Waclaw Szybalski

Mapping of Deletions and Substitutions in Heteroduplex DNA Molecules of Bacteriophage Lambda by Electron Microscopy

Electron microscopy of heteroduplex DNA molecules, composed of one strand of Escherichia coli phage λ+ DNA annealed to the complementary DNA strand of a λ deletion or substitution mutant, permits visualization, as well as precise measurements and mapping, of the unpaired single-stranded regions of nonhomology in the otherwise double-stranded molecules. In the λb2 mutant, the central segment (13 percent) of the λ+ DNA molecule is shown to be deleted. In the hybrid phages λi434 and λi21 a segment of the right arm of the λ+ genome (5.5 or 7.6 to 9 percent) is replaced by the corresponding immunity regions of phage 434 (3.3 percent or phage 21 (4 percent) DNA. The b5 region in the λb5 mutant appears to be identical to the i21 segment. From these data it is possible to estimate the size and posiion of those λ genes which are replaced by the i434 and i21 segments. The method permits preparing complete physical maps of viral genomes with a precision heretofore unattainable.

A Comprehensive Molecular Map of Bacteriophage LAMBDA

Physical and genetic mapping of deletion mutations has been correlated with the available molecular sizes of the lambda gene products and the DNA base sequence to construct a comprehensive molecular map of the phage lambda genome. The physical length of the DNA making up the left arm from the cos site through gene J is not sufficient to account in a nonoverlapping manner for all the proteins of the sizes reported to be coded, especially in the Nu1--C region. In the right arm all the coding capacity has not been accounted for, and it appears to be oversaturated only in the gam-ral region. The positions of several IS and Tn elements, and of restriction endonuclease cleavage sites are specified.

Class-IIS restriction enzymes--a review.

Class-IIS restriction enzymes (ENases-IIS) interact with two discrete sites on double-stranded DNA: the recognition site, which is 4-7 bp long, and the cleavage site, usually 1-20 bp away from the recognition site. The recognition sequences of ENases-IIS are totally (or partially) asymmetric and all of the characterized ENases-IIS are monomeric. A total of 35 ENases-IIS are described (80, if all isoschizomers are taken into consideration) together with ten related ENases (class IIT), and 15 cognate methyltransferases (MTases-IIS). The physical, chemical, and molecular properties of the ENases-IIS and MTases-IIS are reviewed and many unique applications of this class of enzymes are described, including: precise trimming of DNA; retrieval of cloned fragments; gene assembly; use as a universal restriction enzyme; cleavage of single-stranded DNA; detection of point mutations; tandem amplification; printing-amplification reaction; and localization of methylated bases.

[124] Use of cesium sulfate for equilibrium density gradient centrifugation☆

Publisher Summary This chapter discusses the use of cesium sulfate for equilibrium density gradient centrifugation. Equilibrium density gradient centrifugation is one of the most useful tools for fractionation and characterization of DNA. The two cesium salts most commonly employed in this procedure are CsC1 and Cs 2 SO 4 . The properties of density gradients prepared with these salts are rather different. At similar rotor speeds, Cs 2 SO 4 , which forms approximately twice as steep a gradient as CsCl, is preferable for fractionation of DNAs with widely different buoyant densities, for example, dAT versus dABU or normal unsubstituted versus bromoor iodouracil-labeled DNA. On the other hand, CsCl is better suited for routine determination of the guanine + cytosine (G + C) content of DNA, because in this solvent, there is an almost linear relationship between the percentage of G + C (20–80%) and the buoyant density of DNA. In Cs 2 SO 4 , there is a much less and nonlinear dependence of density on G + C content. DNA is much more heavily hydrated in the Cs 2 SO 4 gradient, with the density averaging 1.4 g/cm 3 as compared with 1.7 g/cm 3 in CsCl. The chapter also explains that RNA can be banded in the Cs 2 SO 4 gradient at a concentration of about 1.6 g/cm and discusses the other specific advantages of Cs 2 SO 4 over CsC1.

Coliphage λnutL−: A unique class of mutants defective in the site of gene N product utilization for antitermination of leftward transcription☆

Abstract A special procedure was developed to isolate a new class of plaque-forming (Spi − ) mutants of phage λ that are unable to express the leftward N -dependent red and gam gene functions by reason of cis -dominant defects located between the p L promoter and the N gene. The p L promoter is intact in these mutants, as the rate of transcription in the p L - N - t L1 region is high. Also, their gene N does not appear to be altered, as shown by genetic complementation. The mutations map near the center of the transcribed interval (about 124 base-pairs long) defined by the left ends of the λdv1 plasmid and the imm434 substitution. Thus, they map near the promoter, but interfere with antitermination at terminators located farther downstream. We conclude that the mutations define a new kind of recognition site (which we designate nut , for N utilization ) that controls an early step in the N-mediated antitermination mode of transcription.

Polar mutations in lac, gal and phage λ consist of a few IS-DNA sequences inserted with either orientation

SummaryIt was found that many insertions which often arise as strongly polar mutations consist of only a few “foreign” DNA sequences. The most commonly observed “short” polar IS1 insertions are all comprised of the same DNA with a duplex length of 750±80 nucleotide pairs. This insertable IS1 DNA can be integrated with either orientation into the genome at various positions in the lac and gal operons of E. coli; however, the inserted sequence is not permuted. The r14 polar insertion in gene cII of phage λ also consists of the same IS1 DNA (Hirsch et al., 1972 b). “Long” insertions (1170 to 1490 nucleotide pairs) were detected in the lac (IS3) and gal (IS2, IS4) operons and in the y and P-Q regions (IS2) of the λ genome and measured as single-strand loops in the l/r heteroduplex DNA. The insertions in the latter two positions are homologous, although those found in the P-Q region do not exhibit any obvious polarity.

Totally asymmetric transcription of coliphage T7 in vivo: Correlation with poly G binding sites☆

In vivo transcription of T7 phage was studied by means of hybridization between phage-specific radioactive RNA and separated complementary strands of T7 DNA. The latter were preparatively separated by CsCl density-gradient centrifugation in the presence of guanine(G)-rich polyribonucleotides, which bind to one strand only. The separated strands failed to renature when annealed separately but renatured when mixed together. Pulse-labeled 3H-RNA, isolated from cultures of Escherichia coli B at various times after T7 infection, hybridized exclusively with the poly G-binding strand H, indicating that only this strand is transcribed in vivo. Non-poly G-binding strand L did not hybridize (less than 0.02%) with the in vivo synthesized T7 RNA, but both the H and L strands hybridized with enzymatically prepared 3H-RNA transcribed from denatured T7 DNA template. The fact that the poly G-binding, deoxycytidylate (dC)-rich clusters are restricted to the in vivo transcribing H strand, together with the absence of thymine-rich clusters in T7 DNA, is compatible with the hypothesis (Szybalski et al., 1966) that pyrimidine-rich clusters are related to RNA transcription, possibly as the initiation and termination sites.

SPECIAL MICROBIOLOGICAL SYSTEMS. II. OBSERVATIONS ON CHEMICAL MUTAGENESIS IN MICROORGANISMS

The chemotherapies of cancer and of infectious diseases are based on a common rationale : destruction, modification, or inhibition of the invader, while the cells of the host are spared. However, the differences between normal and malignant cells are so slight when compared with the great disparity between mammalian and bacterial cells that the search for a sufficiently selective agent becomes a very difficult task. The most significant property of the malignant cell that distinguishes it from the surrounding normal cells is invasiveness, that is, a comparatively high rate of multiplication. Theoretically, this characteristic should make the malignant cell vulnerable to a t least two types of agents: those that are selectively toxic and destructive to actively dividing cells, and those that inhibit nonspecifically all cell division and thereby bring the cancer cells into a stationary quiescent state among the normally nondividing surrounding cells. Unfortunately, both these classes of chemotherapeutic agents would inhibit also the normally rapidly regenerating tissues of the host, in particular the hematopoietic system; this type of toxicity is in fact commonly encountered with the majority of the available antineoplastic drugs. With this bit of theory and great expectations for finding an Ehrlichian “magic bullet” that would inhibit a hypothetical biochemical pathway utilized by the malignant cell, but not by the normal cells of the host, the experimental oncologist generally tends to approach his problem in a rather empirical manner. Although the final test of the efficacy of any antineoplastic agent remains its activity in the diseased human subject, the manner of its preliminary evaluation is not fixed. At present, experimental animal cancers are the initial screening systems of widest usage, but not of greatest facility or economy. Only a few other systems have been evaluated to a comparable extent for preliminary screening of antineoplastic agents. These alternate systems chiefly involve testing for inhibition of microbial growth and cytotoxicity in tissue cultures; they have been discussed elsewhere (Gellhorn and Hirschberg, 1955; Hirschberg, 1955; Reilly, 1953) and are considered in this monograph. This paper is based principally on two considerations: (1) potential antineoplastic agents may occur with considerably greater frequency among a class of compounds known to possess a specific biological activity than within chemicals chosen a t random, and (2) a test for mutagenicity, employing a bacterial mutational system of unusual simplicity, may fill the need for a rapid preliminary screening method. The first contention is supported by the results of a joint evaluation of various biological activities associated with the

The Cs2SO4 equilibrium density gradient and its application for the study of T-even phage DNA: Glucosylation and replication

The buoyant densities of normal native DNAs, containing the usual four bases, depend on their guanine + cytosine (G + C) content. This dependence (density versus G + C increments) is 1.8–4.7 times lower in the Cs2SO4 than in the CsCl gradient. Similarly, the absolute values for buoyant density determined in the Cs2SO4 gradient are lower than the corresponding values determined in the CsCl gradient by 0.25–0.30 g/cm3. Cs2SO4 also establishes a much steeper gradient than CsCl. The buoyant densities of the hydroxymethylcytosine-containing T-even phage DNAs do not conform to the G + C versus buoyant density relationship, and in the Cs2SO4 gradient they are roughly proportional to the quantity of glucose linked to the hydroxymethyl groups. Glucose-deficient T-even phage DNAs, prepared either by enzymatic synthesis or by growing T6 phages in UDPG-deficient host cells, band in the Cs2SO4 gradient at a position only slightly higher than that predicted by the glucosylation versus density relationship. In the CsCl gradient this relationship was found to be less pronounced and of a more complex nature. The natural density variation in the Cs2SO4 gradient provided by a variable degree of glucosylation was utilized for study of T6 DNA replication in wild-type and in UDPG-deficient Escherichia coli cells. In both hosts a glucose-deficient DNA is synthesized in a semiconservative manner until the 9th minute after T6 infection. At this time the glucosylation mechanism becomes operative in the wild-type host, all the phage DNA shifting to the density of fully glucosylated T6 DNA, while the host DNA becomes degraded unless it is protected by timely addition of protein-synthesis inhibitors. In the UDPG-deficient host, only glucose-deficient DNA is synthesized in a semiconservative manner, but with intervening fragmentation and recombination. The glucose-deficient phages eventually produced in this E. coli mutant are not infective for either the same mutant host or other tested strains of E. coli or Shigella paradysenteriae, but plate with high efficiency on the permissive strain Sh-16 of Shigella dysenteriae.

Universal restriction endonucleases: designing novel cleavage specificities by combining adapter oligodeoxynucleotide and enzyme moieties

Class IIS restriction endonucleases cleave double-stranded (ds) DNA at precise distances from their recognition sequences. A method is proposed which utilizes this separation between the recognition site and the cut site to allow a class IIS enzyme, e.g., FokI, to cleave practically any predetermined sequence by combining the enzyme with a properly designed oligodeoxynucleotide adapter. Such an adapter is constructed from the constant recognition site domain (a hairpin containing the ds sequence, e.g., GGATG CCTAC for FokI) and a variable, single-stranded (ss) domain complementary to the ss sequence to be cleaved (at 9 and 13 nucleotides on the paired strands from the recognition sequence in the example of FokI). The ss sequence designated to be cleaved could be provided by ss phage DNA (e.g., M13), gapped ds plasmids, or supercoiled ds plasmids that were alkali denatured and rapidly neutralized. Combination of all three components, namely the class IIS enzyme, the ss DNA target sequence, and the complementing adapter, would result in target DNA cleavage at the specific predetermined site. The target ss DNA could be converted to the precisely cleaved ds DNA by DNA polymerase, utilizing the adapter oligodeoxynucleotide as primer. This novel procedure represents the first example of changing enzyme specificity by synthetic design. A practically unlimited assortment of new restriction specificities could be produced. The method should have many specific and general applications when its numerous ramifications are exploited.