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Achievements at Cetus (1-8) and Elsewhere by Cetus Employees and Members of the Cetus Board of Scientific Advisors (A-I) 1. Development and use of a unique working system for mass screening .of microorganisms in submerged culture. Development and patenting of a new method for cloning Micromonospora. ,for scanning zone areas for certain assays. Development and use ofa unique, dedicated, laser-based instrument t~. ... ... . . i \' .~ ~ . .... . . . ~ _ ..... .. taneous mutation and evoluti=rate - these rates are adjustable: Isolation of a new class of bacterial mutants of accelerated spon- First useful demonstration ofcell fusion among Micromonospora strains. important in detection of minor differences between strains. dented stringency and uniformity of temperature and humidity, so Design and constructiomof unique environmental rooms with unprece- 6. Combining these and other developments to improve aminoglycoside antibiotic yields of Micromonospora production strains. 7. Improvement of penicillin titers of very high yield production strains by cell fusion as well as by mutation. (With respect to items 6 and 7 Cetus' task is complete. Clients' performance will determine the extent of royalties.) 8. Development of proprietary processes of great potential impact on the production of large-scale commodity chemicals. Patents applied for. 9. A. Streptomyces stanford and purification of restriction endonucleases SSTI and SSTII in previously unavailable quantities. Development of new, efficient high-yield procedure for growth of Chairman, Cetus Corporation Board of Scientific Advisors. Professor Donald A. Glaser - University of California, Berkeley; Nobel Prize (1960) in Physics for the discovery of the bubble chamber. Development, since then, of unique facility for screening unprece- dented numbers of bacterial and animal cell colonies at the University of California at Berkeley. This facility, designated a "National Resource," has already accomplished the following: • Isolation ima few months of the world's largest selection of bac- terial mutants aimed at the study of DNA synthesis and control. . .- .'.: .... ~ . _ . .. . 1 .. .: : _~~]. ~~tF~~ `,~~q~yt...~,.~.~F+7~WYwirl~%~~ ~'1E~i14'rF~-Yrbr3.r.iW.~.tl.~•y~i~i.~i.~~v~rw.Y.rO-.t~G~'i~: i+erv.SAL^T ~L:i

Development of a new sensitive assay for insulin that depends on factors. quantitative measurement of the growth rate of mammalian cells in culture. This assay may help to discover new hormones and growth in identifying pathogenic bacteria and subtle changes of human Development of new quantitative high speed methods that are useful cells in culture. Nobel Prize (1958) in Medicine for discoveries concerning the , Professor Joshua Lederberg - President,Rockefeller University. Discovered and developed "transduction" in bacteria. Coined the term. and intraspecies genetic transfer. These studies culminated in: Recent studies in Dr. Lederberg's lab have been devoted to inter- subtilis. Since the normal B. subtilis strains do not contain plasmids, this finding was of fundamental importance for the development of a host/vector system, "Biophore," in the indus- trially relevant species, B. subtilis. 'aureus can be transferred to and be stably maintained in Bacillus The first demonstration that plasmids normally occurring in S. A greater understanding of the mechanism of inter- and intraspecies chromosomal transfer in Bacillus sp. C. Professor Stanley N. Cohen - Stanford University School of Medicine. Developed first plasmid cloning vector and demonstrated ability to isolate specific DNA fragments. This was the "birth" of recom- binant DNA and patent(s) will be issued to Stanford University and the University of California. First to isolate and maintain eukaryotic (non-bacterial) DNA in bacteria. Interests in insertion sequences and transposable genetic elements culminated in the first isolation of a "self-replicating" trans- poson - a powerful tool not only for studies in the mechanism of transposition, but more importantly of particular benefit for mutagenesis and cloning technology. Discoveries regarding natural site-directed recombination were instrumental in showing that Mother Nature can conduct "recombinant DNA research" and this helped alter Senator Kennedy's views vis-a- vis the need for legislation in this area. ~., _ . .

In genetic systems other than E. coli Dr. Cohen's collaboration with Professor David Hopwood resulted in the development of a very high efficiency protoplast fusion system in Streptomyces. This technique can be used for introducing new genes and hence, genetic variability between organisms. formation in Bacillus subtilis 4 orders of magnitude, i.e., 10,000- Recent experiments in Dr. Cohen's lab have led to the development of techniques which have increased the efficiency of plasmid trans- foldt . . i can be used to identify colonies that are synthesizing proteins or In collaboration with Henry Erlich and Hugh.McDevitt, Dr. Cohen developed a sensitive radio-immunoassay screening procedurethat in many industrial programs. proteimfragments, i.e., enzymes, eukaryotic peptide hormones, viral antigens, immunoglobulins, etc. This will be highly useful D. Professor David Hopwood - The John Innes Institute, University of East Anglia, Norwich, England. Developed and characterized the genetics ofStreptomyces. efficiency protoplast fusion system in Streptomyces yielding extra- In collaboration with Dr. Stanley N. Cohen, developed a high- ordinarily high frequency of chromosomal recombinants. This ad- vance is of considerable importance for introducing genetic varia- bility of diverse origin at high frequency without the usual need for selection. biotic biosynthesis. Others have tried to show this, but Dr. Hopwood's experiments are still the only convincing ones. First to demonstrate plasmid location for genes involved in anti- be brought to another "Biophore" species of tremendous practical relevance. Application of this new technique will permit cloning technology to Most recently developed a DNA transformation system~in Streptomyces. E. Dr. Saran A. Narang - National Research Council, Ottawa, Canada. Expertise of long-standing in oligonucleotide synthesis. Associated with Professor H. Gobind Khorana in the elucidation of the genetic code. Developed an improved and highly efficient methodology for oligo- nucleotide synthesis. This method involves the phosphotriester rather than the phosphodiester methodology used by Khorana for his gene synthesis (Dr. Narang and co-workers, including Cetus' Dr. C. Bahl, hold U.S. Patent #,4,059,592 on the key reagent required to practice this invention). #

Using his new methodology of oligonucleotide synthesis, synthesized the lactose operator of E. coli. This synthetic DNA molecule was the first chemically synthesized DNA fragment shown to express its c biological activity at the cellular level. Synthesized a number of oligonucleotides known as adaptors or linkers - useful as tools in molecular cloning experiments. ,Professor Stanley Falkow - University of Washington. First to demonstrate that ampicillin resistance was on a "trans- poson" and could "jump" from one strain to,'another (via plasmids). Interests in plasmid-mediated antibiotic resistance, clinical medicine, and pharmacology have led to the development of rapid diagnostic screening procedures for pathogenic microorganisms resistant to a range of antibiotics. Extensive background in, and knowledge of, broad host range plas- mids of fundamental importance to biophore program. Dr. David H. Gelfand - Director, Recombinant Molecular Research, Cetus Corporation. First to demonstrate total cell-free synthesis of an RNA polymerase using individual purified components in a"coupled system." Boyer's group to protect somatostatin and by Gilbert's group to protect rat insulin. levels of "read through" expression of foreign DNA fragments. This development protects non-bacterial products from degradation by bacterial enzymes and is therefore crucial to useful expression of genes of higher organisms in bacteria. This approach was used by Developed first multicopy cloning vector permitting stable high Developed first ultra-high level plasmid copy number mutant (i.e., 11-500 copies of the plasmid/cell). This is of particular signifi- cance not only as a tool for isolating large amounts of plasmid (S-galactosidase). It is dedicated focus at this level which will be essential if the exciting laboratory curiosities of the 1970's are to become the efficient and economical production processes of the 1980's. sion of plasmid-encoded gene products, i.e., 125-fold, 12,500%, increase in the accumulated amount of one plasmid-encoded enzyme (TEM S lactamase) and 60-70% of the protein synthetic capacity of the cell used for the synthesis of another plasmid-encoded enzyme DNA, but more importantly for obtaining very high levels of expres- First to demonstrate high efficiency recombination in Micromonospora species using a protoplast fusion technique. This is essential to the use of the "new biology" in Micromonospora. . ~ _ . , .`- tl.i-.~.~?'..~.ss _..~.Y..~-v~`iM:: .r~ .-..i•IaC~.K.~~ra_......~ ,.nY+:n91iSi.ea-M~+.•~`w`•'•e'a+rlv~'~lb''~fi~..as, sJ.,~..1i~i ~~a : 'a.•L"b~~i~

H. Dr. Chander Bahl - Scientist, Cetus Corporation nucleotides. Developed efficient methods of oligonucleotide synthesis. Developed improved methods for the sequence analyses of oligo- and in vivo. .bacterial regulatory element (lac operator) can function in vitro Demonstrated for the first time that a chemically synthesized adapting almost any kind of DNA molecule for molecular cloning These include a whole series of synthetic oligonucleotides used for Developed a number of new techniques for recombinant DNA work. work. . oligonucleotides. This method is being used for the analyses of Developed the probing of genes by the use of chemically synthesized yeast cytochome C gene. Dr. Bill Williams - Scientist, Cetus Corporation Developed a set of A phage cloning vectors called the Charon phages, which now include 21 different designs. and RNA polymerase expression. These experiments also showed the power of phage promoters which could be turned on or off to ex- press, potentially, any cloned gene product in high amounts. laboratory, wherein a link was found between the levels of ribosome coli and showing that none successfully colonized the human gut. Cloned ribosomal protein genes of E. coli so that their regulated expression could be studied in collaboration with M. Nomura's possible laboratory escapees by drinking•1011 specially designed E. Constructed and tested four of the six phage vectors now certified by the National Institutes of Health (NIH) as EK2. This included experiments to demonstrate directly the safety of encountering any