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Studies on transformation of Escherichia coli with plasmids

In our view, gene therapy may ameliorate some human genetic diseases in the future. For this reason, we believe that research directed at the development of techniques for gene therapy should continue. For the foreseeable future, however, we oppose any further attempts at gene therapy in human patients because (i) our understanding of such basic processes as gene regulation and genetic recombination in human cells is inadequate; (ii) our understanding of the details of the relation between the molecular defect and the disease state is rudimentary for essentially all genetic diseases; and (iii) we have no information on the short-range and long-term side effects of gene therapy. We therefore propose that a sustained effort be made to formulate a complete set of ethicoscientific criteria to guide the development and clinical application of gene therapy techniques. Such an endeavor could go a long way toward ensuring that gene therapy is used in humans only in those instances where it will prove beneficial, and toward preventing its misuse through premature application. Two recent papers have provided new demonstrations of directed genetic modification of mammalian cells. Munyon et al. (44) restored the ability to synthesize the enzyme thymidine kinase to thymidine kinase-deficient mouse cells by infection with ultraviolet-irradiated herpes simplex virus. In their experiments the DNA from herpes simplex virus, which contains a gene coding for thymidine kinase, may have formed a hereditable association with the mouse cells. Merril et al. (45) reported that treatment of fibroblasts from patients with galactosemia with exogenous DNA caused increased activity of a missing enzyme, alpha-D-galactose-l-phosphate uridyltransferase. They also provided some evidence that the change persisted after subculturing the treated cells. If this latter report can be confirmed, the feasibility of directed genetic modification of human cells would be clearly demonstrated, considerably enhancing the technical prospects for gene therapy.

https://science.sciencemag.org/content/sci/175/4025/949.full.pdf

Gene Therapy for Human Genetic Disease

Natural competence is widespread among bacterial species. The mechanism of DNA uptake in both gram-positive and gram-negative bacteria is reviewed. The transformation pathways are discussed, with attention to the fate of donor DNA as it is processed by the competent cell. The proteins involved in mediating various steps in these pathways are described, and models for the transformation mechanisms are presented. Uptake of DNA across the inner membrane is probably similar in gram-positive and gram-negative bacteria, and at least some of the required proteins are orthologs. The initial transformation steps differ, as expected, from the presence of an outer membrane only in the gram-negative organisms. The similarity of certain essential competence proteins to those required for the assembly of type-4 pili and for type-2 protein secretion is discussed. Finally several hypotheses for the biological role of transformation are presented and evaluated.

DNA Uptake in Bacteria

Transformation and conjugation permit the passage of DNA through the bacterial membranes and represent dominant modes for the transfer of genetic information between bacterial cells or between bacterial and eukaryotic cells. As such, they are responsible for the spread of fitness-enhancing traits, including antibiotic resistance. Both processes usually involve the recognition of double-stranded DNA, followed by the transfer of single strands. Elaborate molecular machines are responsible for negotiating the passage of macromolecular DNA through the layers of the cell surface. All or nearly all the machine components involved in transformation and conjugation have been identified, and here we present models for their roles in DNA transport.

/pdf/the-ins-and-outs-of-dna-transfer-in-bacteria-uaqjgjstv4.pdf

The Ins and Outs of DNA Transfer in Bacteria

Organization of the bacterial chromosome.

Efficiently regenerated single colonies from mixed multiply auxotrophic Bacillus subtilis protoplasts, fused with polyethylene glycol, reveal colonies carrying each of the parent types (biparentals) and recombinant colonies. The latter appear in high yields (up to 1% of certain recombinant classes); the yield of biparentals may be as large as 10%, in the range of the indicated physical fusion events. Many of the biparentals are diploids although, contrary to expectation, they are not complementing prototrophs, but show precisely the phenotype of one (either one) of the parent strains. Extensive pedigree analysis and subcloning of diploid lines show that they can propagate with varying stability on the appropriate parental selective medium to reproduce diploid progeny, parental segregants, and late-appearing recombinants, including some prototrophs. Thus, the principal product of intertype protoplast fusion is a diploid carrying two chromosomes, only one of which is expressed in each particular clone. The extinction of one parental genome is especially well demonstrated when it includes suppression of a normally dominant antibiotic sensitivity marker. In transformation experiments, DNA made from a selected diploid clone was able to transfer several of the unexpressed genes. The structural or topological character of DNA associated with the chromosome extinction remains unexplained.

Biparental products of bacterial protoplast fusion showing unequal parental chromosome expression

Bacterial Transformation, with Special Reference to Recombination Process

The DNA plus cell interaction leading to bacterial transformation is an orderly two-particle reaction, dependent upon the first power of cell and DNA concentration,1-4 and of the duration of the extracellular contact.3-' When two physically unlinked markers are incorporated into the same cell, the double transformations become proportional to the second power of DNA concentration4' or duration of exposure.5 The competitive power of other DNA6 and the kinetics3' I suggest an early reversible step, but DNA is eventually physically incorporated.8-'0 The DNA which enters pneumococcal cells is rapidly separated into two equal fractions, high-molecular and degraded,1' and the regions associated with the introduced marker activity appear to be large tracts carrying only a single strand from the introduced donor DNA.12 In Hemophilus'3 and Bacillus subtilis14 cells also, it seems that only one transforming DNA strand survives intact. The reproducibility of the maximal pneumococcal transforming activity for several markers, as well as of various linkages, in DNA prepared without deliberate shear, has suggested that the recovered material may have relatively regular points of rupture when separated from the presumably much longer bacterial chromosome. This could happen if there were fixed discontinuities or regions of relative lability in the chromosome. This report will show that there is a tendency for reproducible sequences of marker entry in transformation by pneumococcal DNA, suggesting that the molecules as isolated from the cells have to some degree uniform terminations. The study makes use of separated single strands of DNA, reactivated by complexing with unmarked homologous DNA. Methods and Materials.-Deoxycholate lysates of pneumococci were used for all preparations of DNA. The preparation of chloroform gel DNA (C) and the methods of transformation have been frequently described.3' 15 The marker strains were multiple mutants and transformants of wild type (R6, derived from R36A), and the competent strain was a single colony derivative (R1-26) of the same strain. Zone centrifugation was done with 1 uAg DNA in 5-20 per cent sucrose gradients in saline at 34,000 rpm for 3 hr in the cold in celluloid tubes, and fractions of 2 drops collected from the bottom in sterile transformation medium and biologically tested. DNA preparations of type C were denatured with 0.1 M NaOH at 250C, then neutralized at 0?C in concentrations of about 25 ug/ml and put on 1-cm columns containing 5 ml of MAK having 0.7 the quantity of methylated albumin previously specified."6 About 0.2-0.6 mg of denatured DNA in 0.6 or 0.7 M NaCl was used; "A" fractions came at about 0.85 M and "B" at about 1.0 M salt. Renaturation was in 1.5 M NaCl at 65?C for 30 min, followed by 1-2 hr cooling; biological activity was measured at concentrations giving linear response. Rescue of denatured DNA was accomplished by renaturation with a double quantity of wild-type DNA and measured per unit of marked DNA present, expressed as per cent of activity given by native marked DNA.

Manifestation of linear organization in molecules of pneumococcal transforming DNA.

The primary heterodiploid bacteria regenerated after Bacillus subtilis fusion, although generally noncomplementing diploids, behave in pedigree analysis as multipotential systems. Individual diploid colonies yielding complete reciprocal recombinant (RR) progeny—often accompanied by one or both parents—constitute 10-30% of the total recombinant-forming units. The RR (reciprocal for 8-11 genes) usually occur in equivalent numbers both among and within individual colonies. Novel for bacteria, they demonstrate that entire parental genomes brought together within a diploid protoplast are retained as two independent replicons able to undergo classical recombination characteristic of eukaryotic gametogenesis. Parental or recombinant genomes are also subject to multiple rounds of recombination without obligate segregation and often not reciprocal. Diploid recombinant clones, sharing streptomycin resistance but reciprocal for auxotrophic markers, have displayed a partial ability to make a facultative shift in chromosome expression. They have also produced two types of prototrophs: a stable one (presumably haploid and recombinant) and an unstable one, (diploid and temporarily complementing at low frequency). It follows that chromosome extinction may affect both parental and recombinant chromosomes and does not interfere with recombination. Analysis of the number and chromosomal distribution of crossovers in all recombinants and those from single diploid clones shows increased frequency of exchange in the regions of the replication origin and terminus, possibly a result of the association of these sites with the cell wall or membrane.

https://www.pnas.org/content/pnas/80/5/1426.full.pdf

Reciprocal and nonreciprocal recombination in diploid clones from Bacillus subtilis protoplast fusion: Association with the replication origin and terminus

Since transformation of Pneumococcus seems to be effected largely by native double stranded DNA (Marmur and Lane, 1960; Roger and Hotchkiss, 1961) which is relatively stiff and rod-like, but yields within the transforming cells 50% degradation to small fragments plus a single stranded half (Lacks, 1962) of considerable integrity and length (Fox and Allen, 1964), it seems likely that entry is endwise. Degradation is so selective, virtually all-or-none, that presumably it is occasioned by an exonuclease attacking either the 5' or conversely the 3' ends; therefore, only one of the strands will survive to be genetically utilized, the particular one depending upon which end of the double stranded DNA makes contact with the cell. It is generally assumed that bacterial chromosomes are long, double strands of DNA interrupted principally by the progression of one, or very few, replication points. The continuity of the molecule may also be interrupted by single strand gaps, or small amounts of non-nucleotide matter; but there is little evidence for this. It is often further assumed that all segments of the chromosome may be considered randomly subject to shear, hydrolysis, etc., despite manifold evidences that there are structural modulations building up messages and that there are specific points involved with punctuation, functional repression, high rates of mutation, and sites for methylation or rare base incorporation. In recovering DNA from bacteria, it is possible to treat the cells and extracts so gently that crude DNA of high particle weight is obtained (Davison, 1959; Berns and Thomas, 1965; Kelly and Pritchard, 1965). These products may be, biologically speaking, quite pure, being devoid of active viruses, enzymes, or ribosomes; but chemically they are impure DNA preparations. The very easily accomplished shear on manipulation of these crude preparations, bringing them down to the in vitro stable size approximating 107 molecular weight-not nearly so subject to further shear-is also for the present taken to be random, since this makes certain calculations possible. Nevertheless, it is predicted and confirmed that shear tends to give successive breaks near the midpoints of long molecules (Burgi and Hershey, 1961; Thomas, 1966), and this would give random products only if the original chromosome had random ends. Moreover, if the gentle shear occurs at predetermined interruptions or weak spots (physical

Magda Gabor

Papers

Biparental products of bacterial protoplast fusion showing unequal parental chromosome expression

Bacterial Transformation, with Special Reference to Recombination Process

Manifestation of linear organization in molecules of pneumococcal transforming DNA.

Reciprocal and nonreciprocal recombination in diploid clones from Bacillus subtilis protoplast fusion: Association with the replication origin and terminus

Evidences of Organization in Transfer and Expression of Bacterial Transforming DNA