72 TIBTECH - MARCH 1991 (Vol. 9]

constitutive expression of Mx pro- teins is deleterious to the organism or whether this would further in- crease the influenza virus resistance of animals with a functional en- dogenous Mx defense system.

A third potential problem is re- lated to the fact that Mx proteins are effective against viruses only if the proteins are abundant in the cells. Mx proteins are not secreted and must therefore be synthesized by those specialized cells of the body in which the virus is able ~o replicate. In the case of influenza virus, these cells represent the surface epithelial cell layers of the respiratory tract. Ideally, one would like to use a strong constitutive promoter that directs transgene expression specifi- cally in these specialized cells. These promoters have yet to be characterized.

Perspective Many other questions remain to be

answered before Mx proteins or other IFN-induced proteins can be used for transgenic livestock pro- duction. Since Mx and other IFN-

induced proteins are typically en- coded by small families of genes, the question of which family member to choose is an important one. In the case of the Mx genes, some genes encode nuclear proteins and others encode cytoplasmic proteins. Would the nuclear murine Mxl protein, which is highly effective in mice, also be suitable for the protection of chicken from influenza virus? Would a cytoplasmic Mx protein be more effective, or would it be better to express an avian Mx protein?

The transgene technology is still quite new and its application towards disease-resistant domestic animals awaits further exploration. Nevertheless, intracellular immuniz- ation, as proposed by Arnheiter and co-workers, seems to have a good chance of becoming a feasible method for introducing resistance genes into valuable livestock.

R~ferences 1 Baltimore, D. (1988) Nature 335,

395-396 2 Friedman, A. D., Triesenberg, S. J.

and McKnight, S. L. (1988) Nature

335,452-454 3 Trono, D., Feinberg, M. B. and

Baltimore, D. (1989) Cell 59,113-120 4 Salter, D. W. and Crittenden, L. B.

(1989) Theor. Appl. Genet. 77, 457-461

5 Abel, P. P., Nelson, R. S., De, B., Hoffmann, N., Rogers, S. G., Fraley, R. T. and Beachy, R. N. (1986) Science 232, 738-743

6 Arnheiter, H., Skuntz, S., Noteborn, M., Chang, S. and Meier, E. (1990) Cell 62, 51-61

7 Staeheli, P., Haller, O., Boll, W., Lindenmann, J. and Weissmann, C. (1986) Cell 44, 147-158

8 Horisberger, M. A., Staeheli, P. and Hailer, O. (1983) Prec. Natl Acad. Sci. USA 80, 1910-1914

9 Staeheli, P. and Hailer, O. (1987) Interferon 8, 1-23

10 Staeheli, P., Grob, R., Meier, E., Sutcliffe, G. and Hailer, O. (1988) Mol. Cell. Biol. 8, 4518-4523

11 Staeheli, P., Yu, Y-X., Grob, R. and Hailer, O. (1989) Mol. Cell. Biol. 9, 3117-3121

PETER STAEHELI

Department of Virology, University of Freiburg, Herman Herder Strasse 11, D-7800 Freiburg, FRG.

[] [] [] [] [] [] [] D D [] [] []

Capturing the light and capturing the market

A recent symposium on biolumin- escence and chemiluminescence* concentrated on advances which are enabling the introduction of new, commercially competitive technol- ogies based on these phenomena to a range of clinical, industrial and environmental applications. Ap- plications are emerging in DNA- and protein-blotting methodologies, diagnostics, biosensors, food hy- giene and environmental monitor- ing, and pharmaceutical and clinical research.

Chemiluminescence (CL) is the emission of radiation, usually visible or near-infrared, caused by the decay of a chemical reaction product from

* Vlth International Symposium on Bio- luminescence and Chemiluminescence, 10-13 September 1990, University of Cambridge, UK.

an electronic excited state to ground state. Bioluminescence (BL) is the CL produced by a wide range of organisms. Although known for sev- eral hundred years, only in the past two decades has there been analyti- cal interest in luminescent reactions, and coupled in assays with enzy- matic or immunological methods, the reactions can be adapted to detect and quantify a variety of analytes with great precision.

Compared with the previous symposia in this series, presentations at this meeting reflected a shift in emphasis from theoretical and mechanistic aspects to clinical and industrial applications. Instrumen- tation was discussed, particularly in concurrent industrial seminars, and this area also received strong support from industrial exhibitors; great interest was shown in the new

(~) 1991, Elsevier Science Publishers Ltd (UK) 0167 - 9430191/$2.00

detection, imaging and software systems now available.

Molecular biology of BL The cloning and requirements for

successful expression of both pro- karyotic and eukaryotic luciferases were discussed and E. Meighen (McGill University, Canada), re- viewed the characteristics of cloned luciferases. Addition of reduced flavin mononucleotide (FMNH2) and aldehyde to either of the two general types of bacterial luciferase ident- ified results in light emission. Bac- terial luciferase is a heteropolymeric protein with the 0b and J3 subunits of the luciferase (encoded by lux A, B genes), having arisen by gene dupli- cation. The lux C, D and E genes encode the fatty acid reductase necessary to generate the aldehyde. G. Stewart (Nottingham University, UK) indicated that a bioluminescent phenotype could be obtained from an E. colt strain into which V. fis- chert lux A, B genes had been cloned, but in which lux E was absent and only derivatives of lux

TIBTECH - MARCH 1991 [Vol. 9] 73

C, D present. V. harveyi and V. fischeri lux genes have now been expressed in a wide range of other bacteria, including Agrobacterium, Bacillus, Erwinia, Escherichia, Pseudomonas, Rhizobium, Ana- baena and Streptomyces.

In contrast with bacterial bio- luminescence, light emission by crustaceans, coelenterates and fire- flies is dependent on ATP rather than pyridine nucleotides. Firefly lucfferase uses benzothiazole as luci- ferin, while crustacean (Vargula), and coelenterate (Aequorea), lucifer- ases both use imidazolopyrazine. Expression of luciferases in eukary- otic systems has, until recently, been more or less limited to firefly lu- ciferase, but there is reasonable promise for bacterial luciferases - provided an appropriate promoter is introduced and the lux A and B genes fused (E. Meighen; A. A. Szalay, University of Alberta, Canada). Bacterial iV. harvey~] luci- ferase has been expressed in plant, mammalian, yeast and also in insect cells (baculovirus system), where very high levels of expression (up to 10% total protein) have been achieved. Interesting evolutionary implications were thrown up by K. Wood (Promega Corp., WI, USA), reporting significant protein se- quencc homology between firefly luciferase and a CoA ligase from plants. The luciferase had previ- ously been shown to possess CoA ligase activity and it appears that oxidase activity of luciferase arose by development of a new catalytic mechanism within the existing pro- tein structure of an ancestral CoA ligase. Cloned firefly luciferases are being used extensively in assays as genetic reporters, and modifications to enzyme structures which cause changes in colour of light emitted may lead to applications using multiple luciferases simultaneously (K. Wood). A reminder that BL was not created for the benefit of biotech- nologists was provided by J. Morin (UCLA, USA), who discussed the natural role of BL in communication in marine organisms.

Many speakers emphasized the importance of considering not only the replication and transcription of cloned lux genes, but also transcript stability and processing, translation and protein stability and folding. A. A. Szalay indicated that the

thermostability of cloned proteins in mammalian cells should be borne in mind - incorporation of bacterial beat-shock chaperonin GroELS could facilitate correct protein folding. A. K. Campbell (University of Wales College of Medicine, Cardiff, UK), reported cloning both luciferase genomic and cDNA, coupled to pro- moters which allow tissue-specific, inducible expression in mammalian cells and whole organisms.

Luminescent labels and substrates The trend away from isotopic

labelling of proteins and other mol- ecules, particularly for diagnostic kits, is increasing. In addition to the health risks, staff training and radio- active waste-disposal problems, iso- tope half-life limits the useful shelf life of diagnostic kits. Luminol was the first luminescent isotope- substitute, and is still widely used in commercial immunoassay kits. Most CL reactions for immunoassay are based on luminol, lucigenin or other cyclic hydrazides, acridinium esters or oxalates. Dioxetanes and acridin- ium esters appear to be the com- pounds targeted for most attention and development. Horseradish per- oxidase (HRP), with luminol as in- dicator, is probably the commonest enzyme label, though glucose and xanthine oxidases, alkaline phos- phatase, and J3-galactosidase are also used. The majority of molecular ma- nipulations are aimed at improving compound stability, light yield, and reaction times compatible with signal reading range of instrumentation.

Dioxetanes, perhaps the most ef- ficient organic compound emitters, are unstable - stability has been achieved by the addition of an adamantyl group. P. Schaap (Lumi- gen Inc., USA), has further devel- oped this approach to produce a highly efficient substrate for alkaline phosphatase (AP) , commercial applications for which have already appeared. The synthesis and com- mercialization of enzyme-activated, stabilized chemiluminescent di- oxetanes is also being hotly pursued by Tropix Inc. I. Bronstein (Tropix, Inc.), presented the uses of dioxetane labels and substrates in sensitive immunoassays and blotting proto- cols. P. Schaap presented a simple, rapid DNA fingerprinting protocol using AP coupled to a synthetic oligo derived from the Jeffreys mini-

satellite sequences. Sensitivity is reported to be as good as that using conventional probes and 32p, and has the advantage over other non- colorimetric signals that filter re- probing is easy.

The main challenge in non- isotopic systems for the detection of DNA and protein has been to achieve sensitivity comparable with that of radioactive labels (D. Pollard-Knight, Amersham, UK). The coupling of enzymes or chemical labels to nucleic acid probes (e.g. HRP/luminol enhanced chemilu- minescence, chemically triggered acridinium esters and AP cleavage of substituted dioxetanes), combined with probe and target amplification strategies, are now approaching the requisite sensitivity, with detec- tion limits claimed to be 0.2-5.0 attomoles. Enhanced chemilumi- nescence (ECL), a well-established method for the detection of HRP labels has recently been applied to filter-based detection of DNA and protein (A. C. Simmonds, Amer- sham, UK), and can be used to detect single-copy genes in Southern blots.

Appropriately derivatized bio- luminescent proteins can replace enzyme labels in solid-phase im- munoassays. Aequorin (derived from recombinant apoaequorin ex- pressed in E. cohl, is highly stable, and may be derivatized with biotin and then coupled to antibodies to produce stable, specific biolumi- nescent detection reagents (M. T. Cormier, University of Georgia, USA and ELA Technologies, GA, USA).

Applications Commercial growth of BL and CL

technologies is undoubtedly follow- ing the progress in developing new luminescent substrates and cloning the genes for bioluminescent, pro- teins. To date, most applications have been based on the firefly- system detection of ATP. E. Schram (Free University, Brussels, Belgium), indicated that, despite the introduc- tion of recombinant luciferase and the use of lipids as protective agents, methodology development (separ- ation methods and automation), has lagged behind that of reagents and instrumentation. Despite its lack of specificity in biomass assays, its rapidity and sensitivity ensures the firefly §ystem remains a popular method in the dairy and food indus-

74 TIBTECH - MARCH 1991 [Vol. 9]

--Fig. 1 (a) Image of microtitre tray containing varying levels of A TP and firefly luciferase/luciferin. Emitted light was measured using a sensitive intensified CCD imaging system, the Biomedical Image Quantifier (BIQ BioView) Image Research Ltd, Cambridge, IlK. Light levels (photon counts) in 96 wells may be quantified rapidly and simultaneously. The kinetic profile of light emission can be followed by measuring successive images of the tray. ApplicPtion of BL.imaging techniques in "rapid micro- biology ~ permits identification of discrete colonies with- out long incubation periods: (b, c) images of light emission from genetically engineered E. coli expressing luciferase (i.e. the lux gene), inoculated onto agar in a 90 mm petri dish• (b) Light emission after 10 h incubation at 30°C; and (©) light emission after overnight incubation (large and sometimes confluent colonies) with computer superposition of colonies seen in (b) as red spots. Photographs b and c are reproduced, with permission, from Trends Anal Chem. (1990) 9, pp. 269-277.

tries, for clinical chemistry and hy- giene and environmental monitoring.

'Rapid microbiology' Problems with applications in

sensitive microbial detection in the food and beverage industry were highlighted by D. A. Stafford (Dyna- tech, USA). Suitable extractants, to overcome variations in the efficiency of ATP extraction from microbial cells in food contamination assays, have been developed. [The use of detergents to liberate ATP, thought to be bound to proteins within cells, was also reported for use with ery- throcytes by M. Kellerman (Univer- sity Medical School, Pecs, Hungary).] Methods proposed for removing somatic ATP and cells involved filtration of milk to leave bacteria on a filter which can then be assayed (E. Schi'am), or destruction of non- bacterial ATP prior to bacterial lysis by treatment with surfactant-sodium iodate mixtures (N. N. Ugarova, Lomonosow University, Moscow,

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USSR). Improvements in signal and enzyme stability by the use of phos- pholipids which protect luciferase enzyme (D. Stafford) were reported, and detection of 10-20 microorgan- isms m1-1 is now claimed to be routinely possible.

However, for acceptance of these techniques on a large scale, both Stafford and Schram pointed out that costs of assays need to be reduced, and that, whereas large labs need automated systems, on-site testing necessitates simple-to-use, portable 'dip-stick' technology.

The lack of appropriate method- ology also probably accounts for the failure of luminescence-based tech- nology to make a major impact on clinical microbiology. J. G. M. Hastings (Queen Elizabeth Hospital, Birmingham, UK), indicated that assays which are easy to use for no great increase in cost, giving quicker results and tangible advantages in patient management, are required. Development of luminometers, for

microtitre plates in particular, will push bioluminescent assays into the lab. Use of CL assays in estimating bacterial ATP in screening for bac- teriuria, microbial antibiotic-suscep- tibility/sensitivity testing and hygiene testing has encountered little com- mercial success: again, the recent development of dipstick luciferin- luciferase assays should help (see Fig. 1).

Clinical research CL is finding applications in clini-

cal chemistry and medical physi- ology. Many assays (mostly im- munoassays), developed for analytes of medical, veterinary and forensic importance (hormones, drugs), were reported.

Altered protein glycosylation, characteristic of certain disease states, may be monitored using bind= ing to plant lectins coupled with use of a recombinant aequorin tag (M. J. Cormier, University of Georgia, USA). It has been known for some

TIBTECH- MARCH 1991 [Vol. 9] 75

years that chloroplasts exhibit endo- genous luminescence. More recently it has been reported that phagocytic cells such as alveolar macrophages luminesce and, because of its high sensitivity, cellular luminescence may be used as a clinical test for activation and function of PMNLs arid other phagocytic cells. An intro- duction to this phenomenon and its applications was provided by P. de Sole (Catholic University, Rome, Italy). I. Cree (University of Dundee, UK), discussed the use of microtitre assays in clinical investigation of opsonization and cellular defects (acquired or inherited) and the study of the effects of cytokines and drugs on phagocyte function in vitro. Data on inherited disorders of phagocyte metabolism (B. Descamps-Latscha, H6pital Necker, Paris, France), treat- ment of asthma (Ph. Godard, Mont- pellier, France; I. Cree), and effec- tiveness of UV treatment of psoriasis by measurement of phagocyte ac- tivity of psoriatic PMNLs (I. Cree), were presented. The sensitivity of luminescence assays means that very little tissue is required: A. Lundin (Karolinska Inst., Sweden), reported measurement of mitochon- drial ATP production rates from individual muscle fibres in detection of various muscle orders, with com- parable results to those obtained using HPLC and spectrophotometry; and in studies on lipid and glucose metabolism, glycerol-utilizing bac- terial luciferases have been used to measure lipolysis rates in fat cells collected by needle biopsy (A. Lundin). P. E. Andreotti (MCL Technologies, FL, USA), reported using microtitre-plate ATP-luciferase assays in selecting chemothera- peutics for cancer patients; to maxi- mize tumour remission, an assay must be capable of predicting both resistance and sensitivity of tumour to agent. Such assays have indicated that dividing and non-dividing cells have different spectra of sensitivity and have even detected some drugs with tumour-stimulating effects.

Hardware and software Parameters to consider in the

selection of appropriate labels and instrumentation for particular appli- cations were discussed in the main symposia and received still greater emphasis in separate, industrially sponsored seminars. Luminescent

reactions can occur very rapidly (<ls), or over longer periods (~lday), with the duration of emission affected by reaction con- ditions. Signal measurement may therefore be endpoint or integrated. This broad range presents a con- siderable challenge to the develop- ment of appropriate instrumentation for monitoring. Light intensity de- pends on the quantum yield - for BL systems (e.g. firefly luciferase), this is often high, resulting in a detection limit of down to 10 -21 mol, whereas the quantum yields of non-biological systems are usually much lower (lO0-fold less). However, the speci- ficity of CL reactions, together with almost non-existant background emission, permits even very inef- ficient reactions to be monitored and therefore used for analytical pur- poses. BL ATP-detecting reactions last several minutes, thus enabling very simple instrumentation to be used. Such systems, however, are unsuitable for rapid reactions and several approaches have been tried to get aro,,md this - flow injection, suitable for liquid chromatographic detection, enables rapid, reproduc- ible mixing (P. J. Worsfold, Ply- mouth Polytechnic, UK}. Advances were reported (Claire Hooper, Robens Institute, University of Surrey, UK} in the development of camera design, software for analysis and imaging systems. Low light level imaging, for biomedical applications in particular, requires sensitivity and needs to detect and determine the position of single photons. Claire Hooper and Richard Ansorge (Image Research Ltd, Cambridge, UK and Dept Physics, Cambridge University, UK), discussed the experimental considerations of coupling assay equipment (microtitre trays, petri dishes, gels, blots, tissue sections), to detectors and imaging equipment. Digital Imaging of each pixel permits quantification of the signal, and either quantitative (e.g. reporter gene expression in single cells), or qualitative/semi-quantitative (e.g. blots), results can be imaged. Con- cerns were voiced as to whether amplification methods used with a real-time, intensified CCD (charge- coupled-device, developed for quan- titative photon imaging in high energy physics), which operates at room temperature might, in effect, limit the dynamic range. However,

camera design and specialized soft- ware was claimed to overcome such problems and to provide similar sensitivity and dynamic range as a cryogenic CCD. A major advantage of the system appeared to be the option of (near)-contact imaging, enabling 80% of photons to be captured, com- pared with lens-based systems in which only 0.05% photons are cap- tured. Improved methods of cali- bration and photometry design of CCD detectors for luciferase and peroxidase assays were discussed (D. Leaback, Biolink Technologies, Cambridge, UK), and the use of CCD cameras was reported for the non- destructive imaging of single live cells in T,leasuring mammalian transient expression of transfected luciferase vectors (M. R. H. White, Amersham Int'l, Bucks., UK).

Prospects for BL- and (L-markets The majority of assays involve

participation of the analyte in well- known CL reactions. Extension of analytical procedures for the (rela- tively) few reactions available will necessitate the development of quite complex reactions. One approach is the screening of analytes for their own CL reactions - usually by treat- ing the analyte with a range of oxidants or reductants. An alter- native, applicable for some analytes, is genetic engineering: protein kinase recognition sites were intro- duced into recombinant firefly luci- ferase and aequorin CA. Campbell). The more widespread use of BL and CL systems will require the intro- duction of user-friendly kits for on- site environmental, hygiene and clinical monitoring, and improved hardware and software - the market will probably split between portable kits and more complex, automated, larger lab-based systems.

Naturally, in a competitive field where the commercial potential of BL- and CL-based monitoring and diagnostic systems is only just begin- ning to be recognized, certain pres- entations at this meeting led to animated discussion of the relative merits of different approaches - further R&D will probably show that different approaches are appropriate for different applications. However, although there are obviously many wrinkles in the methodology still to be ironed out, we can soon expect to see a rapid expansion in the range of

76 TIBTECH- MARCH 1991 [Vol. 9]

BL- and CL-based diagnostic kits, monitoring, analysis and imaging systems on the market.

The next symposium is planned for March 1993, in Banff, Canada (contact: Dr A. A. Szalay, Dept Plant Molecular Genetics, University of Alberta, Edmonton, Canada T6G 2H7). In the meantime however,

there is the proposal to set up a society to promote the science of bioluminescence and chemilumi- nescence. Interested persons should contact either:

Dr A. K. Campbell Fax {44) 222 766276, Te~. (44) 222 742951; or

Dr P. Stanley Fax (44) 223 461777, Tel. (44) 223 460888.

Acknowledgements I am grateful to Philip Stanley and

Claire Hooper for their helpful com- ments on the manuscript, and to Claire Hooper for providing the photographs in Fig. 1.

CLARE ROBINSON

Trends in Biotechnology

Public funding of research and development: a broad picture

Discovery and invention spring from that unstable mixture of curiosity, excitement, ambition and obsession that is nurtured in institutes of higher education and civil research. All industries and most central governments have taken to heart the lesson that today's discovery is tomorrow's economic opportunity. Both are concerned to see invest- ment in research in the public sector. Private and public initiatives in basic research are increasing sub- stantially as we enter the final decade of this century. Last month this column wrung metaphorical hands over the poor showing of the UK in public funding of research and development (R&D) {TIBTECH 9, p. 47, 1991). What is happening elsewhere?

Funding for research and development on a world scale

Figures from the Organization for Economic Co-operation and Devel- opment (OECD) are the most com- plete and, although a little dated, reflect the current league positions of major scientific nations (Table 1).

In 1991, the spending on R&D as a percentage of GDP (gross domestic product) is rising significantly, with the UK still at the bottom of the league table. It is particularly worry- ing, as described in last month's Biotopics (op. cit) that although most industrially developed nations are increasing their funding of civil research well beyond the threshold of inflation, that of the UK remains static. The figures m Table 1 are

dominated by the contribution of private enterprise, but the invest- ment by the USA in civil research makes an eloquent statement (if any- one needs it), about the importance of US basic science on a world scale.

Despite a huge Federal budget defi- cit, US scientists have good cause to celebrate, wi th average increases of civil research spending of 10% in this year's budget: NIH £4.61) (£4.89m for human genome research programme); NASA £7.7b; DoE £2.0b; NSF £1.3b; (NB £1 : US$1.8) (b = billion = 109; m = million = 106). The increase in the NIH budget has a lot to do with the popularity of biomedical science research in Washington through the effective lobbying of groups such as the Association of American Univer- sities and the American Association of Medical Colleges. The NSF has also done well (despite a last-minute cut of $22m), nearly doubling its

budget in five years. This excellent provision for civil research has brought difficult choices {choices that European science base organiz- ations nevertheless would dearly love to be able to make ) in that pressures are strong to renew the infrastructure - buildings and facili- ties - as well as the inevitable demand of many small, deserving programmes and projects.

The NIH budget represents the ]crgest single source of funding for basic science research in the world and the politics of spending it should create its own momentum to end the hiatus in the NIH adminis- tration by the appointment of a new director.

Whatever the domestic politics, the continued strong US science funding situation will increase the movement of trained scientists to the USA from Europe and elsewhere in the world. Not much of this money will be effective outside the US, but there are indications that private foundations are beginning to look overseas. The Howard Hughes Medical Institutes are said to be considering the option of overseas grant applications in one or two year's time.

Money for civil research is well down in the league table of one of

--Table I

OECD a figures for scientific research funding in the major nations

Country Total R&D a Public funding (% GDP) a (£ bill ions) b (£ billions) b

Japan 2.9 73 5 USA 2.86 41 15.6 Germany 2.83 20 7 France 2.29 13 5 UK 2.23 10 4

a Abbreviations: OECD, Organization for Economic Co-operation and Devel- opment; GDP, gross domestic product; R&D, research and development.

1 billion - 10 ~.

~) 1991. Elsevier Science Publishers Ltd (UK) 0167 -9430/91152.00