Scientific activity

Because of a shortage of the chromatography-grade aluminum oxide during World War II, Waclaw Szybalski was „forced" to invent paper chromatography as a substitute for the aluminum oxide chromatography. He did it single-handedly at the Department of Petroleum Technology, (Prof. S. Pilat, Chairman), when he was an undergraduate student at the Institute of Technology (Politechnika Lwowska), Lwow, Poland, during Soviet occupation of Lwow. He completed this work in 1944/1945 when he was in charge of the Agricultural Laboratory, Konskie, Poland. He published some of the applications of his filter paper chromatography studies only after World War II:

• Szybalski, W., 1950. Oznaczanie aminokwasów przy pomocy dwuwymiarowej chromatografii na bibule. (Determination of amino acids by the two-dimensional paper chromatography). Przemysł Chemiczny 5, 32-37 (in Polish, English summary).
  (It is amusing that paper (partition) chromatography was concurrently developed in England (first publication in 1941), and the inventors were awarded the 1952 Nobel Prize).

As a graduate student, Waclaw Szybalski carried out kinetic studies leading to the complete elucidation of the mechanism of the five-step reaction between iodine and azides, as catalyzed by carbon disulfide and other S2' compounds:

•   Hofman-Bang, N., Szybalski, W., 1949. Determination of carbon disulphide in aqueous solution. Acta Chem. Scand. 3, 926-929.
•   Hofman-Bang, N., Szybalski, W., 1949. The iodine-azide reaction. II. The catalytic effect of carbon disulphide. Acta Chem. Scand. 3, 1418-1429.
•   Szybalski, W., 1950. Kinetyka i mechanizm reakcji chemicznych (Kinetics and mechanism of chemical reactions). Wiadomosci Chemiczne 4, 193-213 (in Polish).

Influenced by the 1941-1942 lectures and laboratory course by Prof. Adolf Joszt [the Chairman of the Department of Industrial Fermentation and Biotechnology, Division of Chemical Engineering, Lwow Institute of Technology (Politechnika Lwowska), Lwow, Poland], Dr. W. Szybalski's scientific interest shifted from chemistry to microbial genetics, and he carried out pedigree analysis of the genetic crosses between Saccharomyces cerevisiae and Saccharomyces chevalieri.

These studies were carried out in 1947-1949 at the Carlsberg Laboratory in Copenhagen in the laboratory of Prof. O. Winge, the pioneer of the yeast genetics.

W. Szybalski also studied the genus Pseudomonas, and isolated and classified a new subspecies of Pseudomonas perolens var. Gdansk (listed in Bergey's Manual). This work was done in Gdansk Institute of Technology (Politechnika Gdanska) and was completed at the Danish Institute of Technology, Copenhagen:
Szybalski, W., 1950. A comparative study of bacteria causing mustiness in eggs. Nature 165, 733-734.

At the same time, W. Szybalski studied the causes of corrosion of steel water pipes in the Copenhagen water system. He found that this type of corrosion was caused by iron bacteria (Leptothrix ochracea). He elucidated the mechanism of the corrosion by being able to reproduce it in laboratory by constructing differential-aeration electric cells, where iron bacteria caused an 0.4 volt electric potential. He developed a method of blocking this corrosion by flushing the system with polyphosphates. This saved the water system of Copenhagen from the serious problems of corrosion and pipe perforation. These studies were first published in 1949-1950:

• Olsen, E., Szybalski, W., 1949. Aerobic microbiological corrosion of water pipes. I—II. Acta Chem. Scand. 3, 1094-1116.
• Mansa, J. L., Szybalski, W., 1950. Corrosion due to tuberculation in water systems. I. The effect of Calgon on the potential of iron electrodes in differential aeration cells with running tap water. Acta Chem. Scand. 4, 1275-1292.
• Mansa, J. L., Szybalski, W., 1950. Corrosion due to tuberculation in water systems. II. The effect of Calgon on the potential of zinc and copper electrodes in differential aeration cells with running tap water. Acta Chem. Scand. 4,1293-1299.
  Subsequently these papers were re-published by request of the Editor of the journal „Corrosion" and with the author's permission in the USA:
• Olsen, E., Szybalski, W., 1950. Aerobic microbial corrosion of water pipes (revised reprint from Acta Chem. Scand. 3, 1094-1116, with addendum; published by invitation). Corrosion 6, 405-414, 1950.
• Mansa, J. L., Szybalski, W., 1952. Corrosion due to tuberculation in water systems. Parts I and II (revised reprint from Acta Chem. Scand. 4, 1094-1292, 1950, with addendum; published by invitation). Corrosion 8, 381-390.
• Szybalski, W., 1951. Reply to the discussion of F.N. Speller pertaining to the paper of E. Olsen and W. Szybalski: „Aerobic microbiological corrosion of water pipes" -Corrosion 6, 405-414, 1950. Corrosion 7, 109.
  A review of this work was published in Poland in the journal “Industrial Chemistry".
• Szybalski, W., 1951. Korozja punktowa w roztworach wodnych (Spot corrosion in aequous sections). Przemysl Chemiczny 7, 88-93 (in Polish, English summary).

Dr. Szybalski received his D. Sc. degree in 1949 at the Gdansk Institute of Technology (Politechnika Gdanska), Gdansk, Poland.

7 (1). RESISTANCE, CROSS-RESISTANCE AND COLLATERAL SENSITIVITY In the summer of 1951, just after joining Cold Spring Harbor Laboratories, Dr. W. Szybalski invented the gradient-plate technique for rapid determination of even small differences in antibiotic susceptibilities for various bacterial strains:

• Szybalski, W., 1952. Gradient plate technique for study of bacterial resistance. Science 116,46-48. Making use of this technique, he isolated many drug-resistant mutants and initiated genetics studies of antibiotic resistance, cross resistance and collateral sensitivity in E. coli, Streptococcus aureus, Mycobacteria and Bacilli:
• Szybalski, W., Bryson, V., 1952. Genetic studies on microbial cross resistance to toxic agents. I. Cross resistance of Escherichia coli to fifteen antibiotics. J. Bacteriol. 64, 489-499.
• Szybalski, W., 1953. Genetic studies on microbial cross resistance to toxic agents. II. Cross resistance of Micrococcus pyogenes var. aureus to thirty-four antimicrobial agents. Antibiotics &c Chemother. 3,1095-1103.
• Szybalski, W., Bryson, V., 1954. Genetic studies on microbial cross-resistance to toxic agents. III. Cross-resistance of Mycobacterium ranae to twenty-eight antimycobacterial agents. Am. Rev. Tubercul. 69, 267-279.
• Szybalski, W., 1954. Genetic studies on microbial cross resistance to toxic agents. IV. Cross resistance of Bacillus magaterium to forty-four antimicrobial agents. Applied Microbiol. 2, 57-63.
• Szybalski, W., Bryson, V., 1955. Origin of drug resistance in microorganisms. In: Origin of Resistance to Toxic Agents. Sevag, M. G., Reid, R. D., Reynolds, O. E., Eds. Academic Press, New York, pp.

7(2). MULTIPLE DRUG THERAPY Dr. W. Szybalski proposed and strongly advocated the use of suitably designed “multiple antibiotic therapy” based on his study of the rate of resistance development to two or more antibiotics. Multiple chemotherapy was very successful in the treatment of tuberculosis and childhood Ieukemias:

• Grace, E. J., Bryson, V., Szybalski, W., Demerec, M., 1952. Potential danger of isoniazid resistance through failure to use multiple chemotherapy in treatment of tuberculosis. J. Amer. Med. Assoc. 149, 1241.
• Szybalski, W., Bryson, V., 1953. One step resistance development to isoniazid and sodium-p-aminosalicylate. J. Bacteriol. 66, 468—469.
• Szybalski, W., 1953. Multiple chemotherapy and antagonism between antimicrobial agents. Riassunti delle Comunicazione, VIth Congresso Int. Microbial., Roma, Italy, 1,318-319.
• Szybalski, W., 1956. Theoretical basis of multiple chemotherapy. Tuberculo-logy 15, 82-85.

Dr. Szybalski was among the first to demonstrate genetic exchange in antibiotic-producing Streptomyces (heterokaryons, recombinants):

• Braendle, D. H., Szybalski, W., 1957. Genetic interaction among Streptomycetes: heterokaryosis and synkaryosis. Proc. Nad. Acad. Sci. 43, 947-955.
• Szybalski, W., 1958. Genetics of Streptomyces fradiae. In: Neomycin, Its Nature and Practical Application. Waksman, S. A., Ed. Williams and Wilkins, Baltimore, pp. 44-48.
• Braendle, D. H., Gardiner, B., Szybalski, W., 1959. Heterokaryotic compatibility of Streptomyces. J. Gen. Microbiol. 20, 442—450.
• Braendle, D. H., Szybalski, W. 1959. Heterokaryotic compatibility, metabolic cooperation and genie recombinations in Streptomyces. Ann. N. Y. Acad. Sci. 81, 824-851. He organized and published (edited) the first ever International Conference on the „Genetics of Streptomyces":
• Szybalski, W., Editor, 1959. Genetics of Streptomyces and other antibiotic-producing microorganisms. N. Y. Acad. Sci. 81, Art. 4, 805-1016.

9 (1). SPOT TEST FOR MUTAGENS Dr. Szybalski and his collaborators studied the mechanism of mutagenesis. First, they developed the „paper disc mutagenicity test", which was later adopted in so-called „Ames tests":

• Iyer, V. N., Szybalski, W., 1958. Two simple methods for the detection of chemical mutagens. Appl. Microbiology 6, 23-29. Using these methods, Dr. Szybalski has surveyed 431 chemical compounds, and found that 5.1% of them were effective mutagens.
• Szybalski, W., 1958. Observations on chemical mutagenesis in micro-organisms. Annals N. Y. Acad. Sci. 76, 475-489.

9 (2). MECHANISMS OF CHEMICAL MUTAGENESIS His studied the mechanism of chemical mutagenesis:

• Iyer, V. N., Szybalski, W., 1958. The mechanisms of chemical mutagenesis. I. Kinetic studies on the action of triethylene melamine (TEM) and azaserine. Proc. Nad. Acad. Sci. 44, 446-456.
• Steinman, I.D., Iyer, V. N., Szybalski, W., 1958. The mechanism of chemical mutagenesis. II. Interaction of selected compounds with manganous chloride. Arch. Biochem. Biophys. 76, 78-86.
• Szybalski, W., Pitzurra, M., 1959. The mechanism of chemical mutagenesis. III. Induced mutations in spheroplasts of Escherichia coli. J. Bact. 77, 621-622.
• Iyer, V. N., Szybalski, W., 1959. Mutagenic effect of azaserine in relation to azaserine resistance in Escherichia coli. Science 129, 839-840.
• Szybalski, W., 1960. The mechanism of chemical mutagenesis with special reference to triethylene melamine action. In: Developments in Industrial Microbiology. Miller, B. M., Ed. Plenum Press, New York 1, 231-241.
• Lorkiewicz, Z., Szybalski, W. 1960. Genetic effects of halogenated thymidine analogs incorporated during thymidylate synthetase inhibition. Biochem. Biophys. Res. Comm. 2, 413-418.
• Lorkiewicz, Z., Szybalski, W., 1961. Mechanism of chemical mutagenesis. IV. Reaction between triethylene melamine and nucleic acid components. J. Bacteriol. 82, 195-201.

9 (3). MUTAGENESIS IN SPHEROPLASTS Dr. Szybalski studied the formation of spheroplasts and mutagenesis in spheroplasts:

• Kohn, A., Szybalski, W., 1959. Lysozyme spheroplasts from thawed Escherichia coli cells. Bacteriol. Proc, 126-127.
• Pitzurra, M., Szybalski, W., 1959. Formation and multiplication of spheroplasts of Escherichia coli in the presence of lithium chloride. J. Bact. 77, 614-620.
• Szybalski, W., Pitzurra, M., 1959. The mechanism of chemical mutagenesis. III. Induced mutations in spheroplasts of Escherichia coli. J. Bact. 77, 621-622.

In these studies it was shown that proximal carcinogens are mutagenic in vitro toward transforming DNA. This provided the first direct proof for causative relationship between mutagenesis and carcinogenesis.

• Maher, Sr. V. M., Miller, E. C, Miller, J. A., Szybalski, W., 1968. Mutations and decreases in density of transforming DNA produced by derivatives of the carcinogens 2-acetylaminofluorene and N-methyl-4-aminoazobenzene. Molec. Pharmacol. 4, 411^26.

Dr. Szybalski also studied mutagenesis in human cell lines:

• Szybalski, W., Smith, M. J., 1959. Effect of ultraviolet light on mutation to 8-—azaguanine resistance in a human cell line. Fed. Proc. 18, 336.
• Szybalski, W., Ragni, G., Cohn, N. K., 1964. Mutagenic response of human somatic cell lines. In: Cytogenetics of Cells in Culture. Harris, R. J. C, Ed. Symposia of the International Society for Cell Biol., Academic Press 3, 209-221.

While studying mutagenic interacions between radiation and thymidine analogues, Dr. Szybalski discovered radiosensitization of bacterial and also human cells by the incorporation of 5—bromo- or 5—iododeoxyuridine into cell DNA. This principle was used for many purposes, including enhancement of radiotherapy of cancers:

• Szybalski, W., Djordjevic, B., 1960. Radiosensitization of human cells by partial 5-bromodeoxyuridine substitution of the DNA component. Proc. Amer. As-soc. Cancer Res. 3, 155.
• Djordjevic, B., Szybalski, W., 1960. Genetics of human cell lines. III. Incorporation of 5-bromo-, and 5-iododeoxyuridine into the deoxyribonucleic acid of human cells and its effect on radiation sensitivity. J. Exper. Med. 112, 509-531.
• Erikson, R. L., Szybalski, W., 1961. Molecular radiobiology of human cell lines. I. Comparative sensitivity to X-rays and ultraviolet light of cells containing halogen-substituted DNA. Biochem. Biophys. Res. Comm. 4, 258-261.
• Ragni, G., Szybalski, W., 1962. Molecular radiobiology of human cell lines. II. Effects of thymidine replacement by halogenated analogues on cell inactivation by decay of incorporated radiophosphorus. J. Mol. Biol. 4, 338-346.
• Erikson, R. L., Szybalski, W., 1963. Molecular radiobiology of human cell lines. III. Radiation sensitizing properties of 5-iododeoxyuridine. Cancer Res. 23, 122-130.
• Erikson, R. L., Szybalski, W., 1963. Molecular radiobiology of human cell lines. V. Comparative radiosensitizing properties of 5-halodeoxycytidines and 5-halo-deoxyuridines. Radiation Res. 20, 252-262.
• Szybalski, W., 1962. Properties and applications of halogenated deoxyribonucleic acids. In: The Molecular Basis of Neoplasia (A Collection of Papers Presented at the Fifteenth Annual Symposium on Fundamental Cancer Research, 1961). University of Texas Press, Austin, pp. 147-171. The principle of radiosensitization was used to study the mechanism of DNA replication in phage 0X-174:
• Kozinski, A. W., Szybalski, W., 1959. Dispersive transfer of the parental DNA molecule to the progeny of phage aX-174. Virology 9, 260-274.
• Szybalski, W., 1959. Discussion (Mode of DNA replication in phage aX-174), Structure and Function of Genetics Elements. Brookhaven Symposia in Biology No. 12, 32-34.

Dr. Szybalski also elucidated the mechanism of „thymine-less death", due to single-strand breakage of DNA, which was found to lose its transforming activity:

• Mennigmann, H. D., Szybalski, W., 1962. Transforming activity of DNA isolated from bacteria undergoing „thymine-less" death. Bacteriol. Proc, 43.
• Mennigmann, H. D., Szybalski, W., 1962. Molecular mechanism of thymine-less death. B. B. Res. Comm. 9, 398-404.

Dr. Szybalski showed that transforming DNA labelled bifilarly (in both strands) by 5-bromodeoxyuridine retained its biological transforming activity:

• Szybalski, W., Opara-Kubinska, Z., Lorkiewicz, Z., Ephrati-Elizur, E., Zamen-hof, S., 1960. Transforming activity of deoxyribonucleic acid labelled with 5-bromouracil. Nature 188, 743-745.

Using the above principle, Dr. Szybalski provided definitive proof that DNA is a principal target of radiation, by showing that in vivo incorporation of 5-bromodeoxyuridine into DNA results in a parallel increase in radiosensitivity of: (1) isolated transforming DNA, as assayed by radiosensitivity of its transforming activity, and (2) entire cells, as measured by their survival:

• Szybalski, W., Opara-Kubinska, Z., 1961. DNA as principal determinant of cell radiosensitivity. Radiation Res. 14, 508-509.
• Opara-Kubinska, Z., Lorkiewicz, Z., Szybalski, W., 1961. Genetic transformation studies. II. Radiation sensitivity of halogen labeled DNA. Biochem. Biophys. Res. Comm. 4, 288-291.
• Szybalski, W., Lorkiewicz, Z., 1962. On the nature of the principal target of lethal and mutagenic radiation effects. In: Strahleninduzierte Mutagenese - Erwin-Baur-Gedachtnisvorlesungen II, 1961. Stubbe, H. (Ed.), Abhandlungen d. Deutschen Akademie d. Wissenschaften zu Berlin, Klasse fur Medizin 1, 63-71.
• Opara-Kubinska, Z., Borowska, Z., Szybalski, W., 1963. Genetic transformation studies. III. Effect of UV light on the molecular properties of normal and halogenated DNA. Biochim. Biophys. Acta 72, 298-309.

The 1961/1962 experiments of Litman and Szybalski were the first demonstration that the crude E. coli DNA polymerase, as prepared by Dr. R. Litman, can synthesize in vitro the biologically active transforming DNA. Bifilarly 5-bromo-deoxy-uridine (BUdR)-labeled transforming DNA was used in vitro as a template in enzymatic replication using only normal triphosphates. After two rounds of replication, the totally BUdR-free DNA was isolated by CsCl-density gradient cen-trifugation, and this template-free product was shown to have transforming activity. [John Cairns, when summarizing the 33th Cold Spring Harbor Symposium, said that biology should be divided into two periods: one before and one after the Litman & Szybalski (1963) experiment]:

• Litman, R. M., Szybalski, W., 1963. Enzymatic synthesis of transforming DNA. Biochem. Biophys. Res. Comm. 10, 473-481.

After deciding to study the genetics of human bone-marrow cell lines (D98 or Detroit 98), Dr. W. Szybalski made the following contributions: 17(1). MUTANTS AND MUTATION RATES He had isolated many mutant lines and determined mutation rates. He also developed a colorimetric procedure, based on methylene blue binding and elution, to follow the cell growth on the dish surface, and improved some methods for maintaining the human cell lines:

• Szybalski, W., Smith, M. J., 1959. Genetics of human cell lines. I. 8-Azaguani-ne resistance, a selective „single-step" marker. Proc. Soc. Exp. Biol. Med. 101, 662-666.
• Szybalski, W., 1959. Genetics of human cell lines. II. Method for determination of mutation rates to drug resistance. Exp. Cell Res. 18, 588-591.
• Ragni, G., Szybalski, W., Borowski, E., Schaffner, C. P., 1961. N-acetylcandidin, a water-soluble polyene antibiotic for prophylaxis and decontamination of tissue cultures. Antibiotics & Chemotherapy 11, 797-799.

17(2). HPRT- MUTANTS; BIOCHEMICAL BASIS For 8-azahypoxanthine-resistant mutants, he showed that they were missing hypoxanthine phosphoribosyl transferase [HPRT (or old name IMPPase)]:

• Szybalski, W., Szybalska, E. H., Brockman, R. W., 1961. Biochemical basis of sequential mutations toward resistance to purine analogs in human cell lines. Proc. Amer. Assoc. Cancer Res. 3, 272.

17(3). HAT MEDIUM Based on the pathways of purine synthesis, Dr. Szybalski developed a novel kind of selection, and named it HAT (hypoxanthine + aminopterine + thymidine). HAT medium permits one to isolate even rare HPRT+ cells among HPRT" cells:

• Szybalski, W., Szybalska, E. H., 1961. Selective systems for measuring forward and reverse mutation rates involving loss and gain of enzyme in human cell lines. V International Congress of Biochemistry. Abstracts and Communications. Moscow, 10-16 August 1961. Pergamon Press Ltd., Oxford, and Panstwowe Wydawnictwo Nauko-we, Warsaw 411.
• Szybalski, W., Szybalska, E. H., Ragni, G., 1962. Genetic studies with human cell lines. National Cancer Institute Monograph No. 7, 75-89.
• Szybalski, W., 1983. Old HAT. Science News 124, 147. HAT medium is widely used, and led to the development of monoclonal antibodies by Kohler and Millstein, for which they were awarded the 1984 Nobel Prize.

17(4). GENETIC TRANSFORMATION OF HUMAN CELLS, HYBRIDOMAS AND GENE THERAPY To demonstrate for the first time the DNA-mediated transformation of human cells (from HPRT" to HPRT+ phenotype and genotype), Dr. Szybalski has used HAT selection and DNA carrying parts of the HPRT+ gene. These experiments were seminal for the future gene therapy (the term coined and used by Dr. Szybalski since 1962):

• Szybalska, E. H., Szybalski, W., 1962. Genetics of human cell lines. IV. DNA mediated heritable transformation of a biochemical trait. Proc. Nad. Acad. Sci. 48, 2026-2034.
• Szybalski, W., 1963. DNA-mediated genetic transformation of human cell lines. Proc. 12th Ann. Session Nad. Poultry Breeder's Roundtable, Kansas City, pp. 90-109. The paper above described the effect of Ca-phosphate precipitation on the transformation of mammalian cells. He also demonstrated HPRT4 hybridoma formation between HPRT" D98/AH-2 cells and freshly explanted human skin cells:
• Szybalski, W., 1964. Drug resistance as a genetic marker. In: Somatic Cell Genetics. Krooth, R. S., Ed. Fourth Macy Conference on Genetics, Univ. of Michigan Press, Ann Arbor, pp. 226-252.
• Szybalski, W., Szybalska, E. H., 1962. Drug sensitivity as a genetic marker for human cell lines. Univ. of Michigan Medical Bulletin 23 (1962) 227-293; and In: Approaches to the Genetic Analysis of Mammalian Cells. Michigan Conference in Genetics. Merchant, D. and Neel, J. V., Eds. The University of Michigan Press, Ann Arbor, MI, pp. 11-27. The experiments described above are considered to be forerunners of monoclonal antibody-producing hybridomas and gene therapy:
• Szybalski, W., 1992. Roots: Use of the HPRT gene and the HAT selection technique in DNA-mediated transformation of mammalian cells: first steps toward developing hybridoma techniques and gene therapy. BioEssays 14, 495-500.
• Szybalski, W., 1991. A forerunner of monoclonal antibodies and human gene therapy (This Week's Citation Classic. Szybalska, E. H., Szybalski, W., Genetics of human cell lines. IV. DNA-mediated heritable transformation of a biochemical trait. Proc. Natl. Acad. Sci., U.S.A. 48, 2026-2034. Current Contents 34 No. 46, 11,1991.

17 (5). SEMICONSERVATIVE MODE OF HUMAN DNA REPLICATION Dr. Szybalski has demonstrated that the replication of human DNA is semiconservative:

• Djordjevic, B., Szybalski, W., 1960. Genetics of human cell lines. II. Incorporation of 5-bromo-, and 5-iododeoxyuridine into the deoxyribonucleic acid of human cells and its effect on radiation sensitivity. J. Exper. Med. 112, 509-531.

Dr. W. Szybalski showed (for the first time) that some antibiotics (mitomycin C and porfiromycin) can crosslink complementary DNA chains in vivo. He unraveled the chemistry of mitomycin activation by its enzymatic or chemical reduction, and of DNA crosslinking (mediated by carbonium ions):

• Iyer, V. N., Szybalski, W., 1963. A molecular mechanism of mitomycin action: linking of complementary DNA strands. Proc. Natl. Acad. Sci. 50, 355-362.
• Iyer, V. N., Szybalski, W., 1964. Mitomycins and porfiromycin: chemical mechanism of activation and crosslinking of DNA. Science 145, 55-58.
• Szybalski, W., Iyer, V.N., 1964. Crosslinking of DNA by enzymatically or chemically activated mitomycins and porfiromycins, bifunctionally „alkylating" antibiotics. Federation Proc. 23, 946-957.
• Szybalski, W., 1964. Structural modifications of DNA: crosslinking, circula-rization and single-strand interruptions. Abhandlungen d. Deutschen Akad. d. Wissenschaft. zu Berlin, Klasse Med., Nr. 4, 1-19.

Dr. Szybalski also developed automated equipment to study the melting transitions (temperature) for DNA:

• Szybalski, W., Mennigmann, H. D., 1962. The recording thermospectrophoto-meter, an automatic device for determining the thermal stability of nucleic acids. Analyt. Biochem. 3, 267-275.
• Szybalski, W., 1967. Effect of elevated temperatures on DNA and some poly-nucleotides: Denaturation, renaturation and cleavage of glycosidic and phosphate ester bonds. In: Thermobiology. Rose, A. H., Ed. Chapter 4, Academic Press, London, pp. 73-121.

Dr. W. Szybalski perfected various aspects of CsCl density gradient centrifugation (including the development of special 4-cell optics for the analytical ultra-centrifuge) and developed Cs2SO4 density gradient centrifugation for RNA and DNA:

• Szybalski, W., 1960. Sampling of virus particles and macromolecules sedimen-ted in an equilibrium density gradient. Experientia 16, 164.
• Lozeron, H. A., Szybalski, W., 1966. Suppression of RNA precipitation during Cs2SO4 density gradient centrifugation. Biochem. Biophys. Res. Commun., 23, 612-618.
• Szybalski, W., Szybalska, E. H., 1971. Equilibrium density gradient centrifugation. In: Procedures in Nucleic Acid Research. Cantoni, G. L., Davies, D. R., Eds. Vol. 2, Harpei; and Row, New York, pp. 311-354. Using these techniques he demonstrated the following:

20 (1). INTERCALATION OF ANTIBIOTICS Dr. Szybalski showed that the antibiotics of the tetracycline group and ethidium bromide could intercalate into DNA, even at high (7 M) salt concentration:

• Kersten, W., Kersten, H., Szybalski, W., 1966. Physicochemical properties of complexes between DNA and antibiotics which affect RNA synthesis (acti-nomycin, daunomycin, cinerubin, nogalamycin, chromomycin, mithramycin, and olivomycin). Biochemistry 5, 236-244.

20 (2). PLASMIDISOLATION Dr. Szybalski, together with Dr. H. Bujard, was the first to apply the above finding for the separation of covalently closed DNA circles (papilloma DNA) from the nicked circles and linear DNA. This method became a very important and widely used technique for plasmid isolation. 20 (3). SEPARATION OF COMPLEMENTARY DNA STRANDS Dr. Szybalski showed that poly(IG) or poly(UG) binds preferentially to one of the two strands of phage T7 and lambda DNA, which permitted efficient separation of the complementary DNA strands using CsCl + poly(UG) density gradient centrifugation:

• Opara-Kubinska, Z., Kubinski, H., Szybalski, W., 1964. Interaction between denatured DNA, polyribonucleotides, and ribosomal RNA: attempts at preparative separation of the complementary DNA strands. Proc. Nad. Acad. Sci. U.S. 52, 923-930.
• Sheldrick, P., Szybalski, W., 1967. Distribution of pyrimidine “clusters" between the complementary DNA strands of certain Bacillus bacteriophages. J. Mol. Biol. 29, 217-228.
• Hradecna, Z., Szybalski, W., 1967. Fractionation of the complementary strands of coliphage 1 DNA based on the asymmetric distribution of the poly IG-binding sites. Virology 32, 633-643.
• Summers, W. C, Szybalski, W., 1968. Size, number, and distribution of poly G binding sites on the separated DNA strands of coliphage T7. Biochim. Biophys. Acta 166, 371-378.

20(4). REPLICATION OF GLUCOSYLATED DNA Dr. Szybalski showed that glucosylated DNA can be separated from non-modified DNA; using this technique he was able to study the mechanism of DNA modification and replication in phages containing glucosylated DNA:

• Erikson, R. L., Szybalski, W., 1964. The Cs2SO4 equilibrium density gradient and its application for the study of T-even phage DNA: glucosylation and replication. Virology 22, 111-124.

5-iododeoxyuridine incorporation was determined not only for cells, but also for plants and animals:

• Smith, H. H., Kugelman, B. H., Commerford, S. L, Szybalski, W., 1963. Incorporation of 5-iododeoxyuridine into DNA of plant cells. Proc. Nad. Acad. Sci. 49,451-457.
• Clifton, K. H., Szybalski, W., Heidelberger, C, Golin, F. F., Ansfield, F. J., Vermund, H., 1963. Incorporation of I125iabeled iododeoxyuridine into the de-oxyribonucleic acid of murine and human tissues following therapeutic doses. Cancer Res. 23, 1715-1723.

Preparative separation of DNA strands of the T7 and lambda (1) phages permitted Dr. Szybalski to study transcription from individual strands, at the time when there was a general belief that there is only one “sense" strand of DNA. Dr. Szybalski found that for T7 phage, only one strand is transcribed:

• Summers, W. C, Szybalski, W., 1968. Totally asymmetric transcription of coliphage T7 in vivo: correlation with poly G binding sites. Virology 34, 9-16. However, Dr. Szybalski found that for lambda DNA both strands were transcribed (this finding was iconoclastic at that time and very important for determining the orientation of genes and operons by physical means):
• Hradecna, Z., Szybalski, W., 1967. Fractionation of the complementary strands of coliphage 1 DNA based on the asymmetric distribution of the poly I, G-binding sites. Virology, 32, 633-643.
• Taylor, K., Hradecna, Z., Szybalski, W., 1967. Asymmetric distribution of the transcribing regions on the complementary strands of the coliphage 1 DNA. Proc. Nad. Acad. Sci., U.S.A. 57, 1618-1625. Dr. Szybalski rapidly extended these studies to preparing a detailed transcriptional map of DNA by: (1) Determining the orientation of transcription, by hybridization to separated DNA strands:
• Szybalski, W., Bavre, K., Fiandt, M., Guha, A., Hradecna, Z., Kumar, S., Lozeron, H. A., Maher, V. M., Sr., Nijkamp, H. J. J., Summers, W. C, Taylor, K., 1969. Transcriptional controls in developing bacteriophages. J. Cell. Physiol. 74, Supplement 1, 33-70. (2) Determining which region is transcribed, by using (a) DNA fragments obtained by shear, and (b) various deletion mutants of phage lambda:
• Nijkamp, H. J. J., B0vre, K., Szybalski, W., 1970. Controls of rightward transcription in coliphage 1. J. Mol. Biol. 54, 599-604.
• Kumar, S., Boejvre, K., Guha, A., Hradecna, Z, Maher, V. M., Sr., Szybalski, W., 1969. Orientation and control of transcription in E. coli phage 1. Nature 221, 823-825.

23(1). GENOME MAPPING (1) In collaboration with Dr. H. Ris's laboratory, Dr. Szybalski developed heteroduplex mapping by electron microscopy of DNA in 50% formamide, permit ting the measuring of both the single-stranded and double-stranded region of heteroduplexes:

• Westmoreland, B. C, Szybalski, W., Ris, H., 1969. Mapping of deletions and substitutions in heteroduplex DNA molecules of bacteriophage lambda by electron microscopy. Science 163, 1343-1348.

(2) This technique permitted the most precise at that time physical mapping of deletions, insertions, and substitution, and thus provided correlation between genetic maps and physical or transcriptional maps for phage lambda and other genomes:

• Hradecna, Z., Szybalski, W., 1969. Electron micrographic maps of deletions and substitutions in the genomes of transducing coliphages ldg and Ibio. Virology 38, 473^77; and 40, 178, 1970.

(3) Heteroduplex mapping permitted one to compare the physical and transcriptional maps of phages 1 and o80:

• Lozeron, H. A., Szybalski, W., 1969. Congruent transcriptional controls and heterology of base sequences in coliphage 1 and o8O. Virology 39, 373-388.
• Fiandt, M., Hradecna, Z., Lozeron, H. A., Szybalski, W., 1971. Electron micrographic mapping of deletions, insertions, inversions, and homoiogies in the DNAs of coliphages lambda and phi80. In: The Bacteriophage Lambda. Hershey, A. D., Ed. Cold Spring Harbor Laboratories, Cold Spring Harbor, pp. 329-354.

23 (2). VISUALIZATION OF EVOLUTION

• Szybalski, W., Szybalski, E. H. Visualization of the evolution of viral genomes. In: E. Kurstak and K. Maramorosch (Eds.), Viruses, Evolution and Cancer, pp. 563-582. New York: Academic Press, 1974.

Heteroduplex analysis permitted one to classify the IS insertion sequences and to show that these do not consist of any DNA, but that there are very specific .Jumping genes". The IS2, IS2, IS3, IS4 and IS5 elements were defined:

• Fiandt, M., Szybalski, W., Malamy, M. H., 1972. Polar mutations in lac, gal, and phage 1 consist of a few IS-DNA sequences inserted with either orientation. Mol. Gen. Genetics 119, 223-231.
• Szybalski, W., 1977. IS elements in Escherichia coli, plasmids and bacteriophages. In: DNA Insertion Elements, Plasmids and Episomes. Bukhari, A. I., Shapiro, J. S., Adhya, S. L. (Eds.). Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 583-590.

Dr. Szybalski contributed significantly to the pioneering findings in the molecular genetics of phage lambda, a model for subsequent analogous studies with other viruses. 25 (1). TRANSCRIPTION FROM BOTH STRANDS IN THE B2 REGION For the first time, it was shown that not only both DNA strands are transcribed, but that even the DNA strands opposite of each other, in the bl region of 1, could be transcribed. The regulatory role for the convergent and overlapping transcription was considered:

• Bavre, K., Szybalski, W., 1969. Patterns of convergent and overlapping transcription within the b2 region of coliphage 1. Virology 38, 614-626.

25 (2). ANTI-SENSE MRNA Based on the convergent transcription in the central region of lambda genome (see above), Dr. Szybalski explored for several years (1969-1980) the possibility of using overlapping convergent transcription as a mode controlling transcription (anti-sense mRNA):

• Hasan, N., Somasekhar, G., Szybalski, W., 1988. Antisense RNA does not significantly affect expression of the galK gene of Escherichia coli or the N gene of coliphage lambda. Gene 72, 247-252.

25 (3). PHYSICAL MAPPING OF TRANSCRIPTIONAL ELEMENTS The physical location of various promoters and operators was determined together with the orientation and physical size of several operons:

• Szybalski, W., 1976. Genetic and molecular map of Escherichia coli bacteriophage lambda (1). In: Handbook of Biochemistry and Molecular Biology. Fasman, G. D., Ed. 3rd Ed., Nucleic Acids, Vol. II. CRC Press, Cleveland, pp. 677-685.
• Szybalska, E. H., Szybalski, W., 1979. A comprehensive molecular map of bacteriophage lambda. Gene 7, 217-270.
• Szybalska, E. H., 1987. This week's citation classic: Szybalska, E. H., Szybalski, W., 1979. A comprehensive molecular map of bacteriophage lambda. Gene 7, 217-220. [McArdle Lab. for Cancer Research, University of Wisconsin, Madison, WI USA] Current Contents 30, No. 34, 15.
• Daniels, D. L., Schroeder, J. L., Szybalski, W., Sanger, F., Blattner, F. R., 1984. A molecular map of coliphage lambda. In: Genetic Maps 1984, Vol. 3, O'Brien, S. J. (Ed.). Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, pp. 1-21.

Dr. Szybalski studied transcription patterns of bacterial genes “naturally cloned" in phage 1. He determined the orientation of the gal, trp, lad and lacZYA operons, the tRNA gene and ribosomal genes, among others:

• Guha, A., Tabaczynski, M., Szybalski, W., 1968. The orientation of transcription for the galactose operon as determined by hybridization of gal mRNA with the separated DNA strands of coliphage ldg. J. Mol. Biol. 35, 207-213.
• Kumar, S., Szybalski, W., 1969. The orientation of transcription of the lac operon and its repressor gene i in Escherichia coli. J. Mol. Biol. 40, 145-151.
• Lozeron, H. A., Szybalski, W., Landy, A., Abelson, J., Smith, J. D., 1969. Orientation of transcription for the amber suppressor gene sum as determined by hybridization between tyrosine tRNA and the separated strands of transducing coliphage o8OdsuIII. J. Mol. Biol. 39, 239-243.
• Fiandt, M., Szybalski, W., Blattner, F. R., Jaskunas, S. R., Lindahl, L., Nomura, M., 1976. Organization of ribosomal protein genes in Escherichia coli. I. Physical structure of DNA from transducing 1 phages carrying genes from the aroE-str region. J. Mol. Biol. 106, 817-835.

The first example of a divergent bacterial operon was the bio operon which is transcribed from both strands (bioA leftward and bioB-F rightward):

• Guha, A., Saturen, Y., Szybalski, W., 1971. Divergent orientation of transcription from the biotin locus. J. Mol. Biol. 56, 53-62.
• Kotval, J., Campbell, A., Konopa, G., Szybalski, W., 1982. Leftward transcription in Escherichia coli bio operon does not require products of the rightward transcript. Gene 17, 219-222 [and Erratum in 18, 366].

A complete map of the bio operon(s) was prepared:

• Szybalska, E. H., Szybalski, W., 1982. A physical map of the Escherichia coli bio operon. Gene 19, 93.

Combining in vivo and in vitro transcription, Dr. W. Szybalski's laboratory for the first time sequenced the 5-terminal 200 nucleotides downstream from the major promoters and found that (a) the promoter is not transcribed, and (b) initiation (first 5' nucleotide) is exactly the same in vivo and in vitro:

• Blattner, F. R., Dahlberg, J. E., Boettiger, J. K., Fiandt, M., Szybalski, W., 1972. Distance from a promoter mutation to an RNA synthesis startpoint on bacteriophage 1 DNA. Nature New Biol. 237, 232-236.
• Dahlberg, J. E., Lozeron, H. A., Szybalski, W., 1972. Identity of in vivo and in vitro initiation of four phage lambda RNAs. Bacteriophage Meeting, Cold Spring Harbor, NY, pp. 86.
• Szybalski, W., 1974. In vivo and in vitro initiation of transcription. In: Control of Gene Expression. Kohn, A., Shatkay, A., Eds. Plenum Press, New York, pp. 23-24; Panel Discussion pp. 404-405,411-412, 415,417.

Two jointly controlled short transcription regions, lit and oop were studied:

• Hayes, S., Szybalski, W., 1973. Control of short leftward transcripts from the immunity and ori regions in induced coliphage lambda. Mol. Gen. Genetics 126, 275-290.

In conjunction with experiments on the initiation of transcription, these experiments led W. Szybalski to propose that two separate regions could be transcribed in the form of a single mRNA molecule, with the region between them being a non-transcribed „dry run". This proposal anticipated by several years the concept of „intron", which would conceptually correspond to „dry run" and the resulting ..repertoire of topologically different mRNAs from a constant segment of DNA". These early concepts of „intron" are discussed on p. 76 in the paper of:

• Szybalski, W., 1972. Transcription and replication in E. coli bacteriophage lambda. In: Uptake of Informative Molecules by Living Cells. Ledoux, L., Ed. North-Holland Publ. Co., Amsterdam, pp. 59-82. And on pp. 234-236 in the paper by:
• Blattner, F. R., Dahlberg, J. E., Boettiger, J. K., Fiandt, M., Szybalski, W., 1972. Distance from a promoter mutation to an RNA synthesis startpoint on bacteriophage 1 DNA. Nature New Biol. 237, 232-236.

Furthermore, Dr. Szybalski's laboratory in collaboration with Dr. S. Adhya has determined the origin and bidirectional nature of 1 DNA replication:

• Stevens, W. F., Adhya, S., Szybalski, W., 1971. Origin and bidirectional orientation of DNA replication in coliphage lambda. In: The Bacteriophage Lambda. Hershey, A. D. (Ed.). Cold Spring Harbor Laboratories, Cold Spring Harbor, pp. 515-533.

Many terminator sequences were identified and cloned in Dr. Szybalski's laboratory. They were and are widely used in many constructs:

• Luk, K.-C, Szybalski, W., 1982. Transcription termination: sequence and function of the rho-independent tL3 terminator in the major leftward operon of bacteriophage lambda. Gene 17, 247-258.
• Luk, K.-C, Dobrzanski, P., Szybalski, W., 1982. Cloning and characterization of the termination site t1 for the gene int transcript in coliphage lambda. Gene 17, 259-262.
• Luk, K.-C, Szybalski, W., 1982. Characterization of the cloned terminators tR1, tL3 and t1 and of the nutR antitermination site of coliphage lambda. Gene 20, 127-134.
• Luk, K.-C, Szybalski, W., 1983. The tL2 cluster of transcription termination sites between genes bet and ral of coliphage lambda. Virology 125, 403-418.
• Luk, K.-C, Szybalski, W., 1983. A cluster of leftward, rho-dependent t} terminators in the J gene of coliphage lambda. Gene 21, 175-191.
• Luk, K.-C, Szybalski, W., 1983. Tandem transcription-termination sites in the late rightward operon of bacteriophage lambda. Mol. Gen. Genet. 189, 289-297.

In Dr. Szybalski's laboratory, several mutants were isolated which define the nuth and nutK antiterminator elements:

• Salstrom, J. S., Szybalski, W., 1978. Coliphage lnutL-: a unique class of mutants defective in the site of gene N product utilization for antitermination of leftward transcription. J. Mol. Biol. 124, 195-221.

Very extensive studies were carried out on N-mediated antitermination showing that the complete nut element directs antitermination of transcription originating at a variety of promoters. Variants of nut (truncated or mutated) can work with only some promoters and some terminators. Rho-independent terminators are generally easier to antiterminate. Many variants of nut sites were chemically synthesized, at that time one of the first cases of chemical synthesis of transcription-controlling elements:

• Salstrom, J. S., Fiandt, M., Szybalski, W., 1979. N-independent leftward transcription in coliphage lambda: deletions, insertions and new promoters bypassing termination functions. Mol. Gen. Genetics 168, 211-230.
• Salstrom, J. S., Fiandt, M., Szybalski, W., 1979. The site controlling the specificity of Naction is outside the promoter-operator: a triple hybrid phage lN21imm434nin5. Gene 5, 305-327.
• Drahos, D., Szybalski, W., 1981. Antitermination and termination functions of the cloned nutL, N, and tu modules of coliphage lambda. Gene 16, 261-274 [and Errata in 17, 354-355, 1982].
• Szybalski, W., Drahos, D., Luk, K.-C, Somasekhar, G., 1983. Modules for termination and antitermination of transcription in coliphage lambda. In: Microbiology-1983, Schlessinger, D. (Ed.). American Society for Microbiology, Washington, D. C, pp. 35-38.
• Drahos, D., Galluppi, G. R., Caruthers, M., Szybalski, W., 1982. Synthesis of the nutL DNA segments and analysis of antitermination and termination functions in coliphage lambda. Gene 18, 343-354.
• Peltz, S. W., Brown, A. L., Hasan, R, Podhajska, A. J., Szybalski, W., 1985. Thermosensitivity of a DNA recognition site: activity of a truncated nuth antiterminator of coliphage lambda. Science, 228, 91-93.
• Hasan, R, Szybalski, W., 1986. Boundaries of the nuth antiterminator of coliphage lambda and effects of mutations in the spacer region between boxA and boxB. Gene 50, 87-96.
• Hasan, R, Szybalski, W., 1986. Effect of the promoter structure on the transcription antitermination function. Gene 50, 97-100.
• Szybalski, W., Brown, A. L., Hasan, R, Podhajska, A. J., Somasekhar, G., 1987. Modular structure of the nut and qut antiterminators of transcription.
• Interactions between control elements of phage lambda and construction of novel regulatory circuits. In: RNA Polymerase and the Regulation of Transcription, Reznikoff, W. S., Burgess, R. R., Dahlberg, J. E., Gross, C. A., Record, M. T., Wickens, M. P. (Eds.). Elsevier, New York, pp. 381-890.

Dr. Szybalski found that the Q protein interacts with the qut site, which includes the -10 region of the pR promoter and about 15 nt of downstream sequence. Thus, the qut site is coupled with the pR promoter which differs from the nut antitermination site which does not have to overlap promoters:

• Somasekhar, G., Szybalski, W., 1983. Mapping of the Q-utilization site (qut) required for antitermination of late transcription in bacteriophage lambda. Gene 26,291-294.
• Szybalski, W., Drahos, D., Luk, K.-C, Somasekhar, G., 1983. Antitermination: the lambda system. Modules for termination and antitermination of transcription in coliphage lambda. In: Microbiology-1983. Schlessinger, D., Ed. Washington, D. C, American Society of Microbiology, pp. 35-38.
• Somasekhar, G., Szybalski, W., 1987. The functional boundaries of Q-utilization site required for antitermination of late transcription in bacteriophage 1. Virology 158, 414-426.
• Kur, J., Hradecna, Z., Hasan, R, Szybalski, W., 1989. Evaluation of the multi-step method for generating BspMI-mediated deletions in studies on the role of the direct 10-bp repeat in the qut-controlled antitermination in phage lambda. Virology 176, 629-632.
• Kur, J., Hradecna, Z., Hasan, R, Szybalski, W., 1990. The role of the direct repeat in g«?-controlled antitermination in phage 1. Virology 176, 629-632.

The mode of processing of the lambda transcripts was determined in Dr. Szybalski's laboratory, including the endonucleolytic cuts and 3'-5' exonucleolytic digestion of lambda mRNA:

• Lozeron, H. A., Dahlberg, J. E., Szybalski, W., 1976. Processing of the major leftward mRNA of coliphage lambda. Virology 71, 262-277.

34(1). CONVERTING CLASS-IIS RESTRICTION ENZYMES TO ENDONUCLEASES WHICH CUTS AT ANY PREDETERMINED UNIQUE SITE The Fokl enzyme which is a class-IIS enzyme which recognizes a specific site (5'-AUGCC>apd cuts DNA at the 9th and 13th nucleotide (nt) away from this site. By designing a special oligodeoxynucleotide adaptor, which has a double-stranded 5'-AUGCC domain and a single-stranded DNA in the 9-13-nt region, one could cut any single-stranded DNA complementary to the single-stranded domain of the adaptor:

• Szybalski, W., 1985. Universal restriction endonucleases: designing novel cleavage specificities by combining adapter oligo-deoxynucleotide and enzyme moieties. Gene 40, 169-173.
• Podhajska, A., Szybalski, W., 1985. Conversion of the Fokl endonuclease to a universal restriction enzyme: cleavage of phage M13mp7 DNA at predetermined sites. Gene 40, 175-181.
• Kim, S. C, Podhajska, A. J., Szybalski, W., 1988. Cleaving DNA at any predetermined site with adapter-primers and class-IIS restriction enzymes. Science 200,504—506.
• Szybalski, W., Kim, S. C, Hasan, N., Podhajska, A. J., 1991. Class-IIS restriction enzymes - a review. Gene 100, 13-26.
• Podhajska, A. J., Kim, S. C, Szybalski, W., 1992. [26] Conferring new specificities on restriction enzymes: cleavage at any predetermined site by combining an adapter oligodeoxynucleotide and a class-IIS enzyme. Meth. Enzymol. 216, part G, 303-309.

  This novel concept was patented:

• Szybalski, W., 1990-patent. Universal restriction endonuclease. U.S. Patent No. 4, 935, 357 (June 19, 1990) 1-14.

34(2). ACHILLE' HELL VERY RARE CLEAVAGE (AC AND RECA-AC) Having the restriction enzyme and cognate methyltransferase (MTase), one could select out of many cut sites only one by covering it with a protein or other complex while inactivating all other sites by methylation with the cognate MTase. The specific covering proteins were various repressors and other proteins which bind to a specific DNA sequence, usually about 20 bp long:

• Koob, M., Grimes, E., Szybalski, W., 1988. Conferring operator specificity on restriction endonucleases. Science 241, 1084-1086. Koob, M., Szybalski, W., 1990. Cleaving yeast and Escherichia colt genomes at a single site. Science 250, 271-273.
• Szybalski, W., 1992. Modifying specificities of restriction enzymes. In: Biotechnology: Bridging Research and Applications. Kamely, D., Chakrabarty, A. M., Korn-guth, S. E. (Eds.). Proceedings of the U.S.-Israel Research Conference on Advances in Applied Biotechnology (June 24-30, 1990, Haifa, Israel). Kluwer Academic Publ., Boston, pp. 371-376.

A complex of a 40-nucleotide long oligodeoxynucleotide (an oligo carrying a specific restriction site) together with the RecA protein and [g-S]ATP, permits one to cover any specific sequence homologous to the oligo which will then be protected from methylation by the cognate MTase. This variant of AC (RecA-AC) is very convenient for physical mapping of genomes by determining the length of DNA fragments (by PFGE) between two adjoining STS'es (sequence-tagged sites):

• Koob, M., Burkiewicz, A., Kur, J., Szybalski, W., 1992. RecA-AC single-site cleavage of plasmids and chromosomes at any predetermined restriction site. Nucleic Acid Res. 20, 5831-5836.
• Szybalski, W., 1997. RecA-mediated Achilles' heel cleavage. Curr. Opin. Biotechnol. 8, 75-81.

This approach permits regulation of the activity of any promoter, including constitutive promoters: 35 (1). AN INVERTIBLE PROMOTER This was first accomplished by placing a promoter between the convergent attB and attP sites (from bacteriophage lambda) and orienting it away from the gene to be expressed (this the OFF orientation). Supplying the Int protein in trans inverts the promoter which results in gene expression (the ON orientation):

• Podhajska, A. J., Hasan, N., Szybalski, W., 1985. Control of cloned-gene expression by promoter inversion in vivo: construction of the att-p-nutL-att-N module. Gene 40, 163-168.
• Hasan, N., Szybalski, W., 1987. Control of cloned gene expression by promoter inversion in vivo: construction of improved vectors with a multiple cloning site and the ptac promoter. Gene 56, 145-151.

35 (2). AN INVERTIBLE GENE (THE INT/ATT AND FLP/FRT SYSTEMS) Still better results were obtained with the inversion of the gene to be expressed, against the stationary promoter. The gene to be expressed was placed between the convergent attB and attP sites and was facing the promoter. In this OFF phase, only the anti-sense mRNA was transcribed from the gene. Supplying the Int protein in trans inverts the gene, which results in massive gene expression in this ON phase. Similarly, the Flp/FRT system (from the 2mm plasmid of yeasts) was selected, because it works in all organisms tested including various bacteria, plants and animals, and because is very tightly regulated. A highly sophisticated plasmid was developed with a two-step control. On the same plasmid, the FLP gene is controlled by the Pm promoter and Tet repressor. In the repressed stage, not enough of Flp protein is produced to cause any Fi?T-mediated inversion of the gene to be expressed, which is (i) oriented against the stationary promoter, (ii) is located between two convergent FRT sites, (iii) has its 5' end is blocked by four terminators, and (iv) in the OFF phase could be transcribed only in the anti-sense direction. - In stage one, the PMt promoter is induced by adding autoclaved chlortetracycline (cTc), which inactivates the Tet repressor, and results in expression of the FLP gene. In stage two, the Flp protein inverts the gene by acting at two convergent FRT sites, which results in the ON phase and massive gene expression from the stationary promoter:

• Hasan, N., Szybalski, W., 1990. Control of cloned gene expression by its inversion in vivo: switch from anti-sense to sense mRNA. Molecular Genetics of Bacteria and Phaggs. Cold Spring Harbor, NY, August 21-26, pp. 64.
• Sektas, M., Szybalski, W., 1998. Tightly controlled two-stage expression vectors employing the Flp/FRT-mediated inversion of cloned genes. Molecular Biotechnology 9, 17-24. [This system was modified by M. Bereta to express genes in mammalian cells.]
• Sektas, M., Hasan, N., and Szybalski, W., 2001. Expression plasmid with very tight two step-control; Int/att-mediated gene inversion with respect to the stationary promoter. Gene 267, 213-220.

Using elements described above (and below) Dr. Szybalski has constructed many regulatory circuits as models for various biological functions and for gene therapy:

• Szybalski, W., 1988. Construction of novel regulatory circuits. 14th International Congress of Biochemistry, Prague, Czechoslovakia, July 10-15, 1988. Abstracts, Vol. TU: S13-4, (July 12, 1988), Videopress IOJ 162 00 Prague 6, 22.

(1) IHF blocks in vitro transcription in the A+T-rich b2 region of phage lambda. That explains the old mystery as to why there is no in vivo transcription from the b2 region of the lambda prophage, whereas up to 50% of in vitro transcription originates in the bl region:

• Kur, J., Hasan, N., Szybalski, W., 1989. Repression of transcription from the b2-att region of the coliphage 1 by the integration host factor. Virology 168, 236-244.
• Kur, J., Hasan, N., Szybalski, W., 1992. Integration host factor (IHF) binds to many sites in the A+T-rich b2 region of phage 1 DNA. Gene 111, 1-9.

  (2) IHF partially represses transcription from the lambda pR promoter:

• Kur, J., Hasan, N., Szybalski, W., 1989. Physical and biological consequences of interactions between the integration host factor (IHF) and the coliphage lambda pR promoter and its mutants. Gene 81, 1-15.
• Kur, J., Hasan, N., Szybalski, W., 1990. Alterations in the late pR promoter of coliphage 1 modify both its activity and interaction with the integration host factor (IHF). Mol. Gen. Genet. 221, 411-420.

(3) IHF was used to convert common restriction enzymes into rare cutters:

• Kur, J., Koob, M., Burkiewicz, A., Szybalski, W., 1992. A novel method for converting common restriction enzymes into rare cutters: integration host factor-mediated Achilles' cleavage (IHF-AC). Gene 110, 1-7.

(1) The cleavage specificity of the FokI enzyme was increased from 5 bp to 7 bp by methylation:

• Posfai, G., Szybalski, W., 1988. Increasing the FokI cleavage specificity from 5 to 7 base pairs by two-step methylation. Nucleic Acids Res. 16, 6245.

(2) A new method was developed to locate methylated C or A:

• Posfai, G., Szybalski, W., 1988. A simple method for locating methylated bases in DNA as applied to detect asymmetric methylation by M FokIA. Gene 69,147-151.
• Posfai, G., Szybalski, W., 1988. A simple method for locating methylated bases in DNA using class-IIs restriction enzymes. Gene 74, 179-191. [Proceedings of the New England Biolabs Workshop on Biological DNA Modification, Gloucester, MA (USA), May 20-23, 1988].

(3) A new simple method of tandem amplification of any gene or sequence was developed:

• Kim, S. C, Szybalski, W., 1988. Amplification of cloned DNA as tandem mul-timers using BspMI-generated asymmetric cohesive ends. Gene 71, 1-8.

(4) A new method for the precise and stepwise trimming of a gene was developed:

• Hasan, N., Kim, S. C, Podhajska, A. J., Szybalski, W., 1986. A novel multistep method for generating precise unidirectional deletions using BspMl, a class-IIS restriction enzyme. Gene 50, 55-62.

(5) A vector for simple gene fusions was constructed:

• Kim, S. C, Posfai, G., Szybalski, W., 1991. A novel gene-fusing vector: construction of a 5'-GGmCC-specific chimeric methyltransferase M-BspRI/M-BsuRI. Gene 100, 45-50.

In 1979, Dr. Szybalski devised a novel method for creating immunity to viral infection by cloning and expressing viral genes in bacteria or in plants. These viral genes carry mutations which are dominant negative toward the infecting virus:

• Szybalski, W., 1991. Protection of plants against viral diseases by cloned viral genes and anti-genes. Gene 107, 177-179.
• Szybalski, W., 1994. Resistance genes: protection from virus infection using dominant-negative mutations in viral genes. Book of Abstracts, Vol. 1, Abstract No. 436. Eighth IUPAC International Congress of Pesticide Chemistry, July 4-9, 1994, Sheraton Washington Hotel, Washington, D. C, p. 492.

A patent for this invention was issued:

• Szybalski, W. T., 1988-Patent. Partially defective foreign gene for conferring immunity on a biological host. U. S. Patent No. 4, 774, 182, Sept. 27, 1988.

Three novel approaches toward this goal, including the sequencing of large eukaryotic genomes, are currently being pursued in Dr. Szybalski's laboratory. These are (1) physical genome mapping using the RecA-AC method,(2) preparation of a large amount of 10- to 100-kb genomic fragments, excised directly from the genome without conventional cloning, and (3) SPEL-6 sequencing by primer walking of the 10- to 100-kb genomic fragments, while preparing primers by ligation of hexamers annealed to the single-stranded template. 40(1). PHYSICAL MAPPING OF THE GENOME. This was already described in the section 34(2) on Achilles' hell cleavage (AC), specifically RecA-AC. All physical distances between STSes up to 10 Mb could be measured and aligned as to generate a precise physical map. This permits one to determine how much DNA is to be sequenced between all the STSes. For the human genome (3000 Mb), there should be at least 600 STSes at average distances of 5 MB:

• Szybalski, W., 1993. From the double-helix to novel approaches to the sequencing of large genomes. Gene 135, 279-290. [translated into Polish and reprinted in: Postepy Mikrobiologii vol. 33 (1994) pp. 393-417, and p. 423].
• Koob, M., Szybalski, W., 1994. Achilles' cleavage: conferring the specificities of DNA-binding molecules on restriction endonu—cleases. In: Eckstein, F., Lilley, D. M. J. (Eds.), Nucleic Acids and Molecular Biology, Vol. 8, 283-296.
• Szybalski, W., 1997. RecA-mediated Achilles' heel cleavage. Curr. Opin. Biotechnol. 8, 75-81.

40(2). LARGE AMOUNTS OF W- TO WO-KB CENOMZC FRAGMENTS Because of artifacts connected with the cloning of large fragments, Dr. Szybalski's laboratory has developed a novel technique which allows in vivo excision and amplification directly from the genome. This is accomplished by placing pairs of parallel FRT sites, together with the oriV sites, at 10-100-kb distances on the genome. When the Flp and TrfA proteins were added in trans, the 10- to 100-kb genomic fragments between the pair of FRT sites were excised and amplified up to 500-fold:

• Szybalski, W., 1993. From the double-helix to novel approaches to the sequencing of large genomes. Gene 135, 279-290. [translated into Polish and reprinted in: Postepy Mikrobiologii vol. 33 (1994) pp. 393-417, and p. 423].
• Posfai, G., Koob, M., Hradecna, Z., Hasan, N., Filutowicz, M., Szybalski, W., 1994. In vivo excision and amplification of large fragments of the Escherichia coli genome. Nucl. Acids Res. 22, 2392-2398.
• Szybalski, W., 1994. In vitro and in vivo excision, amplification and rapid SPEL-6 sequencing of genomic fragments. In: Dujon, B. (Ed.) Workshop on Tools for Genome Mapping, Institut Pasteur, Paris, France, 16 to 18 January 1994, European Commission, EUR 15751 EN, Office for Official Publications of the European Communities, L-2985, Luxemburg, pp. 8-9.
• Wild, J., Hradecna, Z., Posfai, G., Szybalski, W., 1996. A broad-host-range in vivo pop-out and amplification system for generating large quantities of 50- to 100-kb genomic fragments for direct DNA sequencing. Gene 179, 181-188.
• Szybalski, W., Posfai, G., Sektas, M., Wild, J., 1997. In vivo excision of large fragments from bacterial genomes and their amplification: bypassing conventional cloning and subcloning. Genomic Science Series: Small Genomes: Sequencing, Functional Characterization and Comparative Genomics. Hyatt Regency, Hilton Head, SC, January 25-28, 1997. Microbial & Comp. Genomics 1, 385, 1996.
• Wild, J., Sektas, M., Hradecna, Z., Szybalski, W., 1997. Towards construction of a prototype library of strains for in vivo excision and amplification of genomic fragments. Genomic Science Series: Small Genomes: Sequencing, Functional Characterization and Comparative Genomics. Hyatt Regency, Hilton Head, SC, January 25-28, 1997. Microbial & Comp. Genomics 1, 387, 1996.
• Hradecna, Z., Wild, J., Szybalski, W., 1998. Conditionally amplifiable inserts in pBAC vectors. Microbial & Comp. Genomics 3, 58.
• Yoon, Y. G., Posfai, G., Szybalski, W., and Kim, S. C, 1998. Cre//oxP-mediated in vivo excision of large segments from yeast genome and their amplification based on the 2 mm plasmid-derived system. Gene 223, 67-76.
• Wild, J., Sektas, M., Hradecna, Z., Szybalski, W., 1998. Targeting and retrofitting pre-existing libraries of transposon insertions with FRT and oriV elements for in vivo generation of large quantities of any genomic fragment. Gene 223, 55-66.

40(3). SEQUENCING BY SPEL-6 PRIMER WALKING In principle, primer walking is superior to random subcloning and sequencing, because it could easily be automated and it produces the sequence directly, for the 10- to 100-kb genomic fragment, avoiding laborious subcloning and 7- to 10-fold oversequencing. The 5-min assembly of the 18-60 nt primers from a library of hexamers makes the SPEL-6 method very practical and 10- to 100-fold more rapid than present methods, when the full automation becomes available:

• Szybalski, W., 1990. Proposal for sequencing DNA using ligation of hexamers to generate sequential elongation primers (SPEL-6). Gene 90, 177-178. Szybalski, W., 1992. Automated sequencing of large genomes without conventional cloning. [13th International Conference on Biochemical Analysis, Munich (FRG), May 5-8, 1992. Symposium 1: Analysis of the human genome: implications for the diagnosis and management of genetic disease]. Fresenius J. Anal. Chem. 343 (No. 1), p. 4.
• Szybalski, W., 1993. From the double-helix to novel approaches to the sequencing of large genomes. Gene 135, 279-290 [translated into Polish and reprinted in: Postepy Mikrobiologii vol. 33 (1994) pp. 393-417, and p. 423].
• Kaczorowski, T., Szybalski, W., 1994. Assembly of 18-nucleotide primers by ligation of three hexamers: sequencing of large genomes by primer walking. Ana-lyt. Biochemistry 221, 127-135.
• Kaczorowski, T., Szybalski, W., 1996. Cooperativity of hexamer ligation. Gene 179, 189-193.
• Kaczorowski, T., Szybalski, W., 1996. Automated four-color DNA sequencing using primers assembled by hexamer ligation. Gene 179, 195-198.
• Kaczorowski, T., Szybalski, W., 1998. DNA sequencing by SPEL-6 hexamer ligation and primer walking. Gene 223, 83-91.
• Kaczorowski, T., Sektas, M. and Szybalski, W.: (1999). Rapid preparation of denatured double-stranded DNA templates for sequencing. Molecular Biotechnology 11(1999) 199-200.

  40(4). BAC VECTORS AND OTHER AUXILIARY METHODS

• Hradecna, Z., Wild, J., Szybalski, W., 1998. Conditionally amplifiable inserts in pBAC vectors. Microbial & Comp. Genomics 3, 58.
• Szybalski.W., 1999. Conditionally amplifiable BAC vector. US Patent No. 5, 874, 259.
• Sektas, M., Gregorowicz and Szybalski, W. (1999) Transient conversion to RecA+ phenotype to permit PI transduction in any Escherichia coli recA strains. Bio-Techniques 27(1999) 911-914.

41. IN DEFENSE OF RECOMBINANT DNA (REDNA) Dr. Szybalski has served as a founding member of RAC (The NIH Recombinant DNA Advisory Committee) and in addition was very active in «defending» the molecular genetics research from the ill-advised legislative actions, the purpose of which was to restrict or to prohibit the reDNA research in USA and othe countries. - He testified at the Committees of the US Congress and several Foreign Parliaments, including German Bundestag in Bonn, Germany. He published many articles in lay press and in scientific Journals, a few of which are listed below:

• Szybalski, W., 1978. Dangers of legislative and regulatory approaches concerning the hypothetical risks of the recombinant DNA technique. In: H. W. Boyer and S. Nicosia (Eds.), Genetic Engineering, pp. 253-275. Amsterdam: Elsevier/ North Holland Biomedical Press.
• Szybalski, W., 1978. Much ado about recombinant DNA regulations. In: H. H. Fudenberg and V. L. Melnick (Eds.), Biomedical Scientists and Public Policy, Chap. 7, pp. 97-142. New York: Plenum Press.
• Szybalski, W., 1978. Dangers of regulating the recombinant DNA technique. Trends Biochem. Sci. 3, N243-N247.
• Szybalski, W., 1979. Summary and critique of the new NIH Guidelines for recombinant DNA research. Gene 5, 179-196.
• Szybalski, W., 1980. Recommendations against regulations and legislation. In: E. Herwig and S. Hübner (Eds.), Chancen und Gefahren der Genforschung. Protokolle und Materialien zur Anhorung des Bundesministers für Forschung und Technologie in Bonn, 19. bis 21. September 1979, pp. 297-300 and 361-370. R. Oldenbourg Verlag, Munich.
• Szybalski, W., 1980. Asilomar and five years. Trends Biochem. Sci. 5, VI-IX.
• Szybalski, W., 1980. Trying to please both devils and angels. Trends Biochem Sci. 5, XVIII-XIX.
• Szybalski, W., 1982. Benefits and pitfalls of patent policies in academic research employing genetic engineering techniques. In: W. J. Whelan and S. Black (Eds.), From Genetic Engineering to Biotechnology - The Critical Transition pp 223-231. New York: John Wiley & Sons, Ltd.
• Szybalski, W., 1985. (Editorial) Early warning principle offsets the need for regulation of the recombinant DNA technique. BioEssays 2, 147-148.
• Szybalski, W., 1998. Sifting and winnowing: its meaning and significance to faculty and to freedom of research. In: Hansen, W. L. (Ed.). Academic Freedom on Trial. 100 Years of Sifting and Winnowing at the University of Wisconsin-Madison. Office of Univ. Publ., University of Wisconsin-Madison, pp. 235-237.