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an extent that they felt that they could read and respond to very little. They shared a common frustration - companies send them press releases on 'hot ' topics, but do not assign anyone to answer questions related to the announcement. If a company wants to keep the attention of the business press, they need to make sure ' someone is home ' when the press calls.

The financial community was rep- resented by three delegates from the venture-capital sector. Alan Walton (Oxford Partners, Stamford, CT, USA) pointed out that 95% of companies in the USA that went public between 1990 and 1992 were venture-capital-backed firms. How- ever, Wal ton noted that 'venture- capital firms have not made profits in the past few years'. A recent study showed that 80% of the venture capi- talists' money in recent initial public offerings has not yet been sold, mainly because of the small cash value of the stock of these companies. Walton dispelled the common belief that the industry will consolidate via merger of companies. ' I f you take two companies, each losing [US]$10 million per year and put them together, what happens? Then add the egos involved and merger is not that likely. Rather, companies will choose to cut their spending rate and shrink in size. Biotech firms don' t merge and they don' t die'.

Peter Bick (Burr, Egan, Deleage and Co., San Francisco, CA, USA)

followed by saying, 'Venture capital- ists are a very pessimistic group and my pessimism is extended so far as to question the sincerity of other ven- ture capitalists. It is said that venture capitalists have predicted eight out of the last three recessions'. However , each of the venture capitalists present described a wide variety of technical, business and financial data sources that they turn to, but also described using a panel o f expert consultants to help with the assessment o f their potential investments.

Hiroki Yoshihara (Transpect Inc., Yokohama, Japan), the former presi- dent of Genzyme Japan KK, pre- sented his view of biotechnology in Japan. He is convinced that, con- trary to popular opinion, Japanese companies are not focused on the long term. Rather, they are focused on the short term, bypassing the lengthy R & D phase by strategic alliances and moving directly to clini- cal trials with proven products. He presented evidence that Japanese companies are catching up in the biotechnology race and that Japanese regulatory agencies are approving products with a shorter lag time than previously.

The conference ended with a keynote talk from John Hodgson, the senior editor for Europe of Bio/Technolo2y. He navigated the group through the possibility of a unified Europe and enlightened the audience as to the difference between the regulations, directives,

decisions, recommendations and opinions related to biotechnology that are handed out by the European Commission. Among his con- clusions was that Europe was far from being unified, with many member countries having yet to create or implement their gene laws that were mandated by a Commission directive in 1990.

In addition to these information sessions, the delegates, representing nine countries, all pointed to a unique value of the meeting. It was the first time that most of them had met others who work with the com- plex maze of strategic business data. It was the perfect venue to discuss common problems, such as the fact that many companies do not include strategic-information acquisition as a budget item, or that many people seeking information expect it to bc provided at no or little cost. One delegate summed up the general feeling of all present: 'I go to lots of meetings where I know dozens o f others, but I only know two people at this meeting. My network of key information people has grown many-fold' .

Mark D. Dibner Director of tile Institute for Bioteclmolqw

hformatio~t, North Carolii~a Biotechnology Center, 130 Box 13547,

Research Triangle Park, NC 27706, USA

'Touching an elephant in the darkness', or 'How to get the whole

picture of biotechnology'

meeting

report For those who still think ofb io tech- nology ,as an unlikely interface between biological research and the stock market, the message from a recent International Workshop* organized by three biotechnology research centers in Europe* is that all research into viral, bacterial, fungal, insect, plant or animal genetics has the potential, sooner or later, to lead

*The International Workshop on 'Molecular Genetics in Modern Biotechnolobw'was held in Mallorca, Spain, 2 6 October 1993.

tO the development of products and processes for the pharmaceutical, agro-industrial and environmental sectors. This meeting also proved to be extremely valuable to the scien- tists involved as it brought together key researchers from diverse biotech-

*The workshop was organized by Gesellschaft ffir Biotcchnologische Forschung (GBF), Braunschweig, Germany; Centro Nacional de Biotecnologla (CNB), Madrid, Spain; and International Institute of Biotechnology (liB), Kent, UK.

nological disciplines and enabled them to identify common issues and problems. The ability to envisage biotechnology as an integrated whole, rather than as disconnected, unrelated research efforts with little to offer each other, can only bring benefits. The following report is a personal view of those diverse pres- entations of this stimulating work- shop that were new to me or seemed to be highlights. However , all the contributions were of high quality, and the many omissions have been dictated purely by limitations on space.

IBTECH JANUARY 1994 (VOL 12) © 1994, Elsevier Science Ltd

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H o w to express m o r e for less The promoters o f genes from

Escherichia coli still represent the model of choice in the study of how to improve and control transcription in other, disparate systems. The lessons learnt from understanding the con- trol of simple prokaryotic promoters have permitted Hermann Bujard (University of Heidelberg, Germany) to construct a hybrid regulatory system. He has combined the activating domain of VP16 from Herpes virus and the tetracycline repressor of Tn 10, enabling heterol- ogous proteins to be expressed in HeLa calls in response to the addition of minute amounts o f tetracycline. By combining this novel regulatory sequence with bicistronic expression systems (i.e. the gene of interest is transcriptionally coupled to a re- porter gene such as that encoding alkaline phosphatase), Hansj6rg Hauser [Gesellschaft Eir Biotech- nologische Forschung (GBF), Braunschweig, Germany] has been able to generate clones of cells that display tetracycline-regulated ex- pression of the interferon regulatory factor 1 (IP,.F1) gene. These clones were used to study the effect of IR_F1 on the proliferation of tumor cells.

Escherichia coli is, of course, not only a useful model system, but is still a host of major industrial importance,

particularly when considering the search for inducible and economical heterologous expression systems in large-scale fermentations. One promising approach is to use pro- moters that are induced in response to the chemical changes that occur during the late stages of fermen- tation; for example, in response to oxygen depletion (Michael Jarsch, Boehringer Mannheim, Penzberg, Germany). Among eukaryotes, the yeast Saccharomyces cerevisiae has long been a popular host for heterologous gene expression, but Cornelius Hollenberg (Institut Eir Mikro- biologie, Heinrich-Heine Univer- sitat, Dfisseldorf, Germany) told the conference about attractive alterna- tive hosts, such as Hansenula polymorpha and Pichia stipitis.

The problems associated with het- erologous expression are not limited to bioreactors. In situ bioremediation of toxic chemicals using genetically manipulated microorganisms requires the expression ofdetoxifying pheno- types under the conditions prevailing in the field. These are often very different from those that exist in the laboratory or under controlled fermentation• Transposon vectors containing starvation promoters and expression cassettes from biodegra- dative pathways of Pseudomonas spp. open the way to the construction of

ecologically safe and predictable genetically modified organisms (GMOs) [Victor de Lorenzo, Centro de Investigaciones Biologicas (CIB), Madrid, Spain].

The wor ld o f R N A The quest for a way to produce a

valuable peptide or pharmaceutical product using homologous or heterol- ogous expression systems does not end with efficient transcription of the gene(s). To the dismay of many biotechnologists, a myriad of bottle- necks can arise at various post-tran- scriptional steps, such as the process- ing, translation and degradation of the resulting P,_NA. In contrast to work on bacterial systems, progress in this area using higher eukaryotic systems is not so far advanced. John McCarthy (GBF, Braunschweig, Germany) surveyed the multifarious functions of R N A structural el- ements in prokaryotes and eukary- otes and gave examples of the prac- tical relevance of research in this area. For example, fine regulation of the translational efficiency of heterol- ogous genes assembled into multi- cistronic expression systems in E. coli can be used to optimize conditions for the formation o£ recombinant heteromultimeric protein com- plexes. On the other hand, yeast has proven to be an excellent host for

TIBTECH JANUARY 1994 (VOL 12)

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investigating gene regulation via R N A structural elements, whether these are homologous elements con- trolling m R N A translation and/or stability, or heterologous ones de- rived from mammalian regulatory circuits (for example, those involved in metallorcgulation). The construc- tion, for the first time, of an in vitro system that reproduces the in vivo pattern o f degradation o f a yeast m R N A ( S. cerevisiae phosphoglycer- ate kinase) should lead to an improved understanding of the degradation process (Hendrik Raud, Vrije Universiteit, Amsterdam, The Netherlands).

The R N A world encompasses far more than just a minefield for researchers working on gene ex- pression: it is also a treasure trove of exciting new aspects of gene regu- lation. Moreover, the study of R N A is enabling the development of powerful techniques that are relevant to molecular biology and medicine. Of course, RNA-related events can be highly complex and present a major theoretical and experimental chal- lenge to the molecular biologist. One fascinating example is R N A editing in trypanosomes (Rob Benne, E. C. Slater Institute, University of Amsterdam, The Netherlands) which, as yet, has not been harnessed as an experimental 'tool'. On the other hand, generally applicable rules for the design of antisense R N A molecules based on studies of IS10 regulation in E. roll are beginning to emerge (Robert Simons, University of California at Los Angeles, CA, USA). There have also been import- ant advances in our understanding and ability to manipulate catalytic sites in (hammerhead) ribozymes (Kazunari Taira, National Institute of Bioscience and Human Technology, Tsukuba, Japan). The combination of tools based on antisense and ribozyme function promises to pro- vide powerful methods for gene tar- geting resulting, for example, in potential anti-HIV agents. Bernard Ehresmann (Institut de Biologie Moleculaire et Cellulaire, Strasbourg, France) discussed the 5' region of HIV-1 R N A and, in doing so, brought home the important mess- age that an understanding of R N A structure can have important conse- quences both scientifically and in terms of potential medical appli- cations. Clearly, research on R N A structure and function promises to be both exciting and productive over the coming years.

IBTECH JANUARY 1994 (VOL 12)

Translation: how to ensure the message is decoded correctly

Arguments for exercising caution when overexpressing heterologous genes in eukaryotic and prokaryotic expression systems were embodied in a review of translational inaccuracy given by Mick Tuite (University of Kent, Canterbury, UK). The issue of misincorporation of amino acids into a polypeptide chain (which may reach levels of 10% or more if rare codons are used) indicates the im- portance of understanding nonstan- dard m R N A decoding mechanisms. Unexpected events such as t R N A hopping (the translational machinery bypasses regions of a coding se- quence in an m R N A molecule) or codon reassignment (deviations from the normal genetic code) have also been reported. Therefore, it is also essential to understand how ribo- somes work - an issue that seems to be regaining some attention over the past few years. The control o f the activity of eukaryotic ribosomes through phosphorylation of the acidic proteins that form the stalk, a distinct protuberance o f the large subunit [Juan Pedro Garcla-Ballesta, Centro de Biologla Molecular (CBM), Madrid, Spain], illustrates the need to fill the many gaps still left in our understanding of the basic mechanisms underlying translation. In any case, transcriptional and post- transcriptional bottlenecks not- withstanding, the dream of most biotechnologists is, ultimately, to obtain the desired protein in a single purification step. The widespread use o f fusion proteins with heterologous affinity groups has been of great help in some cases, but improvements are still required in the step where affinity handles have to be removed to yield the required final product (Mathias Uhl~n, Royal Institute of Technology, Stockholm, Sweden).

Biotechnology in the hothouse Thermophilic microorganisms are

still a matter of curiosity for many, but bacteria that grow happily at 105°C (Garabed Antranikian, T U Harburg, Hamburg, Germany) can- not go unnoticed as a source of novel activities of biotechnological interest (many more than just the Taq pol- ymerase). What makes the pullu- lanase o f Pyrococcus woesei (optimal growth at 99°C) resistant to boiling, even if it is synthesized in E. coli? How can D N A survive denaturation under such conditions? High GC content is not the answer. Interest-

ingly, the 'toughness' of enzymes from thermophiles is not just restricted to resistance against thermo- denaturation; a serine protease from Whermococcus stetteri was not only per- fectly active at 99°C, but also sur- vived in 6% SDS. How can these properties be explained in light o f the recent development o f general rules and mathematical modeling of pro- tei n folding [Alfonso Valencia, Euro- pean Molecular Biology Laboratory (EMBL), Heidelberg, Germany] remains a major challengc for the near future.

Vaccines: tackling old problems with new approaches

If there is a field in biotechnology where progress has been spectacular during the past few years, it is vac- cine development. Live systems using vaccinia virus, adenovirus or even bacterial vectors to present foreign epitopes to lymphoid tissues to induce neutralizing antibodies may, in the future, supersede immu- nization procedures that involve purified antigens or antigen-carrying fusion proteins. However, one prob- lem with the production o f many antigens in a heterologous host is their frequent inability to fold prop- erly and, thus, induce a protective response against the disease. An in- genious approach to circumvent this problem occurring with a recombi- nant vaccine against whooping cough was described by Rino Rappuoli (hnmunobiological Re - search Institute, Siena, Italy). A strain of Bordetella pertussis was engineered to produce a nontoxic form of the pertussis toxin - only in this particu- lar strain could the molecule bc expressed and folded correctly.

The immunization o f mice with a recombinant influenza virus express- ing an epitope from Plasmodium yoelii, followed by immunization with a vaccinia virus vector express- ing the entire circumsporozoite protein, induced protective im- munity against malaria [Mariano Esteban, Centro Nacional de Biotec- notogla (CNB), Madrid, Spain]. Human adenovirus vectors express- ing portions of the spike (S) protein of the transmissible gastroenteritis vires (TGEV) resulted in production of TGEV-specific antibodies in ham- sters (Luis Enjuanes, CNB, Madrid, Spain). However, the imagination of vaccine developers does not stop here. The same author reported recent data on the construction of transgenic swine secreting TGEV-

fOF/2 /g /

neutralizing antibodies in milk dur- ing lactation. However, engineering heterologous gene expression in the mammary gland under the control of adequate hormonal signals is still being investigated, for the most part, in mice, and the full potential and applicabilities of this technology are still to be assessed (Bruce Whitelaw, Institute of Animal Physiology and Genetics Research, Roslin, UK). A very different approach to vaccine development involves the construc- tion of fusions to the T Y A gene of the yeast retrotransposon Ty (Alan Kingsman, University of Oxford, UK) to produce hybrid proteins con- taining HIV antigenic determinants from highly immunogenic virus-like particles (VLPs).

Progress in vaccine development (and the success of therapeutic approaches in general) depends on knowledge of the basis of virulence. The p60 protein of Listeria monocytogenes is responsible for much of the virulence associated with these bacteria (Werner Goebel, Theodor- Boveri Institut f/.ir Biowissenschaften, Wiirzburg, Germany), since it participates actively in adhesion to animal cells. Monoclonal antibodies against p60 and probes for the p60 gene have been developed and are being used to detect the presence of L. monocytogenes in food samples although, unfortunately, treatment is a major problem once the infection has taken hold.

The hemolysins of the enteric bac- teria are another class of virulence factor that give cause for concern. An export signal located at the C-terminus of the hemolysin protein of E. coil determines its release into the external medium via an unusual transport system that does not require periplasmic intermediates (Barry Holland, Institut de Grn4tique et Microbiologie, Orsay, France). This system provides opportunities for vaccine development by using the same transport mechanism to present foreign antigens fused to the C-terminal domain of hemolysin in gut-associated lymphoid tissues.

In stark contrast to the many con- tributions about the construction and use of highly immunogenic agents, Maximilian Tropschug (Albert- Ludwigs-Universit~it Freiburg, Germany) discussed the current status of research on the mode of action of the clinically important immunosuppressants cyclosporin A and FK506. These drugs bind to intracellular receptors that function

not only as peptidyl-prolyl cis-trans isomerases and chaperones, but also form complexes with other interest- ing proteins, such as calcineurin.

Environmental bioremediation In a gentle reminder to those who

believe that microorganisms are interesting only as long as they pro- duce valuable molecules, several speakers stressed the importance of microorganisms that destroy trouble- some molecules. Novel biodegra- dative pathways of Pseudomonas spp. and other Gram-negative bacteria are being studied and improved in the laboratory to give microorganisms that can degrade a variety of other- wise recalcitrant aromatic com- pounds. However, the ecological unpredictability of such GMOs when released into the field has pre- cipitated major developments in the application of molecular techniques to microbial ecology, primarily in the field of monitoring intergeneric gene transfer (Elizabeth Wellington, Uni- versity of Warwick, Coventry, UK). In order to avoid the problem of recombinant DNA dissemination, Eduardo Diaz (GBF, Braunschweig, Germany) has developed an ingeni- ous system to inhibit any possible DNA transfer. A universal killer function (colicin E3) is attached to the gene(s) of interest so that recipi- ents of the recombinant DNA, other than specialized hosts, are unable to survive.

Most environmental problems, however, are less sophisticated. In many countries, the quality of&ink- ing water is being threatened by the presence of excessive amounts of phosphates and nitrates. Whether genetic engineering will enable bac- teria to perform efficient denitrifi- cation under aerobic conditions remains an open question (Stuart Ferguson, University of Oxford, UK). One possibility that has been considered is augmentation of the

residual aerobic denitrifying activity of certain bacteria, perhaps by alter- ing the transcriptional regulation of the genes responsible for this ac- tivity, and protein engineering of some of the oxygen-sensitive prod- ucts involved. Further good news in the environmental sector is the in- creasing use of bacteria engineered with a variety of gene (reporter) fusions to monitor with great speci- ficity and sensitivity the presence and bioavailability of toxic pollutants such as selenium (Hanna Engelberg-Kulka, Hebrew University, Jerusalem, Israel) or alkyl/haloaromatic compounds.

Antibiotics: revisiting the origins Twenty years ago, one of the few

activities that could he properly termed biotechnology was antibiotic production. Although interest in this field has clearly lost ground to other areas of research, new results hint at a major revival of the subject. The concepts developed in the analysis of the actinorhodin gene cluster of Streptomyces (Francisco Malpartida, CNB, Madrid, Spain) suggest that the development of novel antibiotics for clinical use through the genetic engineering of novel biosynthetic pathways may not he far away. In addition, the production of classical antibiotics such as penicillin has ben- efited from the application of mol- ecular techniques to the analysis of the biosynthetic pathways that pro- duce these useful metabolites, (Miguel Pefialva, CIB, Madrid, Spain, and Juan Martin, Universidad de Le6n, Le6n, Spain).

In summary, this was a valuable, inspiring and wide-ranging meeting that offered new insights into the approaches and possibilities offered by molecular genetics to the field of biotechnology.

Victor de Lorenzo Cemro de Investigaciones Biologicas CSIC,

Vetazquez 144, Madri'd 28006, Spain.

• h Trends in Biotec ,nology

are subject to peer-review, whether they are commissioned or unsolicited

TIBTECH JANUARY 1994 (VOL 12)