoms derived from parental molecules.6

Radioisotopic labels have been employed in experiments bearing on the distribution of parental atoms among progeny molecules in several organisms.6–9 We anticipated that a label which imparts to the DNA molecule an increased density might permit an analysis of this distribution by sedimentation techniques. To this end, a method was developed for the detection of small density differences among macromolecules.10 By use of this method, we have observed the distribution of the heavy nitrogen isotope N15 among molecules of DNA following the transfer of a uniformly N15-labeled, exponentially growing bacterial population to a growth medium containing the ordinary nitrogen isotope N14.

A small amount of DNA in a concentrated solution of cesium chloride is centrifuged until equilibrium is closely approached. The opposing processes of sedimentation and diffusion have then produced a stable concentration gradient of the cesium chloride. The concentration and pressure gradients result in a continuous increase of density along the direction of centrifugal force. The macromolecules of DNA present in this density gradient are driven by the centrifugal field into the region where the solution density is equal to their own buoyant density.11 This concentrating tendency is opposed by diffusion, with the result that at equilibrium a single species of DNA is distributed over a band whose width is inversely related to the molecular weight of that species (Fig. 1).



Fig. 1. 
Ultraviolet absorption photographs showing successive stages …

https://www.pnas.org/content/pnas/44/7/671.full.pdf

The replication of DNA in Escherichia coli

This communication presents a new method for the study of the molecular weight and partial specific volume of macromolecules, with some illustrations based on results with deoxyribonucleic acid (DNA) and several viruses. The method involves observation of the equilibrium distribution of macromolecular material in a density gradient itself at equilibrium. The density gradient is established by the sedimentation of a low-molecular-weight solute in a solution subject to a constant centrifugal field.

https://www.pnas.org/content/pnas/43/7/581.full.pdf

Equilibrium sedimentation of macromolecules in density gradients.

For many organisms, meiotic double crossing over is less frequent than expected on the assumption that exchanges occur at random with respect to each other. This "interference," which can be almost total for nearby intervals, diminishes as the intervals in which the double crossovers are scored are moved farther apart. Most models for interference have assumed, at least implicitly, that the intensity of interference depends inversely on the physical distance separating the intervals. However, several observations suggest that interference depends on genetic distance (Morgans) rather than physical distance (base pairs or micrometers). Accordingly, we devise a model in which interference is related directly to genetic distance. Its central feature is that recombinational intermediates (C9s) have two fates--they can be resolved with crossing over (Cx) or without (Co). We suppose that C9s are distributed at random with respect to each other (no interference); interference results from constraints on the resolution of C9s. The basic constraint is that each pair of neighboring Cx9s must have between them a certain number of Co9s. The required number of intervening Co9s for a given organism or chromosome is estimated from the fraction of gene conversions that are unaccompanied by crossover of flanking markers. The predictions of the model are compared with data from Drosophila and Neurospora.

/pdf/chiasma-interference-as-a-function-of-genetic-distance-41toxsnr3k.pdf

Chiasma interference as a function of genetic distance.

The products of genes recB and recC are responsible for conjugal recombination and for the repair of chromosomal double chain breaks in Escherichia coli. The product of the recD gene, which combines with the RecB and RecC proteins to comprise the RecBCD enzyme, is not required for either recombination or repair. On the contrary, RecBCD enzyme is a potent exonuclease which inhibits recombination by destroying linear DNA. The RecD Ejection model supposes that RecBCD enzyme enters DNA at a double-chain end and travels destructively along the DNA until (typically) it encounters the recombination hotspot sequence chi. Chi then alters the RecBCD enzyme by weakening the affinity of the RecD subunit for the RecBC heterodimer. With the loss of the RecD subunit from the enzyme, the resulting protein, RecBC(D-), becomes deficient for exonuclease activity and proficient as a recombinagenic helicase. Thus, the RecD Ejection model proposes that chi participates in recombination by acting as a toggle to convert RecBCD (a powerful exonuclease) to RecBC(D-) (a recombinase). We review the properties of RecBCD and its cognate site chi, including recent results that support both the RecD Ejection model and the view that chi plays only an indirect role in recombination.

Franklin W. Stahl

Papers

The double-strand-break repair model for recombination

The replication of DNA in Escherichia coli

Equilibrium sedimentation of macromolecules in density gradients.

Chiasma interference as a function of genetic distance.

chi and the RecBC D Enzyme of Escherichia Coli