Bioluminescent novelty items

Systems and apparatus for generating bioluminescence, and combinations of these systems and apparatus with inanimate articles of manufacture to produce novelty items are provided. These novelty items, which are articles of manufacture, are designed for entertainment, recreation and amusement, include, toys, paints, slimy play material, textiles, particularly clothing, bubbles in bubble making toys and other toys that produce bubbles, balloons, personal items, such as bath powders, body lotions, gels, powders and creams, toothpastes and other dentifrices, soaps, body paints, and bubble bath, foods, such as gelatins, icings and frostings, beverages such as beer, wine, champagne, soft drinks, and ice cubes, fountains, including liquid "fireworks" and other such jets or sprays or aerosols of compositions that are solutions, mixtures, suspensions, powders, pastes, particles or other suitable formulation.

FIELD OF INVENTION
 The present invention relates to systems for producing bioluminescent
 light, and to combinations of the systems with articles of manufacture
 including toys, textiles, food and beverages, to produce novelty items. By
 virtue of the combination the novelty items glow or produce or spew a
 bioluminescent composition. Also, provided are compositions, encapsulated
 bioluminescent generating reagents, and methods for producing the
 bioluminescence.
 BACKGROUND OF THE INVENTION
 Luminescence is a phenomenon in which energy is specifically channeled to a
 molecule to produce an excited state. Return to a lower energy state is
 accompanied by release of a photon (hv). Luminescence includes
 fluorescence, phosphorescence, chemiluminescence and bioluminescence.
 Bioluminescence is the process by which living organisms emit light that
 is visible to other organisms. Luminescence may be represented as follows:
 A+B.fwdarw.X*+Y
 X*.fwdarw.X+hv,
 where X* is an electronically excited molecule, hv represents light
 emission upon return of X* to a lower energy state in which creation of
 the excited state derives from an enzyme catalyzed reaction. The color of
 the emitted light in a bioluminescent (or chemiluminescent or other
 luminescent) reaction is characteristic of the excited molecule, and is
 independent from its source of excitation and temperature.
 The essential condition for bioluminescence the use of molecular oxygen,
 either bound or free in the presence of a luciferase. Luciferases, are
 oxygenases, that act on a substrate luciferin in the presence of molecular
 oxygen and transform the substrate to an excited state. Upon return to a
 lower energy level energy is released in the form of light [for reviews
 see, e.g., McElroy et al. (1966) in Molecular Architecture in Cell
 Physiology, Hayashi et al., eds., Prentice-Hall, Inc., Englewood Cliffs,
 N.J., pp. 63-80; Ward et al., Chapter 7 in Chemi-and Bioluminescence,
 Burr, ed., Marcel Dekker, Inc. N.Y., pp.321-358; Hastings, J. W. in (1995)
 Cell Physiology:Source Book, N. Sperelakis (ed.), Academic Press, pp
 665-681; Luminescence, Narcosis and Life in the Deep Sea, Johnson, Vantage
 Press, N.Y., see, esp. pp. 50-56].
 Though rare overall, bioluminescence is more common in marine organisms
 than in terrestrial organisms. Bioluminescence has developed from as many
 as thirty evolutionarily distinct origins and, thus, is manifested in a
 variety of ways so that the biochemical and physiological mechanisms
 responsible for bioluminescence in different organisms are distinct.
 Bioluminescent species span many genera and include microscopic organisms,
 such as bacteria [primarily marine bacteria including Vibrio species],
 fungi, algae and dinoflaggellates, to marine organisms, including
 arthropods, mollusks, echinoderms, and chordates, and terrestrial organism
 including annelid worms and insects.
 Bioluminescence, as well as other types of chemiluminescence, is used for
 quantitative determinations of specific substances in biology and
 medicine. For example, luciferase genes have been cloned and exploited as
 reporter genes in numerous assays, for many purposes. Since the different
 luciferase systems have different specific requirements, they may be used
 to detect and quantify a variety of substances. The majority of commercial
 bioluminescence applications are based on firefly [Photinus pyralis]
 luciferase. One of the first and still widely used assays involves the use
 of firefly luciferase to the detect the presence of ATP. It is also used
 to detect and quantify other substrates or co-factors in the reaction. Any
 reaction that produces or utilizes NAD(H), NADP(H) or long chain aldehyde,
 either directly or indirectly, can be coupled to the light-emitting
 reaction of bacterial luciferase.
 Another luciferase system that has been used commercially for analytical
 purposes is the Aequorin system. The purified jellyfish photoprotein,
 aequorin, is used to detect and quantify intracellular Ca.sup.2+ and its
 changes under various experimental conditions. The Aequorin photoprotein
 is relatively small [.about.20 kDa], nontoxic, and can be injected into
 cells in quantities adequate to detect calcium over the a large
 concentration range [3.times.10.sup.-7 to 10.sup.-4 M].
 Because of their analytical utility many luciferases and substrates
 therefor have been studied and well-characterized and are commercially
 available [e.g., firefly luciferase is available from Sigma, St. Louis,
 Mo., and Boehringer Mannheim Biochemicals, Indianapolis, Ind.;
 recombinantly produced firefly luciferase and other reagents based on this
 gene or for use with this protein are available from Promega Corporation,
 Madison, Wis.; the aequorin photoprotein luciferase from jellyfish and
 luciferase from Renilla are commercially available from Sealite Sciences,
 Bogart, Ga.; coelenterazine, the naturally-occurring substrate for these
 luciferases, is available from Molecular Probes, Eugene, Oreg.]. These
 luciferases and related reagents are used as reagents for diagnostics,
 quality control, environmental testing and other such analyses. These
 reagents have not been used in connection with entertainment and
 recreation for the glow, illumination and color produced upon generation
 of bioluminescence.
 Thus, it is an object herein to exploit bioluminescence for use as a
 recreational product in combination with articles of manufacture to
 produce novelty items, including toys, personal items, foods, fountains,
 beverages, coating compositions, such as paints and inks, textiles,
 including clothing, and other such items. It is also an object herein to
 provide such combinations and to provide means for producing and using
 such combinations.
 SUMMARY OF THE INVENTION
 Systems and apparatus for generating bioluminescence, and combinations of
 these systems and apparatus with inanimate articles of manufacture to
 produce novelty items are provided. These novelty items, which are
 articles of manufacture, are designed for entertainment, recreation and
 amusement, include, but are not limited to: toys, particularly squirt
 guns; finger paints and other paints, slimy play material; textiles,
 particularly clothing, such as shirts, hats and sports gear suits, threads
 and yarns; bubbles in bubble making toys and other toys that produce
 bubbles; balloons; personal items, such as bath powders, body lotions,
 gels, powders and creams, nail polishes, make-up, toothpastes and other
 dentifrices, soaps, body paints, and bubble bath; items such as inks,
 paper; foods, such as gelatins, icings and frostings; and beverages, such
 as beer, wine, champagne, soft drinks, and ice cubes; fountains, including
 liquid "fireworks" and other such jets or sprays or aerosols of
 compositions that are solutions, mixtures, suspensions, powders, pastes,
 particles or other suitable form.
 Thus, the novelty items provided herein include but are not limited to:
 textiles that glow, ink that glows, paints, particularly fingerpaints,
 that glow, paper products that glow, toys, particularly squirt guns that
 eject a bioluminescent fluid, dolls and dummies with internal organs or
 parts that glow; foods and beverages that glow, soapy compositions for
 blowing bubbles that produce bubbles that glow, bubble bath compositions
 that produce bubbles that glow, fountains that spew glowing fluid,
 bioluminescent "fireworks", sparklers, magic-wand toy, and numerous other
 such items.
 Bioluminescence is advantageously used combination with the such novelty
 items because it can be generated using reagents that are nontoxic,
 noncorrosive and nonstaining. Bioluminescence is also advantageously used
 because it can be sustained to provide a glow that lasts, if desired, from
 minutes up to hours.
 Any article of manufacture that can be combined with a
 bioluminescence-generating system as provided herein and thereby provide
 entertainment, recreation amusement, including use of the items for
 recreation or to attract attention, such as for advertising goods and/or
 services that are associated with a logo or trademark. Such uses may be in
 addition to or in conjunction with or in place of the ordinary or normal
 use of such items. As a result of the combination the items glow or
 produce, such as in the case of squirt guns and fountains, a glowing fluid
 or spray of liquid or particles. The novelty in the novelty item derives
 from its bioluminescence.
 The preferred bioluminescence-generating reactions are performed by adding
 oxygen (or water containing oxygen) or calcium ions to luciferin and
 luciferase mixtures using apparatus and systems as described herein.
 Apparatus, systems and substrates for generating the bioluminescence are
 provided. The systems include matrix materials that are coated with
 bioluminescence generating reagents, capsular vehicles containing the
 reagents, single chamber and multiple chamber apparatus containing the
 reagents.
 Methods and compositions for producing bioluminescence in combination with
 the novelty items are also provided. Micro- and macro-capsular vehicles
 containing components of bioluminescence generating reactions are
 provided. The capsular vehicles are capsules, such as liposomes, isolated
 endosomes, isolated vacuoles, gelatin capsules, and other such delivery
 vehicles, and the apparatus include vessels, and single chambers, dual
 chamber and three chamber or more apparatus. These vehicles encapsulate
 bioluminescent generating system components, and typically contain less
 than all of the components necessary to generate a bioluminescent
 reaction. The capsular vehicles include vehicles often used for drug
 delivery, such as liposomes, and time release capsules; and also capsules
 made glass, plastic and other such materials.
 For example, the bioluminescence generating components may be coated on the
 inside of a glass container, such as a glass capillary tube [see, e.g.,
 U.S. Pat. No. 5,387,526]. Upon addition of a composition containing the
 necessary activating agents, such as molecular oxygen, ATP, a reductase,
 Ca.sup.2+, the coating will be contacted with the activator and will
 produce a glow. The capsular vehicles are intended for use in combination
 with the articles of manufacture.
 Thus, the micro- or macro-capuslar vehicles, when crushed, opened,
 dissolved or otherwise placed under conditions that cause delivery of the
 contents, release material that glows upon contact with air and/or
 moisture or other activator(s). These vehicles vary in size [in the
 largest dimension] from as small as less than 0.1 .mu.m up to 0.1 cm or
 more.
 Matrix materials, such as glass, plastics, cotton and other textile
 material, that contain linked bioluminescence-generating reagents are also
 provided. For example, one or more components of the bioluminescence
 generating system is (are) linked to a matrix material. Matrix materials,
 such as textiles, glass, a plastic or ceramic surfaces or particles are
 combined with at least one component of the bioluminescent reagent,
 particularly the luciferin, luciferase, or, where the components are
 amenable, the luciferin and luciferase. The component(s) such as the
 luciferase are linked to the matrix, such as cotton, using methods known
 to those of skill in the protein synthesis art for linking peptides or
 proteins to solid substrates [see, e.g., Eichler et al. (1993)
 Biochemistry 32:11035-11041; Merrifield (1964) Biochemistry 3:1385-1390]
 Linkage is effected either covalently or non-covalently and can be direct
 or via linkers. Such methods and linkers are well known to those of skill
 in the chemical arts. The matrix materials with linked bioluminescent
 generating system components are contacted with an article of manufacture
 resulting in a novelty item that, when appropriately treated, such as by
 spraying on a composition that contains the remaining components of the
 reactions, glows or produces bioluminescence.
 Also provided are single and multi-chamber, particularly dual chamber,
 apparatus for producing bioluminescence, and combinations of these
 apparatus with bioluminescence generating reagents are also provided. Such
 apparatus include at least one chamber that contains all but at least one
 reagent or component required to produce bioluminescence. Upon addition of
 the component either to the chamber or after ejection of some or all of
 the contents of the chamber a bioluminescent glow or glowing fluid, spray
 or jet is produced.
 Articles of manufacture containing one or more components of a
 bioluminescence generating system or a composition, such as a composition
 containing ATP or Ca.sup.2+ or other activator, within the packaging
 material, and a label that indicates that the contents is used for
 generating bioluminescence are also provided.
 Kits containing an article of manufacture and appropriate reagents for
 generating bioluminescence are also provided.
 DESCRIPTION OF THE DRAWINGS
 In the accompanying drawings:
 FIG. 1 is a side elevation, with portions cut away, of a squirt gun
 incorporating the dual chamber structure;
 FIG. 2 is a sectional view taken on line 2--2 of FIG. 1;
 FIG. 3 is a sectional view taken on line 3--3 of FIG. 1;
 FIG. 4 is a side elevation view, with portions cut away, of a gas powered
 toy gun with dual chamber detachable fluid reservoir;
 FIG. 5 is a top plan view of the toy gun of FIG.4, with portions cut away;
 FIG. 6 is a side elevation view, partially cut away of a gas-charged fluid
 dispensing apparatus incorporating the dual chamber system;
 FIG. 7 is a sectional view taken on line 7--7 of FIG. 6;
 FIG. 8 is a top plan view of the structure of FIG. 6, partially cut away;
 FIG. 9 is a side elevation view of a fountain type configuration of the
 gas-charged dual chamber fluid dispensing apparatus, with portions cut
 away;
 FIG. 10 is a sectional view taken on line 10--10 of FIG. 9;
 FIG. 11 is a side elevation view, partially cut away, of a dual chamber
 compressible dispensing container;
 FIG. 12 is a side elevation view, partially cut away of a bottle/bladder
 apparatus designed for use with bubble-blowing compositions;
 FIG. 13 is a view similar to FIG. 12, with the components mixed and the
 bubble blowing wand detached for use; and
 FIG. 14 is a side elevation view, partially cut away, of beverage container
 with a bladder apparatus actuated by opening of the beverage container.
 FIG. 15 is a side elevation view, partially cut away of a single use, dual
 chamber fluid packaging apparatus adapted for use with bubble-blowing
 compositions.
 FIG. 16 is a side elevation view, partially cut away of a cap apparatus
 operated by depression of the plunger assembly to rupture the capsule
 contained within the cork cap.
 FIG. 17 is a side elevation view, partially cut away of a cap apparatus
 operated by screwing the plunger assembly into the cork cap to rupture the
 capsule contained therein.
 FIG. 18 is a side elevation view, partially cut away of a cap apparatus
 operated by screwing the screw-cap onto the top of the bottle forcing the
 plunger assembly against the capsule contained within the neck of the
 bottle, thereby rupturing the capsule membranes.
 FIG. 19 is a view similar to the view of FIG. 18, with the cap apparatus
 tightly secured against the top of the bottle and the capsule membranes
 ruptured.

DETAILED DESCRIPTION OF THE INVENTION
 TABLE OF CONTENTS
 A. DEFINITIONS
 B. Bioluminescence generating systems
 1. General description
 a. Luciferases
 b. Luciferins
 c. Activators
 d. Reactions
 2. Ctenophore and coelenterate systems
 a. The aequorin system
 (1) Aequorin photoprotein
 (2) Luciferin
 b. The Renilla system
 3. Crustacean, particular Cyrpidina [Vargula], systems
 a. Vargula luciferase
 (1) Purification from Cypridina
 (2) Preparation by Recombinant Methods
 b. Vargula luciferin
 c. Reaction
 4. Insect bioluminescent systems including fireflies, click beetles, and
 other insect system
 a. Luciferase
 b. Luciferin
 c. Reaction
 5. Bacterial systems
 a. Luciferases
 b. Luciferins
 c. Reactions
 6. Other systems
 a. Dinoflagellate bioluminescence generating systems
 b. Systems from molluscs, such as Latia and Pholas
 c. Earthworms and other annelids
 d. Australian glow worms
 C. Practice of the reactions in combination with articles of manufacture
 D. Packaging of Bioluminescence Systems
 1. Dispensing and Packaging Apparatus for Combination with the
 Bioluminescent System Components
 2. Capsules, pellets liposomes, micronized particles
 a. Encapsulating vehicles-in general
 b. Encapsulating vehicles -liposomes
 c. Encapsulating vehicles -gelatin and polymeric vehicles
 d. Micronized particles
 3. Apparatus and substrates
 a. Matrix materials
 b. Immobilization and activation
 4. Apparatus containing a single chamber, housing or a vessel
 5. Dual and multiple chamber fluid dispensing apparatus
 a. Mechanical pump dispensing apparatus
 b. Gas-charged dispensing apparatus
 c. Compressible dispensing apparatus
 6. Other fluid dispensing and packaging apparatus particularly designed for
 single use
 a. Bottle-type single chamber container/bladder apparatus
 b. Dual chambered bottle type container/bladder apparatus for use with
 foods and beverages
 c. Can type container/bladder apparatus for use with foods and beverages
 7. Cap Apparatus for use a single chamber vessel
 E. Combinations of articles of manufacture and bioluminescence
 1. Personal care products, including bath powders, bubble baths, products
 for use on the nails, hair, skin, lips and elsewhere
 a. Bath powders
 b. Glowing dust or powder
 c. Lotions, gels and other topical application formulations
 (1) Lotions
 (2) Creams
 (3) Solutions and suspensions for topical application
 (4) Gels
 (5) Solids
 2. Glowing toys and other items
 a. Single chamber toy guns and other toy weapons that shoot pellets or
 liquid
 b. Bubble-making toys
 3. Glowing textiles and paper products
 4. Foods and beverages, including ice cubes
 a. Beverages
 b. Ice cubes
 5. Jewelry, Clothing and Other Items of Manufacture Manufacture
 6. Fountains
 A. Definitions
 Unless defined otherwise, all technical and scientific terms used herein
 have the same meaning as is commonly understood by one of skill in the art
 to which this invention belongs. All patents and publications of any sort
 referred to herein are incorporated by reference in their entirety.
 As used herein, novelty items refer to inanimate articles of manufacture
 that are intended to provide, even for only a few moments, amusement,
 entertainment, decoration or recreation. The use for recreation or
 entertainment may be the items only use or may be in addition to other
 uses or benefits of the items, such as clothing, including as hats and
 T-shirts that are modified as described herein by combination with
 bioluminescence.
 Novelty items are understood by those of skill in manufacture of such items
 as well as by the purchasing public and are intended herein to include
 items such as, toys, including toy guns, dolls, dummies, balloons,
 bubbles, "fairy dust", such as micronized lyophilized particles, puzzles,
 and inks and paints, particularly fingerpaints; theatrical vapors when
 mixed, for example with dry ice or a fog; souvenirs; textiles,
 particularly clothing, including T-shirts, hats, swimsuits, bathing suit,
 wet suits, scuba diving suits, surfing suits, and other water sport or
 sports attire; foods and beverages, including gelatins, ice cubes, beer,
 wine, champagne, soft drinks, ice creams, sorbets, ices, frostings, and
 candy; jewelry, medallions, decorative articles, artificial flowers,
 articles for displaying names, business tradenames, slogans, trademarks on
 promotional or other such items, such as T-shirts, hats, paints, wrapping
 paper, gifts intended to promote business goodwill; personal items, such
 as body paints, body sprays, bubble baths, make-up, body lotions,
 dentifrices; fountains; jets or sprays of particles or fluids, including
 "fireworks", sparklers, and magic-wand toys, and many other such novelty
 items [see, e.g., U.S. Pat. Nos. 5,435,010, 5,460,022, 5,458,931,
 5,435,787, 5,435,010, 5,432,623, 5,421,583, 5,419,558, 5,416,927,
 5,413,454, 5,413,332, 5,411,427, 5,410,962, 5,407,691, 5,407,391,
 5,405,958, 5,405,206, 5,400,698, 5,399,122, 5,398,972, 5,397,609,
 5,396,408, 5,393,580, 5,390,086, 5,389,033, 5,383,684, 5,374,805,
 5,368,518, 5,363,984, 5,360,010, 5,353,378, 5,351,931, 5,346,455,
 5,341,538, 5,323,492, 5,283,911, 5,222,797, 5,177,812, 5,158,349,
 4,924,358, 3,597,877 and many others, which describe types of items are
 considered novelty items]. Any such inanimate item that is combined with
 bioluminescence is intended to be encompassed herein.
 Thus, for purposes herein, a novelty item refers to any inanimate article
 of manufacture that upon combination with bioluminescence provides
 amusement, entertainment, recreation or enjoyment if only for even a few
 moments. Addition of the bioluminescence to the article of manufacture
 does not add to the function of the item, but adds entertainment,
 amusement or recreational aspects to the item so that the resulting
 combination is a novelty item. Therefore, the combinations provided herein
 are novelty items by virtue of the combination an inanimate article of
 manufacture with bioluminescence.
 As used herein, inanimate means that the articles of manufacture are not
 live nor formerly living [i.e., dead] items. Thus, the novelty items
 herein, do not encompass living organisms, such as genetically modified
 fireflies or genetically engineered plants that express luciferase or
 other such organisms] that produce bioluminescence.
 As used herein, personal items include items that are used on the body,
 such as toothpastes, dentifrices, make-up, nail polishes, body lotions,
 body creams and body powders.
 As used herein, chemiluminescence refers to chemical reaction in which
 energy is specifically channeled to a molecule causing it to become
 electronically excited and subsequently to release a photon thereby
 emitting visible light. Temperature does not contribute to this channeled
 energy. Thus, chemiluminescence involves the direct conversion of chemical
 energy to light energy.
 As used herein, "fairy dust" refers to particles, such as light sensitive
 liposomes or micronized powdered particles, that glow upon contact with
 the air, such as "dust" that a child would use when pretending to be
 Tinker Bell or other such character.
 As used herein, luminescence refers to the detectable EM radiation,
 generally, UV, IR or visible EM radiation that is produced when the
 excited product of an exoergic chemical process reverts to its ground
 state with the emission of light. Chemiluminescence is luminescence that
 results from a chemical reaction. Bioluminescence is chemiluminescence
 that results from a chemical reaction using biological molecules [or
 synthetic versions or analogs thereof] as substrates and/or enzymes.
 As used herein, bioluminescence, which is a type of chemiluminescence,
 refers to the emission of light by biological molecules, particularly
 proteins. The essential condition for bioluminescence is molecular oxygen,
 either bound or free in the presence of an oxygenase, a luciferase, which
 acts on a substrate, a luciferin. Bioluminescence is generated by an
 enzyme or other protein [luciferase] that is an oxygenase that acts on a
 substrate luciferin [a bioluminescence substrate] in the presence of
 molecular oxygen and transforms the substrate to an excited state, which
 upon return to a lower energy level releases the energy in the form of
 light.
 As used herein, the substrates and enzymes for producing bioluminescence
 are generically referred to as luciferin and luciferase, respectively.
 When reference is made to a particular species thereof, for clarity, each
 generic term is used with the name of the organism from which it derives,
 for example, bacterial luciferin or firefly luciferase.
 As used herein, luciferase refers to oxygenases that catalyze a light
 emitting reaction. For instance, bacterial luciferases catalyze the
 oxidation of flavin mononucleotide [FMN] and aliphatic aldehydes, which
 reaction produces light. Another class of luciferases, found among marine
 arthropods, catalyzes the oxidation of Cypridina [Vargula] luciferin, and
 another class of luciferases catalyzes the oxidation of Coleoptera
 luciferin.
 Thus, luciferase refers to an enzyme or photoprotein that catalyzes a
 bioluminescent reaction [a reaction that produces bioluminescence]. The
 luciferase enzymes, such as firefly and Renilla luciferases, that are
 enzymes which act catalytically and are unchanged during the
 bioluminescence generating reaction. The luciferase photoproteins, such as
 the aequorin photoprotein to which luciferase is non-covalently bound, are
 changed, such as by release of the luciferin, during bioluminescence
 generating reaction. The luciferase is protein that occurs naturally in an
 organism or a variant or mutant thereof, such as a variant produced by
 mutagenesis that has one or more properties, such as thermal stability,
 that differ from the naturally-occurring protein. Luciferases and modified
 mutant or variant forms thereof are well known.
 Thus, reference, for example, to "Renilla luciferase" means an enzyme
 isolated from member of the genus Renilla or an equivalent molecule
 obtained from any other source, such as from another Anthozoa, or that has
 been prepared synthetically.
 The luciferases and luciferin and activators therefore are referred to as
 bioluminescence generating reagents. Typically, a subset of these reagents
 will be provided or combined with an article of manufacture.
 Bioluminescence will be produced upon contacting the combination with the
 remaining reagents. Thus, as used herein, the component luciferases,
 luciferins, and other factors, such as O.sub.2, Mg.sup.2+, Ca.sup.2+ are
 also referred to as bioluminescence generating reagents [or agents].
 As used herein, not strictly catalytically means that the photoprotein acts
 as a catalyst to promote the oxidation of the substrate, but it is changed
 in the reaction, since the bound substrate is oxidized and bound molecular
 oxygen is used in the reaction. Such photoproteins are regenerated by
 addition of the substrate and molecular oxygen under appropriate
 conditions known to those of skill in this art.
 As used herein, bioluminescence substrate refers to the compound that is
 oxidized in the presence of a luciferase, and any necessary activators,
 generates light. These substrates are referred to as luciferins, which are
 substrates that undergo oxidation in a bioluminescence reaction. These
 bioluminescence substrates include any luciferin or analog thereof or any
 synthetic compound with which a luciferase interacts to generate light.
 Preferred substrates are those that are oxidized in the presence of a
 luciferase or protein in a light-generating reaction. Bioluminescence
 substrates, thus include those compounds that those of skill in the art
 recognize as luciferins. Luciferins, for example, include firefly
 luciferin, Cypridina [also known as Vargula] luciferin [coelentrazine],
 bacterial luciferin, as well as synthetic analogs of these substrates or
 other compounds that are oxidized in the presence of a luciferase in a
 reaction the produces bioluminescence.
 As used herein, capable of conversion into a bioluminescence substrate
 means susceptible to chemical reaction, such as oxidation or reduction,
 that yields a bioluminescence substrate. For example, the luminescence
 producing reaction of bioluminescent bacteria involves the reduction of a
 flavin mononucleotide group (FMN) to reduced flavin mononucleotide
 (FMNH.sub.2) by a flavin reductase enzyme. The reduced flavin
 mononucleotide [substrate] then reacts with oxygen [an activator] and
 bacterial luciferase to form an intermediate peroxy flavin that undergoes
 further reaction, in the presence of a long-chain aldehyde, to generate
 light. With respect to this reaction, the reduced flavin and the long
 chain aldehyde are substrates.
 As used herein, bioluminescence system refers to the set of reagents
 required to conduct a bioluminescent reaction. Thus, the specific
 luciferase, luciferin and other substrates, solvents and other reagents
 that may be required to complete a bioluminescent reaction form a
 bioluminescence system. Thus a bioluminescence system refers to any set of
 reagents that, under appropriate reaction conditions, yield
 bioluminescence. Appropriate reaction conditions refers to the conditions
 necessary for a bioluminescence reaction to occur, such as pH, salt
 concentrations and temperature. In general, bioluminescence systems
 include a bioluminescence substrate luciferin, a luciferase, which
 includes enzymes luciferases and photoproteins, and one or more
 activators. A specific bioluminescence system may be identified by
 reference to the specific organism from which the luciferase derives; for
 example, the Vargula [also called Cypridina] bioluminescence system (or
 Vargula system) includes a Vargula luciferase, such as a luciferase
 isolated from the ostracod, Vargula or produced using recombinant means or
 modifications of these luciferases. This system would also include the
 particular activators necessary to complete the bioluminescence reaction,
 such as oxygen and a substrate with which the luciferase reacts in the
 presence of the oxygen to produce light.
 As used herein, ATP, AMP, NAD+ and NADH refer to adenosine triphosphate,
 adenosine monophosphate, nicotinamide adenine dinucleotide (oxidized form)
 and nicotinamide adenine dinucleotide (reduced form), respectively.
 As used herein, production by recombinant means by using recombinant DNA
 methods means the use of the well known methods of molecular biology for
 expressing proteins encoded by cloned DNA.
 As used herein, substantially identical to a product means sufficiently
 similar to so that the property of interest is sufficiently unchanged so
 that the substantially identical product can be used in place of the
 product.
 As used herein, substantially pure means sufficiently homogeneous to appear
 free of readily detectable impurities as determined by standard methods of
 analysis, such as thin layer chromatography (TLC), gel electrophoresis and
 high performance liquid chromatography (HPLC), used by those of skill in
 the art to assess such purity, or sufficiently pure such that further
 purification would not detectably alter the physical and chemical
 properties, such as enzymatic and biological activities, of the substance.
 Methods for purification of the compounds to produce substantially
 chemically pure compounds are known to those of skill in the art. A
 substantially chemically pure compound may, however, be a mixture of
 stereoisomers. In such instances, further purification might increase the
 specific activity of the compound.
 As used herein equivalent, when referring to two sequences of nucleic acids
 means that the two sequences in question encode the same sequence of amino
 acids or equivalent proteins. When "equivalent" is used in referring to
 two proteins or peptides, it means that the two proteins peptides have
 substantially the same amino acid sequence with only conservative amino
 acid substitutions [see, e.g., Table 2, below] that do not substantially
 alter enzymatic activity. When "equivalent" refers to a property, the
 property does not need to be present to the same extent [e.g., two
 peptides can exhibit different rates of the same type of enzymatic
 activity], but the activities are preferably substantially the same.
 "Complementary," when referring to two nucleotide sequences, means that
 the two sequences of nucleotides are capable of hybridizing, preferably
 with less than 25%, more preferably with less than 15%, even more
 preferably with less than 5%, most preferably with no mismatches between
 opposed nucleotides. Preferably the two molecules will hybridize under
 conditions of high stringency.
 As used herein: stringency of hybridization in determining percentage
 mismatch is as follows:
 1) high stringency: 0.1x SSPE, 0.1% SDS, 65.degree. C.
 2) medium stringency: 0.2x SSPE, 0.1% SDS, 50.degree. C.
 3) low stringency: 1.0x SSPE, 0.1% SDS, 50.degree. C.
 It is understood that equivalent stringencies may be achieved using
 alternative buffers, salts and temperatures.
 The term "substantially" varies with the context as understood by those
 skilled in the relevant art and generally means at least 70%, preferably
 means at least 80%, more preferably at least 90%, and most preferably at
 least 95%.
 As used herein, biological activity refers to the in vivo activities of a
 compound or physiological responses that result upon administration of a
 compound, composition or other mixture. Biological activities may be
 observed in in vitro systems designed to test or use such activities.
 Thus, for purposes herein the biological activity of a luciferase is its
 oxygenase activity whereby upon oxidation of a substrate light is
 produced.
 As used herein, a composition refers to a any mixture. It may be a
 solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or
 any combination thereof.
 As used herein, a combination refers to any association between two or
 among more items.
 As used herein, fluid refers to any composition that can flow. Fluids thus
 encompass compositions that are in the form of semi-solids, pastes,
 solutions, aqueous mixtures, gels, lotions, creams and other such
 compositions.
 B. Bioluminescence generating systems
 A bioluminescence generating system refers to the components that are
 necessary and sufficient to generate bioluminescence. These include a
 luciferase, luciferin and any necessary co-factors or conditions.
 Virtually any bioluminescent system known to those of skill in the art
 will be amenable to use in the apparatus, systems, combinations and
 methods provided herein. Factors for consideration in selecting a
 bioluminescent-generating system, include, but are not limited to: the
 item used in combination with the bioluminescence; the medium in which the
 reaction is run; stability of the components, such as temperature or pH
 sensitivity; shelf life of the components; sustainablity of the light
 emission, whether constant or intermittent; availability of components;
 desired light intensity; and other such factors.
 1. General description
 In general, bioluminescence refers to an energy-yielding chemical reaction
 in which a specific chemical substrate, a luciferin, undergoes oxidation,
 catalyzed by an enzyme, a luciferase. Bioluminescent reactions are easily
 maintained, requiring only replenishment of exhausted luciferin or other
 substrate or cofactor or other protein, in order to continue or revive the
 reaction. Bioluminescence generating reactions are well-known to those of
 skill in this art and any such reaction may be adapted for use in
 combination with articles of manufacture as described herein.
 There are numerous organisms and sources of the bioluminescence generating
 systems and some representative genera and species that exhibit
 bioluminescence are set forth in the following table [reproduced in part
 from Hastings in (1995) Cell Physiology:Source Book, N. Sperelakis (ed.),
 Academic Press, pp 665-681]:
 TABLE 1
 Representative luminous organism
 Type of Organism Representative genera
 Bacteria Photobacterium
 Vibrio
 Xenorhabdus
 Mushrooms Panus, Armillaria
 Pleurotus
 Dinoflagellates Gonyaulax
 Pyrocystis
 Noctiluca
 Cnidaria (coelenterates)
 Jellyfish Aequorea
 Hydroid Obelia
 Sea Pansy Renilla
 Ctenophores Mnemiopsis
 Beroe
 Annelids
 Earthworms Diplocardia
 Marine polychaetes Chaetopterus
 Syllid fireworm Odontosyllis
 Molluscs
 Limpet Latia
 Clam Pholas
 Squid Heteroteuthis
 Crustacea
 Ostracod Vargula (Cypridina)
 Shrimp (euphausids) Meganyctiphanes
 Copepods
 Insects
 Coleopterids (beetles)
 Firefly Photinus, Photuris
 Click beetles Pyrophorus
 Railroad worm Phengodes, Phrixothrix
 Diptera (flies) Arachnocampa
 Echinoderms
 Brittle stars Ophiopsila
 Sea cucumbers Laetmogone
 Chordates
 Tunicates Pyrosoma
 Fish
 Cartilaginous Squalus
 Bony
 Ponyfish Leiognathus
 Flashlight fish Photoblepharon
 Angler fish Cryptopsaras
 Midshipman Porichthys
 Midwater fish
 Cyclothone
 Neoscopelus
 Tarletonbeania
 It is understood that bioluminescence generating system may be isolated
 from natural sources, such as those in the above Table or may be produced
 synthetically. In addition, for uses herein, the components need only be
 sufficiently pure so that mixture thereof under appropriate reaction
 conditions produces a glow. Thus, in some embodiments, a crude extract or
 merely grinding up the organism may be adequate. Generally, however,
 substantially pure components are used. Also, components may be synthetic
 components that are not isolated from natural sources. DNA encoding
 luciferases is available [see, e.g., SEQ ID Nos. 1-13] and has been
 modified [see, e.g., SEQ ID Nos. 3 and 10-13] and synthetic and
 alternative substrates have been devised. The DNA provided listed herein
 is only representative of DNA encoding luciferases that is available.
 Any bioluminescence generating system, whether synthetic or isolated form
 natural sources, such as those set forth in Table 1, elsewhere herein or
 known to those of skill in the art is intended for use in the
 combinations, systems and methods provided herein. Chemiluminescence
 systems per se, which do not rely on oxygenases [luciferases] are not
 encompassed herein.
 a. Luciferases
 Luciferases refer to any compound that, in the presence of any necessary
 activators, catalyze the oxidation of a bioluminescence substrate
 [luciferin] in the presence of molecular oxygen, whether free or bound,
 from a lower energy state to a higher energy state such that the
 substrate, upon return to the lower energy state, emits light. For
 purposes herein, luciferase is broadly used to encompass enzymes that act
 catalytically to generate light by oxidation of a substrate and also
 photoproteins, such as aequorin, that acts, though not strictly
 catalytically [since such protein is exhausted in the reaction], in
 conjunction with a substrate in the presence of oxygen to generate light.
 These luciferases, including photoproteins, such as aequorin, are herein
 also included among the luciferases. These reagents include the
 naturally-occurring luciferases [including photoproteins], proteins
 produced by recombinant DNA, and mutated or modified variants thereof that
 retain the ability to generate light in the presence of an appropriate
 substrate, co-factors and activator or any other such protein that acts as
 a catalyst to oxidize a substrate, whereby light is produced.
 Generically the protein that catalyzes or initiates the bioluminescent
 reaction is referred to as a luciferase, and the oxidizable substrate is
 referred to as a luciferin. The oxidized reaction product is termed
 oxyluciferin and certain luciferin precursors are termed etioluciferin.
 Thus, for purposes herein bioluminescence encompasses light produced by
 reactions that are catalyzed by [in the case of luciferases that act
 enzymatically] or initiated by [in the case of the photoproteins, such as
 aequorin, that are not regenerated in the reaction]a biological protein or
 analog, derivative or mutant thereof.
 For clarity herein, these catalytic proteins are referred to as luciferases
 and include, such as enzymes such the luciferases that catalyze the
 oxidation of luciferin, emitting light and releasing oxyluciferin. Also
 included among luciferase are photoproteins, which catalyze the oxidation
 of luciferin to emit light but are changed in the reaction and must be
 reconstituted to be used again. The luciferases may be naturally occurring
 or may be modified, such as by genetic engineering to improve or alter
 certain properties. As long as the resulting molecule retains the ability
 to catalyze the bioluminescent reaction, it can be used herein.
 Any protein that has luciferase activity [a protein that catalyzes
 oxidation of a substrate in the presence of molecular oxygen to produce
 light as defined herein] may be used herein. The preferred luciferases are
 those that are described herein or that have minor sequence variations.
 Such minor sequence variations include, but are not limited to, minor
 allelic or species variations and insertions or deletions of residues,
 particularly cysteine residues. Suitable conservative substitutions of
 amino acids are known to those of skill in this art and may be made
 generally without altering the biological activity of the resulting
 molecule. Those of skill in this art recognize that, in general, single
 amino acid substitutions in non-essential regions of a polypeptide do not
 substantially alter biological activity (see, e.g., Watson et al.
 Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings
 Pub. co., p.224). Such substitutions are preferably made in accordance
 with those set forth in TABLE 2 as follows:
 TABLE 2
 Original residue Conservative substitution
 Ala (A) Gly; Ser
 Arg (R) Lys
 Asn (N) Gln; His
 Cys (C) Ser; neutral amino acid
 Gln (Q) Asn
 Glu (E) Asp
 Gly (G) Ala; Pro
 His (H) Asn; Gln
 Ile (I) Leu; Val
 Leu (L) Ile; Val
 Lys (K) Arg; Gln; Glu
 Met (M) Leu; Tyr; Ile
 Phe (F) Met; Leu; Tyr
 Ser (S) Thr
 Thr (T) Ser
 Trp (W) Tyr
 Tyr (Y) Trp; Phe
 Val (V) Ile; Leu
 Other substitutions are also permissible and may be determined empirically
 or in accord with known conservative substitutions. Any such modification
 of the polypeptide may be effected by any means known to those of skill in
 this art.
 The luciferases may be obtained commercially, isolated from natural
 sources, expressed in host cells using DNA encoding the luciferase, or
 obtained in any manner known to those of skill in the art. For purposes
 herein, crude extracts obtained by grinding up selected source organisms
 may suffice. Since large quantities of the luciferase may be desired,
 isolation of the luciferase from host cells is preferred. DNA for such
 purposes is widely available as are modified forms thereof.
 Examples of luciferases include, but are not limited to, those isolated
 from the ctenophores Mnemiopsis (mnemiopsin) and Beroe ovata (berovin),
 those isolated from the coelenterates Aequorea (aequorin), Obelia
 (obelin), Pelagia, the Renilla luciferase, the luciferases isolated from
 the mollusca Pholas (pholasin), and from the ostracods, such as Cypridina
 (also referred to as Vargula). Preferred luciferases for use herein are
 the Aequorin protein Renilla luciferase, and Cypridina [also called
 Vargula] luciferase [see, e.g., SEQ ID Nos. 1, 2, and 4-13].
 b. Luciferins
 The substrates for the reaction include any molecule(s) with which the
 luciferase reacts to produce light. Such molecules include the
 naturally-occurring substrates, modified forms thereof, and synthetic
 substrates [see, e.g., U.S. Pat. Nos. 5,374,534 and 5,098,828]. Exemplary
 luciferins include those described herein, as well as derivatives thereof,
 analogs thereof, synthetic substrates, such as dioxetanes [see, e.g., U.S.
 Pat. Nos. 5,004,565 and 5,455,357], and other compounds that are oxidized
 by a luciferase in a light-producing reaction [see, e.g., U.S. Pat. Nos.
 5,374,534, 5,098,828 and 4,950,588]. Such substrates also may be
 identified empirically by selecting compounds that are oxidized in
 bioluminescent reactions.
 c. Activators
 The bioluminescent generating systems also require additional components
 discussed herein and known to those of skill in the art. All
 bioluminescent reactions require molecular oxygen in the form of dissolved
 or bound oxygen. Thus, molecular oxygen, dissolved in water or in air or
 bound to a photoprotein, is the activator for bioluminescence reactions.
 Depending upon the form of the components, other activators include, but
 are not limited to, ATP [for firefly luciferase], flavin reductase
 [bacterial systems] for regenerating FMNH.sub.2 from FMN, and Ca.sup.2+ or
 other suitable metal ion [aequorin].
 Most of the systems provided herein will generate light when the luciferase
 and luciferin are mixed and exposed to air or water. The systems that use
 photoproteins that have bound oxygen, such as aequorin, however, will
 require exposure to Ca.sup.2+ [or other suitable metal ion], which can be
 provided in the form of an aqueous composition of a calcium salt. In these
 instances, addition of a Ca.sup.2+ [or other suitable metal ion] to a
 mixture of luciferase [aequorin] and luciferase [such as coelentrazine]
 will result in generation of light. The Renilla system and other Anthozoa
 systems also require Ca.sup.2+ [or other suitable metal ion].
 If crude preparations are used, such as ground up Cypridina [shrimp] or
 ground fireflies, it may be necessary to add only water. In instances in
 which fireflies [or a firefly or beetle luciferase] are used the reaction
 may only require addition ATP. The precise components will be apparent, in
 light of the disclosure herein, to those of skill in this art or may be
 readily determined empirically.
 It is also understood that these mixtures will also contain any additional
 salts or buffers or ions that are necessary for each reaction to proceed.
 Since these reactions are well-characterized those of skill in the art
 will be able to determine precise proportions and requisite components.
 Selection of components will depend upon the apparatus, article of
 manufacture and luciferase. Various embodiments are described and
 exemplified herein; in view of such description other embodiments will be
 apparent.
 d. Reactions
 In all embodiments, up to all but one component of a bioluminescence
 generating system will be mixed with or packaged with or otherwise
 combined with a selected article of manufacture to produce the novelty
 item. When bioluminescence is desired, the remaining component(s) will be
 added and light will be produced.
 In general, since the result to be achieved is the production of light
 visible to the naked eye for entertainment, amusement or recreation, for
 the purposes herein, the precise proportions and amounts of components of
 the bioluminescence reaction need not be stringently determined or met.
 They must be sufficient to produce light. Generally, an amount of
 luciferin and luciferase sufficient to generate a visible glow is used;
 this amount can be readily determined empirically and is dependent upon
 the selected system and selected application.
 For purposes herein, such amount is preferably at least the concentrations
 and proportions used for analytical purposes by those of skill in the such
 arts. Higher concentrations may be used if the glow is not sufficiently
 bright. Also because the conditions in which the reactions are used are
 not laboratory conditions and the components are subject to storage,
 higher concentration may be used to overcome any loss of activity.
 Typically, the amounts are 1 mg of a luciferase per liter of reaction
 mixture or 1 mg coated on portion of a T-shirt or other textile or paper.
 Such coating may be produced by drying a composition containing at least
 about 0.01 mg/l, typically 0.1, 1 mg/l, 10 mg/l or more of each component
 on the item. The amount of luciferin is also between about 0.1 and 10
 mg/l, preferably between 0.1 and 1 mg/l, additional luciferin can be added
 to many of the reactions to continue the reaction. In embodiments in which
 the luciferase acts catalytically and does not need to be regenerated,
 lower amounts of luciferase can be used. In those in which it is changed
 during the reaction, it also can be replenished; typically higher
 concentrations will be selected. Ranges of concentration per liter [or the
 amount of coating on substrate the results from contacting with such
 composition] of each component on the order of 0.1 to 20 mg, preferably
 0.1 to 10 mg, more preferably between about 1 and 1 0 mg of each component
 will be sufficient. When preparing coated substrates, as described herein,
 greater amounts [or coating compositions containing higher concentrations
 of the luciferase or luciferin may be used.
 It is understood, that concentrations and amounts to be used depend upon
 the selected article of manufacture and they may be readily determined
 empirically. Proportions, particularly those used when commencing an
 empirical determination, are generally those used for analytical purposes,
 and amounts or concentrations are at least those used for analytical
 purposes, but the amounts can be increased, particularly if a sustained
 and brighter glow is desired.
 2. Ctenophore and coelenterate systems
 Ctenophores, such as Mnemiopsis (mnemiopsin) and Beroe ovata (berovin),
 coelenterates, such as Aequorea (aequorin), Obelia (obelin), Pelagia,
 produce bioluminescent light using similar chemistries [see, e.g.,
 Stephenson et al. (1981) Biochimica et Biophysica Acta 678:65-75; Hart et
 al. (1979) Biochemistry 18:2204-2210; International PCT Application No.
 WO94/18342, which is based on U.S. application Ser. No. 08/017,116 and
 other references and patents cited herein]. The Aequorin and Renilla
 systems are representative and are described in detail herein as exemplary
 and as among the presently preferred systems The Aequorin and Renilla
 systems can use the same luciferin and produce light using the same
 chemistry, but each luciferase is different. The Aequorin luciferase
 aequorin, as well as, for example, the luciferases mnemiopsin and berovin,
 is a photoprotein that includes bound oxygen and bound luciferin, requires
 Ca.sup.2+ [or other suitable metal ion] to trigger the reaction, and must
 be regenerated for repeated use; whereas, the Renilla luciferase acts as a
 true enzyme because it is unchanged during the reaction and it requires
 dissolved molecular oxygen.
 a. The aequorin system
 The aequorin system is well known [see, e.g., Tsuji et al. (1986)
 "Site-specific mutagenesis of the calcium-binding photoprotein aequorin,"
 Proc. Natl. Acad. Sci. USA 83:8107-8111; Prasher et al. (1985) "Cloning
 and Expression of the cDNA Coding for Aequorin, a Bioluminescent
 Calcium-Binding Protein," Biochemical and Biophysical Research
 Communications 126:1259-1268; Prasher et al. (1986) Methods in Enzymology
 133:288-297; Prasher, et al. (1987) "Sequence Comparisons of cDNAs
 Encoding for Aequorin Isotypes," Biochemistry 26:1326-1332; Charbonneau et
 al. (1985) "Amino Acid Sequence of the Calcium-Dependent Photoprotein
 Aequorin," Biochemistry 24:6762-6771; Shimomura et al. (1981) "Resistivity
 to denaturation of the apoprotein of aequorin and reconstitution of the
 luminescent photoprotein from the partially denatured apoprotein,"
 Biochem. J. 199:825-828; Inouye et al. (1989) J. Biochem. 105:473-477;
 Inouye et al. (1986) "Expression of Apoaequorin Complementary DNA in
 Escherichia coli," Biochemistry 25:8425-8429; Inouye et al. (1985)
 "Cloning and sequence analysis of cDNA for the luminescent protein
 aequorin," Proc. Natl. Acad. Sci. USA 82:3154-3158; Prendergast, et al.
 (1978) "Chemical and Physical Properties of Aequorin and the Green
 Fluorescent Protein Isolated from Aequorea forskalea" J. Am. Chem. Soc.
 17:3448-3453; European Patent Application 0 540 064 A1; European Patent
 Application 0 226 979 A2, European Patent Application 0 245 093 A1 and
 European Patent Specification 0 245 093 B1; U.S. Pat. No. 5,093,240; U.S.
 Pat. No. 5,360,728; U.S. Pat. No. 5,139,937; U.S. Pat. No. 5,422,266; U.S.
 Pat. No. 5,023,181; U.S. Pat. No. 5,162,227; and SEQ ID Nos. 5-13, which
 set forth DNA encoding the apoprotein; and a form, described in U.S. Pat.
 No. 5,162,227, European Patent Application 0 540 064 Al and Sealite
 Sciences Technical Report No. 3 (1994), is commercially available from
 Sealite, Sciences, Bogart, Ga. as AQUALITE.RTM.].
 This system is among the preferred systems for use herein. As will be
 evident, since the aequorin photoprotein includes noncovalently bound
 luciferin and molecular oxygen, it is suitable for storage in this form as
 a lyophilized powder or encapsulated into a selected delivery vehicle. The
 system can be encapsulated into pellets, such as liposomes or other
 delivery vehicles, or stored in single chamber dual or other multiple
 chamber apparatus. When used, the vehicles are contacted with a
 composition, even tap water, that contains Ca.sup.2+ [or other suitable
 metal ion], to produce a mixture that glows. This system is preferred for
 use in numerous embodiments herein, such as in any embodiment that uses
 pellets. These embodiments include, squirt guns, fairy dust, bubble toys,
 bubble baths, soaps, linked to textiles, for addition to beverages and
 foods.
 (1) Aequorin photoprotein
 The photoprotein, aequorin, isolated from the jellyfish, Aequorea, emits
 light upon the addition of Ca.sup.2+ [or other suitable metal ion]. The
 aequorin photoprotein, which includes bound luciferin and bound oxygen
 that is released by Ca.sup.2+, does not require dissolved oxygen.
 Luminescence is triggered by calcium, which releases oxygen and the
 luciferin substrate producing apoaqueorin.
 The bioluminescence photoprotein aequorin is isolated from a number of
 species of the jellyfish Aequorea. It is a 22 kilodalton [kD] molecular
 weight peptide complex [see, e.g., Shimomura et al. (1962) J. Cellular and
 Comp. Physiol. 59:233-238; Shimomura et al. (1969) Biochemistry
 8:3991-3997; Kohama et al. (1971) Biochemistry 10:4149-4152; and Shimomura
 et al. (1972) Biochemistry 11:1602-1608]. The native protein contains
 oxygen and a heterocyclic compound coelenterazine, a luciferin, [see,
 below] noncovalently bound thereto. The protein contains three calcium
 binding sites. Upon addition of trace amounts Ca.sup.2+ [or other suitable
 metal ion, such as strontium] to the photoprotein, it undergoes a
 conformational change the catalyzes the oxidation of the bound
 coelentrazine using the protein-bound oxygen. Energy from this oxidation
 is released as a flash of blue light, centered at 469 nm. Concentrations
 of calcium ions as low as 10.sup.-6 M are sufficient to trigger the
 oxidation reaction.
 Naturally-occurring apoaequorin is not a single compound but rather is
 mixture of microheterogeneous molecular species. Aequoria jellyfish
 extracts contain as many as twelve distinct variants of the protein [see,
 e.g., Prasher et al. (187) Biochemistry 26:1 326-1 332; Blinks et al.
 (1975) Fed. Proc. 34:474]. DNA encoding numerous forms has been isolated
 [see, e.g., SEQ ID Nos. 5-9 and 13].
 The photoprotein can be reconstituted [see, e.g., U.S. Pat. No. 5,023,181]
 by combining the apoprotein, such as protein recombinantly produced in E.
 coli with a coelentrazine, such as a synthetic coelentrazine, in the
 presence of oxygen and a reducing agent [see, e.g., Shimomura et al.
 (1975) Nature 256:236-238; Shimomura et al. (1981) Biochemistry J.
 199:825-828], such as 2-mercaptoenthanol, and also EDTA or EGTA
 [concentrations between about 5 to about 100 mM or higher for applications
 herein] tie up any Ca.sup.2+ to prevent triggering the oxidation reaction
 until desired. DNA encoding a modified form of the apoprotein that does
 not require 2-mercaptoethanol for reconstitution is also available [see,
 e.g., U.S. Pat. No. U.S. Pat. No. 5,093,240]. The reconstituted
 photoprotein is also commercially available [sold, e.g., under the
 trademark AQUALITE.RTM., which is described in U.S. Pat. No. 5,162,227].
 The light reaction is triggered by adding Ca.sup.2+ at a concentration
 sufficient to overcome the effects of the chelator and achieve the
 10.sup.-6 M concentration. Because such low concentrations of Ca.sup.2+
 can trigger the reaction, for use in the methods and apparatus herein,
 higher concentrations of chelator may be included in the compositions of
 photoprotein. Accordingly, higher concentrations of added Ca.sup.2+ in the
 form of a calcium salt will be required. Precise amounts may be
 empirically determined. For use herein, it may be sufficient to merely add
 water to the photoprotein, which is provided in the form of a concentrated
 composition or in lyophilized or powdered form. Thus, for purposes herein,
 addition of small quantities of Ca.sup.2+, such as those present in most
 tap water or in phosphate buffered saline (PBS) or other suitable buffers
 or possible in the moisture on the skin, should trigger the
 bioluminescence reaction.
 Numerous isoforms of the aequorin apoprotein been identified isolated. DNA
 encoding these proteins has been cloned, and the proteins and modified
 forms thereof have been produced using suitable host cells [see, e.g.,
 U.S. Pat. Nos. 5,162,227, 5,360,728, 5,093,240; see, also, Prasher et al.
 (1985) Biophys. Biochem. Res. Commun. 126:1259-1268; Inouye et al. (1986)
 Biochemistry 25: 8425-8429]. U.S. Pat. No. 5,093,240; U.S. Pat. No.
 5,360,728; U.S. Pat. No. 5,139,937; U.S. Pat. No. 5,288,623; U.S. Pat. No.
 5,422,266, U.S. Pat. No. 5,162,227 and SEQ ID Nos. 5-13, which set forth
 DNA encoding the apoprotein; and a form is commercially available form
 Sealite, Sciences, Bogart, Ga. as AQUALITE.RTM.]. DNA encoding apoaequorin
 or variants thereof is useful for recombinant production of high
 quantities of the apoprotein. The photoprotein is reconstituted upon
 addition of the luciferin, coelentrazine or an analog thereof, and
 molecular oxygen [see, e.g., U.S. Pat. No. 5,023,181]. The apoprotein and
 other constituents of the photoprotein and bioluminescence generating
 reaction can be mixed under appropriate conditions to regenerate the
 photoprotein and concomitantly have the photoprotein produce light.
 Reconstitution requires the presence of a reducing agent, such as
 mercaptoethanol, except for modified forms, discussed below, that are
 designed so that a reducing agent is not required [see, e.g., U.S. Pat.
 No. 5,093,240].
 For use herein, it is preferred aequorin is produced using DNA, such as
 that set forth in SEQ ID Nos. 5-13 and known to those of skill in the art
 or modified forms thereof. The DNA encoding aequorin is expressed in a
 host cell, such as E. coli, isolated and reconstituted to produce the
 photoprotein [see, e.g., U.S. Pat. Nos. 5,418,155, 5,292,658, 5,360,728,
 5,422,266, 5,162,227].
 Of interest herein, are forms of the apoprotein that have been modified so
 that the bioluminescent activity is greater than unmodified apoaequorin
 [see, e.g., U.S. Pat. No. 5,360,728, SEQ ID Nos. 10-12]. Modified forms
 that exhibit greater bioluminescent activity than unmodified apoaequorin
 include proteins having sequences set forth in SEQ ID Nos. 10-12, in which
 aspartate 124 is changed to serine, glutamate 135 is changed to serine,
 and glycine 129 is changed to alanine, respectively. Other modified forms
 with increased bioluminescence are also available.
 For use in certain embodiments herein, the apoprotein and other components
 of the aequorin bioluminescence generating system are packaged or provided
 as a mixture, which, when desired is subjected to conditions under which
 the photoprotein reconstitutes from the apoprotein, luciferin and oxygen
 [see, e.g., U.S. Pat. No. 5,023,181; and U.S. Pat. No. 5,093,240].
 Particularly preferred are forms of the apoprotein that do not require a
 reducing agent, such as 2-mercapto-ethanol, for reconstitution. These
 forms, described, for example in U.S. Pat. No. 5,093,240 [see, also Tsuji
 et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83:8107-8111], are modified by
 replacement of one or more, preferably all three cysteine residues with,
 for example serine. Replacement may be effected by modification of the DNA
 encoding the aequorin apoprotein, such as that set forth in SEQ ID No. 5,
 and replacing the cysteine codons with serine.
 In general for use herein, the components of the bioluminescence are
 packaged or provided so that there is insufficient metal ions to trigger
 the reaction. When used, the trace amounts of triggering metal ion,
 particularly Ca.sup.2+ is contacted with the other components. For a more
 sustained glow, aequorin can be continuously reconstituted or can be added
 or is provided in high excess.
 (2) Luciferin
 The aequorin luciferin is coelentrazine and analogs therein, which include
 molecules have the structure [formula (I)]:
 ##STR1##
 in which R.sub.1 is CH.sub.2 C.sub.6 H.sub.5 or CH.sub.3 ; R.sub.2 is
 C.sub.6 H.sub.5, and R.sub.3 is p-C.sub.6 H.sub.4 OH or CH.sub.3 or other
 such analogs that have activity. Preferred coelenterazine has the
 structure in which R.sup.1 is p-CH.sub.2 C.sub.6 H.sub.4 OH, R.sub.2 is
 C.sub.6 H.sub.5, and R.sub.3 is p-C.sub.6 H.sub.4 OH, which can be
 prepared by known methods [see, e.g., Inouye et al. (1975) Jap. Chem.
 Soc.. Chemistry Lttrs. pp 141-144; and Halt et al. (1979) Biochemistry
 18:2204-221 0]. The preferred coelentrazine has the structure (formula
 (II)):
 ##STR2##
 The reaction of coelenterazine when bound to the aequorin photoprotein with
 bound oxygen and in the presence of Ca.sup.2+ can represented as follows:
 ##STR3##
 The photoprotein aequorin [which contains apoaequorin bound to a
 coelenterate luciferin molecule] and Renilla luciferase, discussed below,
 can use the same coelenterate luciferin. The aequorin photoprotein
 catalyses the oxidation of coelenterate luciferin [coelenterazine] to
 oxyluciferin [coelenteramide] with the concomitant production of blue
 light [lambda.sub.max =469 nm].
 Thus, the bioluminescent system of Aequorea is particularly suitable for
 use in the methods and apparatus herein. The particular amounts and the
 manner in which the components are provided depends upon the selected
 combination of article of manufacture. This system can be provided in
 lyophilized form, that will glow upon addition of Ca.sup.2+, It can be
 encapsulated, linked to matrices, such as porous glass, or in as a
 compositions, such as a solution or suspension, preferably in the presence
 of sufficient chelating agent to prevent triggering the reaction. The
 concentration of the aequorin photoprotein will vary and can be determined
 empirically. Typically concentrations of at least 0.1 mg/l, more
 preferably at least 1 mg/l and higher, will be selected.
 As described herein, blue light is produced using the Aequorea photoprotein
 in the presence of Ca.sup.2+ or the Renilla luciferase and the
 coelentrazine luciferin or analog thereof. This light can be converted
 into a green light if a green fluorescent protein is added to the
 reaction. Green fluorescent proteins, which have been purified [see, e.g.,
 Prasher et al. (1992) Gene 111:229-233] and also cloned [see, e.g.,
 International PCT Application No. WO 95/07463, which is based on U.S.
 application Ser. No. 08/119,678 and U.S. application Ser. No. 08/192,274,
 which are herein incorporated by reference], is used by cnidarians as
 energy-transfer acceptors. GFPs fluoresce in vivo upon receiving energy
 from a luciferase-oxyluciferein excited-state complex or a Ca.sup.2+
 -activated photoprotein. The chromophore is modified amino acid residues
 within the polypeptide. The best characterized GFPs are those of Aequorea
 and Renilla [see, e.g., Prasher et al. (1992) Gene 111:229-233; Hart, et
 al. (1979) Biochemistry 18:2204-2210]. For example, a green fluorescent
 protein [GFP] from Aequorea Victoria contains 238 amino acids, absorbs
 blue light and emits green light. Thus, inclusion of this protein in a
 composition containing the aequorin photoprotein charged with
 coelentrazine and oxygen, can in the presence of calcium result in the
 production of green light.
 It is contemplated that GFPs may also be included in the bioluminescence
 generating reactions that employ the aequorin or Renilla luciferases or
 other suitable luciferase in order to enhance or alter color of the
 resulting bioluminescence.
 b. The Renilla system
 Representative of coelenterate systems is the Renilla system. Renilla, also
 known as sea pansies, are members of the class of coelenterates Anthozoa,
 which includes other bioluminescent genera, such as Cavarnularia,
 Ptilosarcus, Stylatula, Acanthoptilum, and Parazoanthus. Bioluminescent
 members of the Anthozoa genera contain luciferases and luciferins that are
 similar in structure [see, e.g., Cormier et al. (1973) J. Cell. Physiol.
 81:291-298; see, also Ward et al. (1975) Proc. Natl. Acad. Sci. U.S.A.
 72:2530-2534]. The luciferases and luciferins from each of these
 anthozoans crossreact with one another and produce a characteristic blue
 luminescence.
 Renilla luciferase and the other coelenterate and ctenophore luciferases,
 such as the aequorin photoprotein, use imidazopyrazine substrates,
 particularly the substrates generically called coelenterazine [see,
 formulae (I) and (II), above]. Other genera that have luciferases that use
 a coelenterazine include: squid, such as Chiroteuthis, Eucleoteuthis,
 Onychoteuthis, Watasenia; cuttlefish, Sepiolina; shrimp, such as
 Oplophorus, Sergestes, and Gnathophausia; deep-sea fish, such as
 Argyropelecus, Yarella, Diaphus, Neoscopelus.
 Renilla luciferase does not, however, have bound oxygen, and thus requires
 dissolved oxygen in order to produce light in the presence of a suitable
 luciferin substrate. Since Renilla luciferase acts as a true enzyme [i.e.,
 it does not have to be reconstituted for further use] the resulting
 luminescence can be long-lasting in the presence of saturating levels of
 luciferin. Also, Renilla luciferase is relatively stable to heat.
 Renilla luciferase, DNA encoding Renilla luciferase, and use of the DNA to
 produce recombinant luciferase, as well as DNA encoding luciferase from
 other coelenterates, are well known and available [see, e.g., SEQ ID No.
 1, U.S. Pat. Nos. 5,418,155 and 5,292,658; see, also, Prasher et al.
 (1985) Biochem. Biophys. Res. Commun. 126:1259-1268; Cormier (1981)
 "Renilla and Aequorea bioluminescence" in Bioluminescence and
 Chemiluminescence, pp. 225-233; Charbonneau et al. (1979) J. Biol. Chem.
 254:769-780; Ward et al. (1979) J. Biol. Chem. 254:781-788; Lorenz et al.
 (1981) Proc. Natl. Acad. Sci. U.S.A. 88: 4438-4442; Hori et al. (1977)
 Proc. Natl. Acad. Sci. U.S.A. 74:4285-4287; Hori et al. (1975)
 Biochemistry 14:2371-2376; Hori et al. (1977) Proc. Natl. Acad. Sci.
 U.S.A. 74:4285-4287; Inouye et al. (1975) Jap. Soc. Chem. Lett. 141-144;
 and Matthews et al. (1979) Biochemistry 16:85-91]. The DNA encoding
 Renilla luciferase and host cells containing such DNA provide a convenient
 means for producing large quantities of the enzyme [see, e.g., U.S. Pat.
 Nos. 5,418,155 and 5,292,658, which describe recombinant production of
 Renilla luciferase and the use of the DNA to isolate DNA encoding other
 luciferases, particularly those from related organisms].
 When used herein, the Renilla luciferase can be packaged, such as in an
 toy, in lyophilized form, encapsulated in a vehicle, either by itself or
 in combination with the luciferin substrate. Prior to use the mixture is
 contacted with an aqueous composition, preferably a phosphate buffered
 saline pH 7-8; dissolved O.sub.2 will activate the reaction. For use
 herein, final concentrations of luciferase in the glowing mixture will be
 on the order of 0.01 to 1 mg/l or more. Concentrations of luciferin will
 be at least about 10.sup.-8 M, but 1 to 100 or more orders of magnitude
 higher to produce a long lasting bioluminescence.
 Lyophilized mixtures, and compositions containing the Renilla luciferase
 are also provided. The luciferase or mixtures of the luciferase and
 luciferin may also be encapsulated into a suitable delivery vehicle, such
 as a liposome, glass particle, capillary tube, drug delivery vehicle,
 gelatin, time release coating or other such vehicle. Kits containing these
 mixtures, compositions, or vehicles and also a selected article of
 manufacture, such as a toy gun, bubble composition, balloon, item of
 clothing, personal item, are also provided. The luciferase may also be
 linked to a substrate, such as cotton, polyester, polyester-cotton blends,
 polypropylene, polyvinyltoluene, polyvinyl propylene, glass, ceramic, or
 plastics are provided in combination with or as part of an article of
 manufacture.
 3. Crustacean, particularly Cyrpidina [Vargula], systems
 The ostracods, such as Vargula [also known as Cypridina] serratta,
 hilgendorfil and noctiluca are small marine crustaceans, sometimes called
 sea fireflies. These sea fireflies are found in the waters off the coast
 of Japan and emit light by squirting luciferin and luciferase into the
 water, where the reaction, which produces a bright blue luminous cloud,
 occurs. The reaction involves only luciferin, luciferase and molecular
 oxygen, and, thus, is very suitable for application herein.
 The systems, such as the Vargula [Cypridina] bioluminescent systems, are
 particularly preferred herein because the components are stable at room
 temperature if dried and powdered and will continue to react even if
 contaminated. Further, the bioluminescent reaction requires only the
 luciferin/luciferase components in concentrations as low as 1:40 parts per
 billion to 1:100 parts per billion, water and molecular oxygen [i.e.,
 dissolved air or oxygen] to proceed. An exhausted system can renewed by
 addition of luciferin.
 a. Vargula luciferase
 Vargula luciferase is a 555-amino acid polypeptide that has been produced
 by isolation from Vargula and also using recombinant technology by
 expressing the DNA in suitable bacterial and mammalian host cells [see,
 e.g., Thompson et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:6567-6571;
 Inouye et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:9584-9587; Johnson
 et al. (1978) Methods in Enzymology LVII:331-349; Tsuji et al. (1978)
 Methods Enzymol. 57:364-72; Tsuji (19740 Biochemistry 13:5204-5209;
 Japanese Patent Application No. JP 3-30678 Osaka; and European Patent
 Application No. EP 0 387 355 A1].
 (1) Purification from Cypridina
 Methods for purification of Vargula [Cypridina] luciferase are well known.
 For example, crude extracts containing the active can be readily prepared
 by grinding up or crushing the Vargula shrimp. In other embodiments, a
 preparation of Cypridina hilgendorfi luciferase can be prepared by
 immersing stored frozen C. hilgendorfi in distilled water containing,
 0.5-5.0 M salt, preferably 0.5-2.0 M sodium or potassium chloride,
 ammonium sulfate, at 0-30.degree. C., preferably 0-10.degree. C., for 1-48
 hr, preferably 10-24 hr, for extraction followed by hydrophobic
 chromatography and then ion exchange or affinity chromatography [TORAY IND
 INC, Japanese patent application JP 4258288, published September 14, 1993;
 see, also, Tsuji et al. (1978) Methods Enzymol. 57:364-72 for other
 methods].
 The luciferin can be isolated from ground dried Vargula by heating the
 extract, which destroys the luciferase but leaves the luciferin intact
 [see, e.g., U.S. Pat. No. 4,853,327].
 (2) Preparation by Recombinant Methods
 The luciferase is preferably produced by expression of cloned DNA encoding
 the luciferase [European Paten Application NO. 0 387 355 A1; International
 PCT Application No. WO9/001542; see, also SEQ ID No. 5, which sets forth
 the sequence from Japanese Patent Application No. JP 3-30678 and Thompson
 et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:6567-6571] DNA encoding the
 luciferase or variants thereof is introduced into E. coli using
 appropriate vectors and isolated using standard methods.
 b. Vargula luciferin
 The natural luciferin in a substituted imidazopyrazine nucleus, such a
 compound of formula (III):
 ##STR4##
 Analogs thereof and other compounds that react with the luciferase in a
 light producing reaction also may be used.
 Other bioluminescent organisms that have luciferases that can react with
 the Vargula luciferin include, the genera Apogon, Parapriacan thus and
 Porichthys.
 c. Reaction
 The luciferin upon reaction with oxygen forms a dioxetanone intermediate
 [which includes a cyclic peroxide similar to the firefly cyclic peroxide
 molecule intermediate]. In the final step of the bioluminescent reaction,
 the peroxide breaks down to form CO.sub.2 and an excited carbonyl. The
 excited molecule then emits a blue to blue-green light.
 The optimum pH for the reaction is about 7. For purposes herein, any pH at
 which the reaction occurs may be used. The concentrations of reagents are
 those normally used for analytical reactions or higher [see, e.g.,
 Thompson et al. (1990) Gene 96:257-262]. Typically concentrations of the
 luciferase between 0.1 and 10 mg/l, preferably 0.5 to 2.5 mg/l will be
 used. Similar concentrations or higher concentrations of the luciferin may
 be used.
 4. Insect bioluminescent systems including fireflies, click beetles, and
 other insect system
 The biochemistry of firefly bioluminescence was the first bioluminescent
 system to be characterized [see, e.g., Wienhausen et al. (1985)
 Photochemistry and Photobiology 42:609-611; McElroy et al. (1966) in
 Molecular Architecture in cell Physiology, Hayashi et al., eds. Prentice
 Hall, Inc., Englewood Cliffs, N.J., pp. 63-80] and it is commercially
 available [e.g.., from Promega Corporation, Madison, Wis., see, eg., Leach
 et al. (1986) Methods in Enzymology 133:51-70, esp. Table 1]. Luciferases
 from different species of fireflies are antigenically similar. These
 species include members of the genera Photinus, Photurins and Luciola.
 Further, the bioluminescent reaction produces more light at 30.degree. C.
 than at 20.degree. C., the luciferase is stabilized by small quantities of
 bovine albumin serum, and the reaction can be buffered by tricine.
 a. Luciferase
 DNA clones encoding luciferases from various insects and the use to produce
 the encoded luciferase is well known. For example, DNA clones that encode
 luciferase from Photinus pyralis, Luciola cruciata [see, e.g., de Wet et
 al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:7870-7873; de We et al. (1986)
 Methods in Enzymology 133:3; U.S. Pat. No. 4,968,613, see, also SEQ ID No.
 3] are available. The DNA has also been expressed in Saccharomyces [see,
 e.g., Japanese Application No. JP 63317079, published Dec. 26, 1988,
 KIKKOMAN CORP] and in tobacco.
 In addition to the wild-type luciferase modified insect luciferases have
 been prepared. For example, heat stable luciferase mutants, DNA-encoding
 the mutants, vectors and transformed cells for producing the luciferases
 are available. A protein with 60% amino acid sequence homology with
 luciferases from Photinus pyralis, Luciola mingrelica, L. cruciata or L.
 lateralis and having luciferase activity is available [see, e.g.,
 International PCT Application No. WO95/25798]. It is more stable above
 30.degree. C. than naturally-occurring insect luciferases and may also be
 produced at 37.degree. C. or above, with higher yield.
 Modified luciferases that generate light at different wavelengths [compared
 with native luciferase], and thus, may be selected for their
 color-producing characteristics. For example, synthetic mutant beetle
 luciferase(s) and DNA encoding such luciferases that produce bioluminesce
 at a wavelength different from wild-type luciferase are known [Promega
 Corp., International PCT Application No. WO95/18853, which is based on
 U.S. application Ser. No. 08/177,081 Jan. 3, 1994]. The mutant beetle
 luciferase has an amino acid sequence differing from that of the
 corresponding wild-type Luciola cruciata [see, e.g., U.S. Pat. Nos.
 5,182,202, 5,219,737, 5,352,598, see, also SEQ ID No.3] by a
 substitution(s) at one or two positions. The mutant luciferase produces a
 bioluminescence with a wavelength of peak intensity that differs by at
 least 1 nm from that produced by wild-type luciferases.
 Other mutant luciferase have also been produced. Mutant luciferases with
 the amino acid sequence of wild-type luciferase, but with at least one
 mutation in which valine is replaced by isoleucine at the amino acid
 number 233, valine by isoleucine at 239, serine by asparagine at 286,
 glycine by serine at 326, histidine by tyrosine at 433 or proline by
 serine at 452 are known [see, eg., U.S. Pat. Nos. 5,219,737, and
 5,330,906]. The luciferases are produced by expressing DNA-encoding each
 mutant luciferase in E. coli and isolating the protein. These luciferases
 produce light with colors that differ from wild-type. The mutant
 luciferases catalyze luciferin to produce red [.lambda.609 nm and 612 nm],
 orange[.lambda.595 and 607 nm] or green [.lambda.558 nm] light. The other
 physical and chemical properties of mutant luciferase are substantially
 identical to native wild type-luciferase. The mutant luciferase has the
 amino acid sequence of Luciola cruciata luciferase with an alteration
 selected from Ser 286 replaced by Asn, Gly 326 replaced by Ser, His 433
 replaced by Tyr or Pro 452 replaced by Ser. Thermostable luciferases are
 also available [see, e.g., U.S. Pat. No. 5,229,285; see, also
 International PCT Application No.@) 95/25798, which provides Photinus
 luciferase in which the glutamate at position 354 is replaced lysine and
 Luciola luciferase in which the glutamate at 356 is replaced with lysine].
 These mutant luciferases as well as the wild type luciferases are among
 those preferred herein, particularly in instances when a variety of colors
 are desired or when stability at higher temperatures is desired. It is
 also noteworthy that firefly luciferases have alkaline pH optima
 [7.5-9.5], and, thus, are suitable for use in combination with articles of
 manufacture, such as the bubble compositions that have alkaline pH.
 b. Luciferin
 The firefly luciferin is a benzothiazole:
 ##STR5##
 Analogs of this luciferin and synthetic firefly luciferins are also known
 to those of skill in art [see, e.g., U.S. Pat. No. 5,374,534 and
 5,098,828]. These include compounds of formula (IV) [see, U.S. Pat. No.
 5,098,828]:
 ##STR6##
 in which:
 R.sup.1 is hydroxy, amino, linear or branched C.sub.1 -C.sub.20 alkoxy,
 C.sub.2 -C.sub.20 alkyenyloxy, an L-amino acid radical bond via the
 .alpha.-amino group, an oligopeptide radical with up to ten L-amino acid
 units linked via the .alpha.-amino group of the terminal unit;
 R.sup.2 is hydrogen, H.sub.2 PO.sub.3, HSO.sub.3, unsubstituted or phenyl
 substituted linear or branched C.sub.1 -C.sub.20 alkyl or C.sub.2
 -C.sub.20 alkenyl, aryl containing 6 to 18 carbon atoms, or R.sup.3
 --C(O)--; and
 R.sup.3 is an unsubstituted or phenyl substituted linear or branched
 C.sub.1 -C.sub.20 alkyl or C.sub.2 -C.sub.20 alkenyl, aryl containing 6 to
 18 carbon atoms, a nucleotide radical with 1 to 3 phosphate groups, or a
 glycosidically attached mono- or disaccharide, except when formula (IV) is
 a D-luciferin or D-luciferin methyl ester.
 c. Reaction
 The reaction catalyzed by firefly luciferases and related insect
 luciferases requires ATP, Mg.sup.2+ as well as molecular oxygen. Luciferin
 must be added exogenously. Firefly luciferase catalyzes the firefly
 luciferin activation and the subsequent steps leading to the excited
 product. The luciferin reacts with ATP to form a luciferyl adenylate
 intermediate. This intermediate then reacts with oxygen to form a cyclic
 luciferyl peroxy species, similar to that of the coelenterate intermediate
 cyclic peroxide, which breaks down to yield CO.sub.2 and an excited state
 of the carbonyl product. The excited molecule then emits a yellow light;
 the color, however, is a function of pH. As the pH is lowered the color of
 the bioluminescence changes from yellow-green to red.
 Different species of fireflies emit different colors of bioluminescence so
 that the color of the reaction will be dependent upon the species from
 which the luciferase is obtained. Additionally, the reaction is optimized
 at pH 7.8.
 Addition of ATP and luciferin to a reaction that is exhausted produces
 additional light emission. Thus, the system, once established, is
 relatively easily maintained. Therefore, it is highly suitable for use
 herein in embodiments in which a sustained glow is desired or reuse of the
 item is contemplated. Thus, the components of a firefly system can be
 packaged with the item of manufacture, such as a toy gun, and then
 combined with the article before use. For example, the luciferin and ATP
 can be added to a mild bubble solution that contains luciferase each time
 the bubbles are used.
 5. Bacterial systems
 Luminous bacteria typically emit a continuous light, usually blue-green.
 When strongly expressed, a single bacterium may emit 10.sup.4 to 10.sup.5
 photons per second. Bacterial bioluminescence systems include, among
 others, those systems found in the bioluminescent species of the genera
 Photobacterium, Vibrio and Xenorhabdus. These systems are well known and
 well characterized [see, e.g., Baldwin et al. (1984) Biochemistry
 23:3663-3667; Nicoli et al. (1974) J. Biol. Chem. 249:2393-2396; Welches
 et al. (1981) Biochemistry 20:512-517; Engebrecht et al. (1986) Methods in
 Enzymology 133:83-99; Frackman et al. (1990) J. of Bacteriology
 172:5767-5773; Miyamoto et al. (1986) Methods in Enzymology 133:70; U.S.
 Pat. No. 4,581,335].
 a. Luciferases
 Bacterial luciferase, as exemplified by luciferase derived from Vibrio
 harveyi [EC 1.14.14.3, alkanol reduced-FMN-oxygen oxidoreductase
 1-hydroxylating, luminescing], is a mixed function oxidase, formed by the
 association of two different protein subunits .alpha. and .beta.. The
 .alpha.-subunit has an apparent molecular weight of approximately 42,000
 kD and the .beta.-subunit has an apparent molecular weight of
 approximately 37,000 kD [see, e.g.., Cohn et al. (1989) Proc. Natl. Acad.
 Sci. U.S.A. 90:102-123]. These subunits associate to form a 2-chain
 complex luciferase enzyme, which catalyzes the light emitting reaction of
 bioluminescent bacteria, such as Vibrio harveyi [U.S. Pat. No. 4,581,335;
 Belas et al. (1982) Science 218:791-793], Vibrio fischeri [Engebrecht et
 al. (1983) Cell 32:773-781; Engebrecht et al. (1984) Proc. Natl. Acad.
 Sci. U.S.A. 81:4154-4158] and other marine bacteria.
 Bacterial luciferase genes have been closed [see, e.g., U.S. Pat. No.
 5,221,623; U.S. Pat. No. 4,581,335; European Patent Application No. EP 386
 691 A]. Plasmids for expression of bacterial luciferase, such as Vibrio
 harveyi, include pFIT001 (NRRL B-18080), pE001 (NRRL B-18082) and pMR19
 (NRRL B-18081)] are known. For example the sequence of the entire lux
 regulon from Vibiro fisheri has been determined [Baldwin et al. (1984),
 Biochemistry 23:3663-3667; Baldwin et al. (1981) Biochem. 20: 512-517;
 Baldwin et al. (1984) Biochem. 233663-3667; see, also, e.g., U.S. Pat.
 Nos. 5,196,318, 5,221,623, and 4,581,335]. This regulon includes luxl
 gene, which encodes a protein required for autoinducer synthesis [see,
 e.g., Engebrecht et al. (1984) Proc. Natl. Acad. Sci. U.S.A.
 81:4154-4158], the luxC, luxD, and luxE genes, which encode enzymes that
 provide the luciferase with an aldehyde substrate, and the luxA and luxB
 genes, which encode the alpha and beta subunits of the luciferase.
 Lux genes from other bacteria have also been cloned and are available [see,
 e.g., Cohn et al. (1985) J. Biol. Chem. 260:6139-6146; U.S. Pat. No.
 5,196,524, which provides a fusion of the luxA and luxB genes from Vibrio
 harveyl]. Thus, luciferase alpha and beta subunit-encoding DNA is provided
 and can be used to produce the luciferase. DNA encoding the .alpha.[1065
 bp] and .beta. [984 bp] subunits, DNA encoding a luciferase gene of 2124
 bp, encoding the alpha and beta subunits, a recombinant vector containing
 DNA encoding both subunits and a transformed E. coli and other bacterial
 hosts for expression and production of the encoded luciferase are
 available. In addition, bacterial luciferases are commercially available.
 b. Luciferins
 Bacterial luciferins include:
 ##STR7##
 in which the tetradecanal with reduced flavin mononucleotide are considered
 luciferin since both are oxidized during the light emitting reaction.
 c. Reactions
 The bacterial systems require, in addition to reduced flavin, five
 polypeptides to complete the bioluminescent reaction: two subunits,
 .alpha. and .beta., of bacterial luciferin and three units of a fatty acid
 reductase system complex, which supplies the tetradecanal aldehyde.
 Examples of bacterial bioluminescent systems useful in the apparatus and
 methods provided herein include those derived from Vibrio fisheri and
 Vibrio harveyi. One advantage to this system is its ability to operate at
 cold temperatures. It will thus be particularly amenable to use in ice
 cubes. All components of a bacterial system can be frozen into ice cubes.
 As it the ice cubes melt into a warmer beverage, which has dissolved
 O.sub.2, the reaction will proceed, thereby providing a sustained glow.
 Bacterial luciferase catalyzes the flavin-mediated hydroxylation of a
 long-chain aldehyde to yield carboxylic acid and an excited flavin; the
 flavin decays to ground state with the concomitant emission of blue green
 light [.lambda..sub.max =490 nm; see, e.g., Legocki et al. (1986) Proc.
 Natl. 5Acad. Sci. USA 81:9080; see U.S. Pat. No. 5,196,524]:
 ##STR8##
 The reaction can be initiated by contacting reduced flavin mononucleotide
 [FMNH.sub.2 ] with a mixture of the bacterial luciferase, oxygen, and a
 long-chain aldehyde, usually n-decyl aldehyde.
 DNA encoding luciferase from the fluorescent bacterium Alteromonas hanedai
 is known [CHISSO CORP; see, also, Japanese application JP 7222590,
 published Aug. 22, 1995]. The reduced flavin mononucleotide [FMNH.sub.2 ;
 luciferin] reacts with oxygen in the presence of bacterial luciferase to
 produce an intermediate peroxy flavin. This intermediate reacts with a
 long-chain aldehyde [tetradecanal] to form the acid and the
 luciferase-bound hydroxy flavin in its excited state. The excited
 luciferase-bound hydroxy flavin then emits light and dissociates from the
 luciferase as the oxidized flavin mononucleotide [FMN] and water. In vivo
 FMN is reduced again and recycled, and the aldehyde is regenerated from
 the acid.
 Flavin reductases have been cloned [see, e.g., U.S. Pat. No. 5,484,723;
 see, SEQ ID No. 14 for a representative sequence from this patent]. These
 as well as NAD(P)H can be included in the reaction to regenerate
 FMNH.sub.2 for reaction with the bacterial luciferase and long chain
 aldehyde. The flavin reductase catalyzes the reaction of FMN, which is the
 luciferase reaction, into FMNH.sub.2 ; thus, if luciferase and the
 reductase are included in the reaction system, it is possible to maintain
 the bioluminescent reaction. Namely, since the bacterial luciferase turns
 over many times, bioluminescence continues as long as a long chain
 aldehyde is present in the reaction system.
 The color of light produced by bioluminescent bacteria also results from
 the participation of a protein blue-florescent protein [BFP] in the
 bioluminescence reaction. This protein, which is well known [see, e.g.,
 Lee et al. (1978) Methods in Enzymology LVII:226-234], may also be added
 to bacterial bioluminescence reactions in order to cause a shift in the
 color.
 6. Other systems
 a. Dinoflagellate bioluminescence generating systems
 In dinoflagellates, bioluminescence occurs in organelles termed
 scintillons. These organelles are outpocketings of the cytoplasm into the
 cell vacuole. The scintillons contain only dinoflagellate luciferase and
 luciferin [with its binding protein], other cytoplasmic components being
 somehow excluded. The dinoflagellate luciferin is a tetrapyrrole related
 to chlorophyll:
 ##STR9##
 or an analog thereof.
 The luciferase is a 135 kD single chain protein that is active at pH 6.5,
 but inactive at pH 8 [see, e.g., Hastings (1981) Bioluminescence and
 Chemiluminescence, DeLuca et al., eds. Academic Press, N.Y., pp.343-360].
 Luminescent activity can be obtained in extracts made at pH 8 by simply
 shifting the pH from 8 to 6. This occurs in soluble and particulate
 fractions. Within the intact scintillon, the luminescent flash occurs for
 .about.100 msec, which is the duration of the flash in vivo. In solution,
 the kinetics are dependent on dilution, as in any enzymatic reaction. At
 pH 8, the luciferin is bound to a protein [luciferin binding protein] that
 prevents reaction of the luciferin with the luciferase. At pH 6, however,
 the luciferin is released and free to react with the enzyme.
 b. Systems from molluscs, such as Latia and Pholas
 Molluscs Latia neritoides and species of Pholas are bioluminescent animals.
 The luciferin has the structure:
 ##STR10##
 and has been synthesized [see, eg., Shimomura et al. (1968) Biochemistry
 7:1734-1738; Shimomura et al. (1972) Proc. Natl. Acad. Sci. U.S.A.
 69:2086-2089]. In addition to a luciferase and luciferin the reaction has
 a third component, a "purple protein". The reaction, which can be
 initiated by an exogenous reducing agent is represented by the following
 scheme:
 ##STR11##
 XH.sub.2 is a reducing agent.
 Thus for practice herein, the reaction will require the purple protein as
 well as a reducing agent.
 c. Earthworms and other annelids
 Earthworm species, such as Diplocardia longa, Chaetopterus and Harmothoe,
 exhibit bioluminescence. The luciferin has the structure:
 ##STR12##
 The reaction requires hydrogen peroxide in addition to luciferin and
 luciferase. The luciferase is a photoprotein.
 d. Australian glow worms
 The luciferase/luciferin system from the glow worms that are found in
 Australian and New Zealand caves is also intended for use herein.
 C. Practice of the reactions in combination with articles of manufacture
 The particular manner in which each bioluminescence system will be combined
 with a selected article of manufacture will be a function of the article
 and the desired effect. In general, however, less than all of the
 components of the reaction will be provided with the article and then
 contact with the remaining component(s) to produce a glow. There are a
 multitude of alternative means for achieving this result; some are
 described herein, and others will be apparent by virtue of the disclosure
 herein.
 In the simplest embodiments, the organisms can be ground up and dried. For
 example, light will be emitted by ground up fireflies when mixed with
 water and ATP. Light will also be emitted merely be combining ground up
 Vargula shrimp and adding water, preferably cool water [room temperature
 or lower]. The only caveat is that the water must not be too hot; high
 temperatures destroy activity of the luciferases.
 In other embodiments, the substantially pure reagents are combined with the
 article of manufacture and the article will glow or spew a glowing spray
 or jet. The reagents may be provided in compositions, such as suspensions,
 as powders, as pastes or any in other suitable form. They may be provided
 as sprays, aerosols, or in any suitable form. The reagents may be linked
 to a matrix and combined with the article of manufacture or formed into
 the article of manufacture. Typically all but one or more, though
 preferably all but one, of the components necessary for the reaction will
 be mixed and provided together; reaction will be triggered contacting the
 mixed component(s) with the remaining component(s), such as by adding
 Ca.sup.2+, FMN with reductase, FMNH.sub.2, ATP, air or oxygen.
 In preferred embodiments the luciferase or luciferase/luciferin, such as
 the aequorin photoprotein, will be provided in combination with the
 article of manufacture or added before use. The article will then be
 contacted with the remaining components. As will become apparent herein,
 there are a multitude of ways in which each system may be combined with a
 selected article of manufacture.
 D. Packaging of Bioluminescence Systems
 Packaging for bioluminescent generating reagents provided herein must be
 chosen according to the article of manufacture with which the reagents are
 to be combined. In general, the packaging is non-reactive with the
 compositions contained therein and must exclude water and or air to the
 degree those substances are required for the luminescent reaction to
 proceed. It will be appreciated, however, that specific uses for the
 bioluminescent systems may require specific packaging. Following are some
 examples of the special packaging requirements of various end uses of the
 bioluminescent systems. These are offered as examples only and are in no
 way intended as limiting.
 The bioluminescence generating reagents may be provided in pellets,
 encapsulated as micro or macro-capsules, linked to matrices and included
 in or on articles of manufacture, or as mixtures in chambers within an
 article of manufacture or in some other configuration. With respect to
 other articles of manufacture that include chambers or vessels, such as
 certain toys, primary considerations are that the bioluminescent system be
 amenable to activation by the user at will and that the container be
 non-reactive and, if desired, translucent to the bioluminescent glow.
 Examples of vessels include beverage holders, plates or other dishes,
 vases, jars, bottles and other containers. In general, vessels for use in
 practicing the methods herein have an enclosed, defined space, that
 contains most of the components of the bioluminescent system, and a
 separate enclosed, defined space containing the remaining necessary
 ingredients; such that, the two spaces are separated by a readily
 removable membrane which, upon removal, permits the components to mix and
 thereby react, resulting in illumination. Alternatively, the vessel can
 have a single compartment containing all but the final ingredients of the
 bioluminescent system and being amenable to addition of the final
 ingredients by the user; for example through an opening in the
 compartment.
 Any toy, vessel or other article of manufacture that is amenable to having
 a generally translucent covering defining a space for containment of the
 bioluminescent system components and that is amenable to simple
 manipulation to permit addition of the final components necessary for the
 illumination reaction is contemplated.
 Thus, whether the item that will glow or produce a glowing fluid, jet or
 spray, is a toy, vessel or other article of manufacture, its general
 design is the same. At least one of the bioluminescent system components
 is separated from the remaining components. The remaining components are
 added prior to use. They can be included in the article of manufacture and
 physically separated from the other components. For example, the physical
 separation means are those that are readily removed by the user, to permit
 mixing, resulting in illumination of the components. For example, an
 article of manufacture may contain a luciferase and substrate in one
 compartment and a bioluminescence activator in an adjacent compartment; or
 alternatively, one compartment may contain the luciferase, and the other
 the substrate luciferin and dissolved oxygen or other requisite
 activators. The compartments are separated by a dividing member, such as a
 membrane, that, upon compression of the article of manufacture, ruptures
 permitting separated components to mix and to thereby glow. For suitable
 embodiments, see EXAMPLES, below [see, also, e.g., containers described in
 U.S. Pat. Nos. 3,539,794 and 5,171,081].
 Other embodiments contemplated herein, include those in which a fluid is
 ejected as a spray or jet and is rendered bioluminescent prior to ejection
 from the device, such as a toy or fountain. In general, the methods will
 involve addition of the bioluminescent system components to the water just
 prior to ejection thereby causing the ejected spray or jet or stream to
 glow. Various apparatus for accomplishing this are provided herein. In
 light of the disclosure herein other apparatus can be adapted for such
 use. Examples include chambers within a toy that inject the components
 into a water chamber just prior to ejection of the water, or a clip-on
 device housing the components, perhaps in pre-measured amounts, which is
 attached to the toy and manually or automatically engaged to inject the
 ingredients into a water chamber. Similarly, the water can be introduced
 into a chamber containing the components and then ejected.
 In other embodiments, the components may be packaged as separate
 compositions, that, upon mixing, glow. For example, a composition
 containing luciferase may be provided separately from, and for use with,
 an a separate composition containing a bioluminescence substrate and
 bioluminescence activator. In another instance, luciferase and luciferin
 compositions may be separately provided and the bioluminescence activator
 may be added after, or simultaneously with, mixing of the other two
 compositions.
 Similarly, the luciferase and bioluminescence substrate may be provided in
 a single packaging apparatus, an composition that is a mixture,
 suspension, solution, powder, paste or other suitable composition, that is
 designed to exclude the necessary bioluminescence activator. Upon addition
 of the bioluminescence activator to the remaining components or upon
 addition of the components to the bioluminesce activator, the reaction
 commences and the mixture glows. One example of such a system is "fairy
 dust". In this embodiment the luciferase and bioluminescence substrate,
 for example, are packaged to exclude water and/or air, the bioluminescence
 activator. Release of the components from the packaging into the air
 and/or moisture in the air activates the components thereby generating
 luminescence. Another example is packaging the luciferase and substrate in
 the cap apparatus of a vessel, such that operation of the cap apparatus
 releases the components into the composition contained in the vessel,
 causing it to glow.
 1. Dispensing and Packaging Apparatus for Combination with the
 Bioluminescent System Components
 In one aspect, the bioluminescent apparatus systems provided herein are
 bioluminescence [or bioluminescent] systems in combination with dispensing
 or packaging apparatus. The bioluminescence systems, described in detail
 elsewhere herein, include three components: a bioluminescence substrate
 [e.g., a luciferin], a luciferase [e.g., a luciferase or photoprotein],
 and a bioluminescence activator or activators [e.g., molecular oxygen or
 Ca.sup.2+ ]. The dispensing and packaging apparatus are configured to keep
 at least one of the three components separate from the other two
 components, until generation of bioluminescence is desired.
 In general, the dispensing and packaging apparatus are non-reactive with
 the bioluminescent system components contained therein and can exclude
 moisture, air or other activators, such as O.sub.2 or Ca.sup.2+, or in
 some manner keep all necessary components that are required for the
 bioluminescent reaction to come into contact until desired.
 It will be appreciated, however, that specific applications and
 configurations of the bioluminescence systems may require specific
 apparatus. Following are exemplary descriptions of various dispensers and
 packages contemplated for use herein. These are offered as examples only
 and are in no way intended as limiting. It is understood that in light of
 the description herein, other apparatus may be modified or devised, that
 would be suitable for use to produce bioluminescence in combination with
 novelty items.
 2. Capsules, pellets, liposomes, endosomes, vacuoles, micronized particles
 Certain embodiments of the novelty item combinations provided herein, such
 as beverage and foods and particles, such as for use as fairy dust or toy
 guns, fountains of particles and other such applications in require
 sequestering the components from the environment prior to use or that
 require particulate form. For example, embodiments in which the
 bioluminescence generating system is manufactured as part of food or
 beverage producing glowing beverages or foods require specific packaging
 considerations. To be amenable to use as an additive to beverages for
 human consumption, the packaging must be non-toxic, and should be easy to
 open to provide for contact of the bioluminescence generating system
 components with the beverage. Examples of suitable packaging for such use
 include encapsulating the bioluminescent system components in one or
 micro- [up to about 100 .mu.m in size] or macroparticles [larger than 100
 .mu.M] of material that permits release of the contents, such as by
 diffusion or by dissolution of the encapsulating material. Liposomes and
 other encapsulating vehicles [see, e.g., U.S. Pat. No. 4,525,306, which
 describes encapsulation of compounds in gelatin; U.S. Pat. Nos. 4,021,364,
 4,225,581, 4,269,821, 4,322,311, 4,324,683, 4,329,332, 4,525,306,
 4,963,368 describe encapsulation of biologically active materials in
 various polymers] known to those of skill in the art, including those
 discussed herein and known to those of skill in the art [such as soluble
 paper, see U.S. Pat. No. 3,859,125]. Likewise, packaging of the system
 components for addition to food products must address the same
 considerations. The components may be added to the food substance
 directly, e.g., by sprinkling the dried and powdered ingredients onto the
 food, or indirectly, e.g., via addition, to the food, of a capsule
 containing the ingredients.
 a. Encapsulating vehicles in general
 All components of the bioluminescence generating system, except for the
 oxygen or water or Ca.sup.2+, depending upon the selected system can be
 incorporated into encapsulating material, such as liposomes, that protect
 the contents from the environment until placed into conditions that cause
 release of the contents into the environment. Encapsulating material
 contemplated for use herein includes liposomes and other such materials
 used for encapsulating chemicals, such as drug delivery vehicles.
 b. Encapsulating vehicles -liposomes
 For example, liposomes that dissolve and slowly release the components into
 the selected beverage, which contains dissolved oxygen or Ca.sup.2+ or
 even ATP for the luciferase system are contemplated herein. They can be
 formulated in compositions, such as solutions, suspensions, gels, lotions,
 creams, and ointments. Liposomes and other slow release encapsulating
 compositions are well known and can be adapted for use in for slow release
 delivery of bioluminescence generating components. Typically the luciferin
 and luciferase will be encapsulated in the absence of oxygen or Ca.sup.2+
 or ATP or other activating component. Upon release into the environment or
 medium containing this component at a suitable concentration, the reaction
 will proceed and a glow will be produced. Generally the concentrations of
 encapsulated components should be relatively high, perhaps 0.1-1 mg/ml or
 more, to ensure high enough local concentrations upon release to be
 visible.
 Liposomes or other sustained release delivery system that are formulated in
 an ointment or sustained release topical vehicle, for example, would be
 suitable for use in a body paint, lotion. Those formulated as a suspension
 would be useful as a spray. Numerous ointments and suitable liposome
 formulations are known [see, e.g., Liposome Technology, Targeted Drug
 Delivery and Biological Interaction, vol. III, G. Gregoriadis ed., CRC
 Press, Inc., 1984; U.S. Pat. Nos. 5,470,881; 5,366,881; 5,296,231;
 5,272,079; 5,225,212; 5,190,762; 5,188,837; 5,188,837; 4,921,757;
 4,522,811]. For example, an appropriate ointment vehicle would contain
 petrolatum, mineral oil and/or anhydrous liquid lanolin. Sustained release
 vehicles such as liposomes, membrane or contact lens delivery systems, or
 gel-forming plastic polymers would also be suitable delivery vehicles.
 Liposomes for topical delivery are well known [see, e.g., U.S. Pat. No.
 5,296,231; Mezei et al. (1980) "Liposomes -A selective drug delivery
 system for the topical route of administration, l. lotion dosage form"
 Life Sciences 26:1473-1477; Mezei et al. (1981) "Liposomes -A selective
 drug delivery system for the topical route of administration: gel dosage
 form" Journal of Pharmacy and Pharmacology 34:473-474; Gesztes et al.
 (1988) "Topical anaesthesia of the skin by liposome -encapsulated
 tetracaine" Anesthesia and Analgesia 67:1079-1081; Patel (1985) "Liposomes
 as a controlled-release system", Biochemical Soc. Trans. 13:513-516;
 Wohirab et al. (1987) "Penetration kinetics of liposomal hydrocortisone in
 human skin" Dermatologica 174:18-22].
 Liposomes are microcapsules [diameters typically on the order of less than
 0.1 to 20.mu.m] that contain selected mixtures and can slowly release
 their contents in a sustained release fashion. Liposomes or other capsule,
 particularly a time release coating, that dissolve upon exposure to
 oxygen, air, moisture, visible or ultraviolet [UV] light or a particular
 pH or temperature [see, e.g., U.S. Pat. No. 4,882,165; Kusumi et al.
 (1989) Chem. Lett. no.3 433-436; Koch Troels et al. (1990) Bioconiugate
 Chem. 4:296-304; U.S. Pat. No. 5,482,719; U.S. Pat. No. 5,411,730; U.S.
 Pat. No. 4,891,043; Straubinger et al. (1983) Cell 32:1069-1079; and
 Straubinger et al. (1985) FEBS Lttrs. 179:148-154; and Duzgunes et al. in
 Chapter 11 of the book CELL FUSION, edited by A. E. Sowers; Ellens et al
 (1984) Biochemistry 23:1532-1538; Yatvin et al. (1987) Methods in
 Enzymology 149:77-87] may be used for example in the squirt guns or toy
 machine guns or fairy dust. Liposome formulations for use in baking [see,
 e.g., U.S. Pat. No. 4,999,208] are available. They release their contents
 when eaten or heated. Such liposomes may be suitable for incorporation
 into food products herein or in embodiments in which release of the
 components by heating is desired.
 Liposomes be prepared by methods known to those of skill in the art [see,
 e.g., Kimm et al. (1983) Bioch. Bioph. Acta 728:339-398; Assil et al.
 (1987) Arch Ophthalmol. 105:400; and U.S. Pat. No. 4,522,811, and other
 citations herein and known to those of skill in the art].
 Liposomes that are sensitive to low pH [see, e.g., U.S. Pat. No. 5,352,448,
 5,296,231; 5,283,122; 5,277,913, 4,789,633] are particularly suitable for
 addition to bath powders or to bubble compositions, just prior to use.
 Upon contact with the low pH detergent or soap composition or a high pH
 composition, the contents of the liposome will be released. Other
 components, particularly Ca.sup.+ or the presence of dissolved O.sub.2 in
 the water will cause the components to glow as they are released.
 Temperature sensitive liposomes are also suitable for use in bath powders
 for release into the warm bath water.
 c. Encapsulating vehicles -gelatin and polymeric vehicles
 Macro or microcapsules made of gelatin or other such polymer that dissolve
 or release their contents in a beverage or food or on contact with air or
 light or changes in temperature may also be used to encapsulate components
 of the bioluminescence generating systems.
 Such microcapsules or macrocapsules may also be incorporated into solid
 soaps, such that as the soap dissolves the incorporated capsules or
 pellets release their contents, which glow upon contact with the water in
 which the soap is placed.
 The aequorin system is particularly suitable for this application. It can
 be encapsulated in suspension or solution or as a paste, or other suitable
 form, of buffer with sufficient chelating agent, such as EDTA, to prevent
 discharge of the bioluminescence. Upon exposure of the capsule
 [microcapsule or macrocapsule] to moisture that contains Ca.sup.2+, such
 as in a food or beverage, a two chamber apparatus or single chamber
 apparatus, such as described herein, or even in a moist environment
 containing Ca.sup.2+, the slowly released components will glow.
 Thus, encapsulated bioluminescence generating components can be used in
 combination with food, beverages, as bullets or pellets, as "fairy dust"
 [pellets that dissolve upon exposure to light and thereby release the
 luciferase/luciferin, such as the Renilla system, which will light upon
 exposure to air], and other such items.
 Other encapsulating containers or vehicles for use with the bioluminescence
 systems are those that dissolve sufficiently in water to release their
 contents, or that are readily opened when squeezed in the hand or from
 which the contents diffuse when mixed with a aqueous mixture. These
 containers can be made to exclude water, so that the bioluminescent system
 components may be desiccated and placed therein. Upon exposure to water,
 such as in an aqueous composition solution or in the atmosphere, the
 vehicle dissolves or otherwise releases the contents, and the components
 react and glow.
 These capsules may be formed from gelatin or similar water soluble
 material. If the packaging is to be added to food or beverage, then it
 should be chosen to be non-toxic, non-reactive and flavorless. To be
 readily opened by hand, the packaging may be constructed of thin plastic
 or may be configured in two halves which form an airtight seal when joined
 but which are readily separated when release of the components is desired.
 In one aspect, these capsular embodiments of the packaging apparatus is
 contemplated for use as an additive to beverages, creams, sauces, gelatins
 or other liquids or semi-solids. In another aspect, it is contemplated
 that the contents of the packaging apparatus is released into the air
 whereby it glows upon contact with the moisture of the atmosphere.
 d. Endosomes and vacuoles
 Vehicles may be produced using endosomes or vacuoles from recombinant host
 cells in which the luciferase is expressed using method known to those of
 skill in the art [see, e.g., U.S. Pat. Nos. 5,284,646, 5,342,607,
 5,352,432, 5,484,589, 5,192,679, 5,206,161, and 5,360,726]. For example,
 aequorin that is produced by expression in a host, such as E. coli, can be
 isolated within vesicles, such as endosomes or vacuoles, after protein
 synthesis. Using routine methods the cells are lysed and the vesicles are
 released with their contents intact. The vesicles will serve as delivery
 vehicles. When used they will be charged with a luciferin, such as a
 coelentrazine, and dissolved oxygen, such as by diffusion, under pressure,
 or other appropiate means.
 e. Micronized particles
 The bioluminescence generating system components that are suitable for
 lyophilization, such as the aequorin photoprotein, the Renilla system, and
 the Vargula systems, can be micronized to form fine powder and stored
 under desiccating conditions, such as with a desiccant. When used the fine
 powder can be combined with the selected article of manufacture, such as a
 personal item, a chamber in a gun or fountain, or used as fairy dust.
 Contact with dissolved oxygen or Ca.sup.2+ in the air or in a mist that
 can be supplied or in added solution will cause the particles to dissolve
 and glow.
 3. Apparatus and substrates
 The combinations herein are produced by combining a selected novelty item
 and combining it with a system and apparatus for producing
 bioluminescence. Selection of the system depends upon factors such as the
 desired color and duration of the bioluminescence desired as well as the
 particular item. Selection of the apparatus primarily depends upon the
 item with which it is combined.
 Among the simplest embodiments herein, are those in which the apparatus
 contains a single chamber [vessel] or matrix material and, if needed,
 ejection means. Components, generally all but at least one necessary
 component, typically the activator as defined herein, of the
 bioluminescence reaction are introduced into the housing or vessel or onto
 the substrate as a mixture in liquid phase or as a powder or other paste
 or other convenient composition. Prior to use the final component(s) is
 added or the other components are contacted with the final component(s).
 a. Matrix materials
 For preparation of combinations of articles of manufacture such as clothing
 , paper, items fabricated from a textile, plastic, glass ceramic or other
 such material, at least one component of the bioluminescence generating
 system is linked to the matrix substrate. When desired, a mixture or
 mixtures(s) containing the remaining component(s(, typically a liquid
 mixture is applied, as by pouring or spraying onto the matrix substrate,
 to produce a glow. For example, the aequorin photoprotein, including
 coelentrazine and oxygen, is linked to the substrate. When desired a
 liquid containing Ca.sup.2+, such as tap water or, preferably, a liquid
 mixture containing the Ca.sup.2+ in an appropriate buffer, is contacted,
 such as by spraying, with the matrix with linked luciferase. Upon
 contacting the material glows.
 In other embodiments, the luciferase, such as a Vargula luciferase, is
 linked to the substrate material, and contacted with a liquid mixture
 containing the luciferin in an appropriate buffer. Contacting can be
 effected by spraying or pouring or other suitable manner. The matrix
 material is incorporated into, onto or is formed into an article of
 manufacture, such as clothing or a ceramic, glass, plastic figurine, toy,
 balloon, flocking agent, such as a Christmas tree flocking agent, or other
 item. The resulting novelty item can be sold as a kit with a container of
 the mixture containing the non-linked components, such as in a canister,
 spray bottle or can, or other suitable format.
 The kits may also include containers containing compositions of the linked
 components which can be provided in a form, such as sprayed on as a liquid
 and air dried, that can be applied to the substrate so that the item can
 be made to glow again. Thus, kits containing a substrate, such as clothing
 or a plastic, ceramic or glass item, and a first composition containing a
 luciferase or a luciferin or both and luciferin, and a second composition
 containing the remaining components. The item as provided in the kit can
 be charged with the first composition, such as having the composition
 applied and dried, or may require charging prior to the first use.
 Alternatively, the item may be sprayed with both compositions when desired
 to produce a glow.
 It is understood that the precise components and optimal means for
 application or storage are a function of the selected bioluminescence
 system. The concentrations of the components, which can be determined
 empirically, are not critical, but must be sufficient to produce a visible
 glow when combined. Typical concentrations are as low as nanomoles/l,
 preferably on the order of mg/l or higher. The concentration on the
 substrate is that produced when a composition containing such typical
 concentration is applied to the material. Again, such ideal concentrations
 can be readily determined empirically by applying the first composition,
 letting it dry, spraying the second composition, and observing the result.
 The matrix material substrates contemplated herein are generally insoluble
 materials used to immobilize ligands and other molecules, and are those
 that used in many chemical syntheses and separations. Such substrates,
 also called matrices, are used, for example, in affinity chromatography,
 in the immobilization of biologically active materials, and during
 chemical syntheses of biomolecules, including proteins, amino acids and
 other organic molecules and polymers. The preparation of and use of
 matrices is well known to those of skill in this art; there are many such
 materials and preparations thereof known. For example, naturally-occurring
 matrix materials, such as agarose and cellulose, may be isolated from
 their respective sources, and processed according to known protocols, and
 synthetic materials may be prepared in accord with known protocols.
 The substrate matrices are typically insoluble materials that are solid,
 porous, deformable, or hard, and have any required structure and geometry,
 including, but not limited to: beads, pellets, disks, capillaries, hollow
 fibers, needles, solid fibers, random shapes, thin films and membranes.
 Thus, the item may be fabricated from the matrix material or combined with
 it, such by coating all or part of the surface or impregnating particles.
 Typically, when the matrix is particulate, the particles are at least about
 10-2000 .mu.M, but may be smaller or larger, depending upon the selected
 application. Selection of the matrices will be governed, at least in part,
 by their physical and chemical properties, such as solubility, functional
 groups, mechanical stability, surface area swelling propensity,
 hydrophobic or hydrophilic properties and intended use.
 If necessary the support matrix material can be treated to contain an
 appropriate reactive moiety or in some cases the may be obtained
 commercially already containing the reactive moiety, and may thereby serve
 as the matrix support upon which molecules are linked. Materials
 containing reactive surface moieties such as amino silane linkages,
 hydroxyl linkages or carboxysilane linkages may be produced by well
 established surface chemistry techniques involving silanization reactions,
 or the like. Examples of these materials are those having surface silicon
 oxide moieties, covalently linked to gamma-aminopropylsilane, and other
 organic moieties; N-[3-(triethyoxysilyl)propyl]phthelamic acid; and
 bis-(2-hydroxyethyl)aminopropyltriethoxysilane. Exemplary of readily
 available materials containing amino group reactive functionalities,
 include, but are not limited to, para-aminophenyltriethyoxysilane. Also
 derivatized polystyrenes and other such polymers are well known and
 readily available to those of skill in this art [e.g., the Tentagel.RTM.
 Resins are available with a multitude of functional groups, and are sold
 by Rapp Polymere, Tubingen, Germany; see, U.S. Pat. No. 4,908,405 and U.S.
 Pat. No. 5,292,814; see, also Butz et al. (1994) Peptide Res. 7:20-23;
 Kleine et al. (1994) Immunobiol. 190:53-66].
 These matrix materials include any material that can act as a support
 matrix for attachment of the molecules of interest. Such materials are
 known to those of skill in this art, and include those that are used as a
 support matrix. These materials include, but are not limited to,
 inorganics, natural polymers, and synthetic polymers, including, but are
 not limited to: cellulose, cellulose derivatives, acrylic resins, glass,
 silica gels, polystyrene, gelatin, polyvinyl pyrrolidone, co-polymers of
 vinyl and acrylamide, polystyrene cross-linked with divinylbenzene or the
 like [see, Merrifield (1964) Biochemistry 3:1385-1390], polyacrylamides,
 latex gels, polystyrene, dextran, polyacrylamides, rubber, silicon,
 plastics, nitrocellulose, celluloses, natural sponges. Of particular
 interest herein, are highly porous glasses [see, e.g., U.S. Pat. No.
 4,244,721] and others prepared by mixing a borosilicate, alcohol and
 water.
 Synthetic matrices include, but are not limited to: acrylamides,
 dextran-derivatives and dextran co-polymers, agarose-polyacrylamide
 blends, other polymers and co-polymers with various functional groups,
 methacrylate derivatives and co-polymers, polystyrene and polystyrene
 copolymers [see, e.g., Merrifield (1964) Biochemistry 3:1385-1390; Berg et
 al. (1990) in Innovation Perspect. Solid Phase Synth. Collect. Pap., Int.
 Symp., 1st, Epton, Roger (Ed), pp. 453-459; Berg et al. (1989) in Pept.,
 Proc. Eur. Pept. Symp., 20th, Jung, G. et al. (Eds), pp. 196-198; Berg et
 al. (1989) J. Am. Chem. Soc. 111:8024-8026; Kent et al. (1979) lsr. J.
 Chem. 17:243-247; Kent et al. (1978) J. Org. Chem. 43:2845-2852; Mitchell
 et al. (1976) Tetrahedron Lett. 42:3795-3798; U.S. Pat. No. 4,507,230;
 U.S. Pat. No. 4,006,117; and U.S. Pat. No. 5,389,449]. Methods for
 preparation of such matrices are well-known to those of skill in this art.
 Synthetic matrices include those made from polymers and co-polymers such as
 polyvinylalcohols, acrylates and acrylic acids such as
 poly-ethylene-co-acrylic acid, polyethylene-co-methacrylic acid,
 polyethylene-co-ethylacrylate, polyethylene-co-methyl acrylate,
 polypropylene-co-acrylic acid, polypropylene-co-methyl-acrylic acid,
 polypropylene-co-ethylacrylate, polypropylene-co-methyl acrylate,
 polyethylene-co-vinyl acetate, polypropylene-co-vinyl acetate, and those
 containing acid anhydride groups such as polyethylene-co-maleic anhydride,
 polypropylene-co-maleic anhydride and the like. Liposomes have also been
 used as solid supports for affinity purifications [Powell et al. (1989)
 Biotechnol. Bioeng. 33:173].
 For example, U.S. Pat. No. 5,403,750, describes the preparation of
 polyurethane-based polymers. U.S. Pat. No. 4,241,537 describes a plant
 growth medium containing a hydrophilic polyurethane gel composition
 prepared from chain-extended polyols; random copolymerization is preferred
 with up to 50% propylene oxide units so that the prepolymer will be a
 liquid at room temperature. U.S. Pat. No. 3,939,123 describes lightly
 crosslinked polyurethane polymers of isocyanate terminated prepolymers
 containing poly(ethyleneoxy) glycols with up to 35% of a
 poly(propyleneoxy) glycol or a poly(butyleneoxy) glycol. In producing
 these polymers, an organic polyamine is used as a crosslinking agent.
 Other matrices and preparation thereof are described in U.S. Pat. Nos.
 4,177,038, 4,175,183, 4,439,585, 4,485,227, 4,569,981, 5,092,992,
 5,334,640, 5,328,603.
 U.S. Pat. No. 4,162,355 describes a polymer suitable for use in affinity
 chromatography, which is a polymer of an aminimide and a vinyl compound
 having at least one pendant halo-methyl group. An amine ligand, which
 affords sites for binding in affinity chromatography is coupled to the
 polymer by reaction with a portion of the pendant halo-methyl groups and
 the remainder of the pendant halo-methyl groups are reacted with an amine
 containing a pendant hydrophilic group. A method of coating a substrate
 with this polymer is also described. An exemplary aminimide is
 1,1-dimethyl-1-(2-hydroxyoctyl)amine methacrylimide and vinyl compound is
 a chloromethyl styrene.
 U.S. Pat. No. 4,171,412 describes specific matrices based on hydrophilic
 polymeric gels, preferably of a macroporous character, which carry
 covalently bonded D-amino acids or peptides that contain D-amino acid
 units. The basic support is prepared by copolymerization of hydroxyalkyl
 esters or hydroxyalkylamides of acrylic and methacrylic acid with
 crosslinking acrylate or methacrylate comonomers are modified by the
 reaction with diamines, aminoacids or dicarboxylic acids and the resulting
 carboxyterminal or aminoterminal groups are condensed with D-analogs of
 aminoacids or peptides. The peptide containing D-amino-acids also can be
 synthesized stepwise on the surface of the carrier.
 U.S. Pat. No. 4,178,439 describes a cationic ion exchanger and a method for
 preparation thereof. U.S. Pat. No. 4,180,524 describes chemical syntheses
 on a silica support.
 Immobilized Artificial Membranes [IAMs; see, e.g., U.S. Pat. Nos. 4,931,498
 and 4,927,879] may also be used. IAMs mimic cell membrane environments and
 may be used to bind molecules that preferentially associate with cell
 membranes [see, e.g., Pidgeon et al. (1990) Enzyme Microb. Technol.
 12:149].
 These materials are also used for preparing articles of manufacture, such
 as toys, balloons, figurines, sponges, knick-knacks, key chains, clothing,
 translucent or transparent soaps, preferably mild soaps, and other items,
 and thus are amenable to linkage of molecules, either the luciferase,
 luciferin, mixtures of both.
 For example, matrix particles may be impregnated into items that will then
 be contacted with an activator. For example, matrix particles with linked
 luciferin, preferably a luciferin/luciferase complex, such as the aequorin
 photoprotein is incorporated into a transparent or translucent soaps [see,
 e.g., U.S. Pat. Nos. 4,081,394, 5,183,429, and 5,141,664, and United
 Kingdom Patent No. GB 2,235,931A], preferably a mild soap. Upon contacting
 the soap with water matrix particles near the surface will glow.
 Kits containing the item including the matrix material with or without the
 coating of the bioluminescence generating components, and compositions
 containing the remaining components are provided.
 b. Immobilization and activation
 Numerous methods have been developed for the immobilization of proteins and
 other biomolecules onto solid or liquid supports [see, e.g., Mosbach
 (1976) Methods in Enzymology 44; Weetall (1975) Immobilized Enzymes,
 Antigens, Antibodies, and Peptides pp. 1-48 Marcel Decker, Inc., N.Y.; and
 Kennedy et al. (1983) Solid Phase Biochemistry, Analytical and Synthetic
 Aspects, Scouten, ed., pp. 253-391; see, generally, Affinity Techniques.
 Enzyme Purification: Part B. Methods in Enzymology, Vol. 34, ed. W. B.
 Jakoby, M. Wilchek, Acad. Press, N.Y. (1974); Immobilized Biochemicals and
 Affinity Chromatography, Advances in Experimental Medicine and Biology,
 vol. 42, ed. R. Dunlap, Plenum Press, N.Y. (1974)].
 Among the most commonly used methods are absorption and adsorption or
 covalent binding to the support, either directly or via a linker, such as
 numerous disulfide linkages, thioether bonds, hindered disulfide bonds,
 and covalent bonds between free reactive groups, such as amine and thiol
 groups, known to those of skill in art [see, e.g, the PIERCE CATALOG,
 ImmunoTechnology Catalog & Handbook, 1992-1993, which describes the
 preparation of and use of such reagents and provides a commercial source
 for such reagents; and Wong (1993) Chemistry of Protein Conjugation and
 Cross Linking, CRC Press; see, also DeWitt et al. (1993) Proc. Natl. Acad.
 Sci. U.S.A. 90:6909; Zuckermann et al. (1992) J. Am. Chem. Soc. 114:10646;
 Kurth et al. (1994) J. Am. Chem. Soc. 116:2661; Ellman et al. (1994) Proc.
 Natl. Acad. Sci. U.S.A. 91:4708; Sucholeiki (1994) Tetrahedron Lttrs.
 35:7307; and Su-Sun Wang (1976) J. Org. Chem. 41:3258; Padwa et al. (1971)
 J. Org. Chem. 41:3550 and Vedejs et al. (1 984) J. Org. Chem. 49:575,
 which describe photosensitive linkers].
 To effect immobilization, a solution of the protein or other biomolecule is
 contacted with a support material such as alumina, carbon, an ion-exchange
 resin, cellulose, glass or a ceramic. Fluorocarbon polymers have been used
 as supports to which biomolecules have been attached by adsorption [see,
 U.S. Pat. No. 3,843,443; Published International PCT Application WO/86
 03840].
 A large variety of methods are known for attaching biological molecules,
 including proteins and nucleic acids, to solid supports [see. e.g., U.S.
 Pat. No. 5,451,683]. For example, U.S. Pat. No. 4,681,870 describes a
 method for introducing free amino or carboxyl groups onto a silica matrix.
 These groups may subsequently be covalently linked to other groups, such
 as a protein or other anti-ligand, in the presence of a carbodiimide.
 Alternatively, a silica matrix may be activated by treatment with a
 cyanogen halide under alkaline conditions. The anti-ligand is covalently
 attached to the surface upon addition to the activated surface. Another
 method involves modification of a polymer surface through the successive
 application of multiple layers of biotin, avidin and extenders [see, e.g.,
 U.S. Pat. No. 4,282,287]; other methods involve photoactivation in which a
 polypeptide chain is attached to a solid substrate by incorporating a
 light-sensitive unnatural amino acid group into the polypeptide chain and
 exposing the product to low-energy ultraviolet light [see, e.g., U.S. Pat.
 No. 4,762,881]. Oligonucleotides have also been attached using a
 photochemically active reagents, such as a psoralen compound, and a
 coupling agent, which attaches the photoreagent to the substrate [see,
 e.g., U.S. Pat. No. 4,542,102 and U.S. Pat. No. 4,562,157].
 Photoactivation of the photoreagent binds a nucleic acid molecule to the
 substrate to give a surface-bound probe.
 Covalent binding of the protein or other biomolecule or organic molecule or
 biological particle to chemically activated solid matrix supports such as
 glass, synthetic polymers, and cross-linked polysaccharides is a more
 frequently used immobilization technique. The molecule or biological
 particle may be directly linked to the matrix support or linked via
 linker, such as a metal [see, e.g., U.S. Pat. No. 4,179,402; and Smith et
 al. (1992) Methods: A Companion to Methods in Enz. 4:73-78]. An example of
 this method is the cyanogen bromide activation of polysaccharide supports,
 such as agarose. The use of perfluorocarbon polymer-based supports for
 enzyme immobilization and affinity chromatography is described in U.S.
 Pat. No. 4,885,250. In this method the biomolecule is first modified by
 reaction with a perfluoroalkylating agent such as
 perfluorooctylpropylisocyanate described in U.S. Pat. No. 4,954,444. Then,
 the modified protein is adsorbed onto the fluorocarbon support to effect
 immobilization.
 The activation and use of matrices are well known and may be effected by
 any such known methods [see, e.g., Hermanson et al. (1992) Immobilized
 Affinity Ligand Techniques, Academic Press, Inc., San Diego]. For example,
 the coupling of the amino acids may be accomplished by techniques familiar
 to those in the art and provided, for example, in Stewart and Young, 1984,
 Solid Phase Synthesis, Second Edition, Pierce Chemical Co., Rockford.
 Other suitable methods for linking molecules to solid supports are well
 known to those of skill in this art [see, e.g., U.S. Pat. No. 5,416,193].
 These include linkers that are suitable for chemically linking molecules,
 such as proteins, to supports and include, but are not limited to,
 disulfide bonds, thioether bonds, hindered disulfide bonds, and covalent
 bonds between free reactive groups, such as amine and thiol groups. These
 bonds can be produced using heterobifunctional reagents to produce
 reactive thiol groups on one or both of the moieties and then reacting the
 thiol groups on one moiety with reactive thiol groups or amine groups to
 which reactive maleimido groups or thiol groups can be attached on the
 other. Other linkers include, acid cleavable linkers, such as
 bismaleimideothoxy propane, acid labile-transferrin conjugates and adipic
 acid diihydrazide, that would be cleaved in more acidic intracellular
 compartments; cross linkers that are cleaved upon exposure to UV or
 visible light and linkers, such as the various domains, such as C.sub.H 1,
 C.sub.H 2, and C.sub.H 3, from the constant region of human IgG.sub.1
 (see, Batra et al. (1993) Molecular Immunol. 30:379-386). Presently
 preferred linkages are direct linkages effected by adsorbing the molecule
 to the surface of the matrix. Other linkages are photocleavable linkages
 that can be activated by exposure to light [see, e.g., Goldmacher et al.
 (1992) Bioconi. Chem. 3:104-107, which linkers are herein incorporated by
 reference]. The photocleavable linker is selected such that the cleaving
 wavelength that does not damage linked moieties. Photocleavable linkers
 are linkers that are cleaved upon exposure to light [see, e.g., Hazum et
 al. (1981) in Pept., Proc. Eur. Pept. Symp.. 16th, Brunfeldt, K (Ed), pp.
 105-110, which describes the use of a nitrobenzyl group as a
 photocleavable protective group for cysteine; Yen et al. (1989) Makromol.
 Chem 190:69-82, which describes water soluble photocleavable copolymers,
 including hydroxypropylmethacrylamide copolymer, glycine copolymer,
 fluorescein copolymer and methylrhodamine copolymer; Goldmacher et al.
 (1992) Bioconi. Chem. 3:104-107, which describes a cross-linker and
 reagent that undergoes photolytic degradation upon exposure to near UV
 light (350 nm); and Senter et al. (1985) Photochem. Photobiol 42:231-237,
 which describes nitrobenzyloxycarbonyl chloride cross linking reagents
 that produce photocleavable linkages]. The selected linker will depend
 upon the particular application and, if needed, may be empirically
 selected.
 Aequorin that is designed for conjugation and conjugates containing such
 aequorin have been produced [see, e.g., International PCT application No.
 WO 94/18342; see, also Smith et al. (1995) in American Biotechnology
 Laboratory]. Vargula luciferase has also been linked to other molecules
 [see, e.g., Japanese application No. JP 5064583, Mar. 19, 1993]. Such
 methods may be adapted for use herein to produce aequorin coupled to
 protein or other such molecules, which are linked to the selected matrix.
 Finally, as an alternative, a component of the bioluminescence generating
 system may be modified for linkage, such as by addition of amino acid
 residues that are particularly suitable for linkage to the selected
 substrate. This can be readily effected by modifying the DNA and
 expressing such modified DNA to produce luciferase with additional
 residues at the N- or C-terminus.
 4. Apparatus containing a single chamber, housing or a vessel
 Examples of vessels include beverage containers, plates or other dishes,
 vases, jars, balloons, bottles and other containers.
 Single chamber housings or vessels will include single chamber water guns,
 inks, paints and other such items, in which one or more components of the
 bioluminescence system up to all of the components except for one of the
 components required for bioluminescence is included in the vessel as a
 mixture, powder or suspension of particles. The remaining component(s)
 is(are) introduced just prior to use. Thus, for example, for a squirt gun
 or a balloon or other such item, the items can be packaged with a powder
 in the chamber or inside the item, or a powder or other composition can be
 added, and then water is added. Alternatively, the luciferase, such as
 Renilla, Vargula, and firefly luciferase, can be linked to the surface of
 the item and water added. Depending upon the bioluminescence generating
 system selected the water can be tap water or water that contains the
 additional component, such as dissolved oxygen, or Ca.sup.2+ or ATP, or
 other suitable composition, and/or appropriate luciferin/bioluminescence
 substrate. Similarly, the luciferase/luciferase can be linked to the
 surface of the item in association with the appropriate
 luciferin/bioluminescence substrate, such that addition of activator alone
 generates luminescence.
 For inks or paints the components are suspended in the ink or paint, and
 then the final component(s) is(are) added. Alternatively, pellets
 containing components of the bioluminescence generating system, such as
 the Renilla or Aequorin system can be added to an ink or paint or other
 such liquid item, and as the pellet dissolves or the contents diffuse out,
 the item will glow.
 Kits containing the item and the bioluminescence generating systems are
 also provided herein.
 5. Dual and multiple chamber fluid dispensing apparatus
 An example of a dispensing apparatus contemplated for use herein is a dual
 chamber fluid dispensing apparatus. In general, this apparatus has two
 chambers thereby maintaining at least one of the bioluminescent system
 components separate from the remaining components until illumination is
 desired. This apparatus may include a mixing chamber to permit mixing of
 the components prior to dispensing from the apparatus. Further, the
 apparatus may be used with fluid or semi-fluid bioluminescence systems;
 for example, water based compositions or cream/lotion systems.
 a. Mechanical pump dispensing apparatus
 Another embodiment of a dual chamber fluid dispensing apparatus employs a
 mechanical pump mechanism in its operation. In this embodiment, the
 dispensing apparatus maintains at least one of the components of the
 bioluminescence reaction, such as the substrate, luciferase or activator,
 in separate chambers. A pump mechanism operates to withdraw the contents
 from each chamber and into a mixing chamber. Within the mixing chamber and
 upon ejection, the mixed composition is activated, for example by the
 oxygen in the air or by reaction of the components that were in one
 chamber, and glows. The pump mechanism may be manually operated, for
 example by pulling the trigger of a toy squirt gun, or it may be
 mechanically operated, for example by a motor which operates the pumping
 mechanism.
 b. Gas-charged dispensing apparatus
 Another example of a dual chamber fluid dispensing apparatus is one that
 uses CO.sub.2 or, preferably a mixture gases containing O.sub.2, or other
 gas, to propel the components of the bioluminescence system, such as the
 bioluminescence substrate and luciferase into a mixing chamber where they
 combine before being ejected through a dispensing nozzle. In such a
 dispensing apparatus, upon mixing of the contents in the mixing chamber
 the contents will glow.
 These apparatus may be configured as, for example, a toy gun, toy cannon or
 other toy weapon, a decorative fountain or volcano or almost any fluid
 squirting or spouting device. A volcano shaped dispensing apparatus may be
 used, for example, as a substitute for conventional, similarly shaped
 fireworks displays.
 Almost any bioluminescent system may be selected for use with the dual
 chamber fluid dispensing apparatus. If air is the bioluminescence
 activator, then the contents glow after mixing and upon ejection from the
 dispensing apparatus. Alternatively, the bioluminescent activator may be
 contained in one of the two chambers along with either the luciferase or
 bioluminescence substrate, or it may be located in a third chamber that is
 also connected to the mixing chamber. Thus, as with all the combinations
 described herein, the critical aspect of these dispensing apparatus is
 that at least one of the bioluminescent system components be maintained
 separate from the other components until reaction is desired.
 c. Compressible dispensing apparatus
 Another embodiment of a dual chamber fluid dispensing apparatus
 contemplated for use herein takes the form of a compressible bottle or
 tube. The bottle has two compartments within it that keep at least two of
 the bioluminescent system components separated. The cap of the bottle can
 serve as a mixing chamber or a mixing chamber may be positioned between
 the two chambers and the cap. The bioluminescent system components are
 forced by compression from the bottle into the mixing chamber. They are
 then dispensed from the mixing chamber. For example, the mixed contents
 may be removed from the bottle by attaching a plunger/syringe apparatus to
 the dispensing end and withdrawing the contents therethrough.
 Such compressible bottle or tube is particularly useful for dispensing
 bioluminescent body creams, gels or lotions, finger paints, dentifrices,
 shampoos, hair gels, cosmetics and other viscous fluids and semi-solids.
 The bottle or tube is preferably constructed of plastic, plastic/metal
 laminate or similar collapsible composite to avoid formation of a vacuum
 within the container as its contents are expelled. See, for example, U.S.
 Pat. No. 4,687,663, which describes a dual chambered tube for use with
 dentifrices and which, as all cited patents and publications herein, is
 incorporated herein in its entirety. This tube may be adapted for use in
 combination with the bioluminescence generating systems provided herein.
 Other tubes and vessels that have dual chambers, such as those used to
 keep components of the final product separate until use, may be used
 herein [see, e.g., U.S. Pat. Nos. 5,405,056, 4,676,406, 4,438,869,
 5,059,417, 4,528,180, 4,849,213, 4,895,721, 5,085,853, see, esp.
 5,038,963]
 6. Other fluid dispensing and packaging apparatus particularly designed for
 single use
 Additional embodiments of the dispensing and packaging apparatus
 contemplated for use herein include fluid packaging apparatus, designed
 for use with bioluminescent fluids. These apparatus maintain at least one
 of the bioluminescent system components separate from the remaining
 components until illumination is desired. Unlike the dual chamber fluid
 dispensing apparatus, however, these apparatus result in illumination of
 the entire contents of the package and therefore are typically intended
 for a single use applications. They can, however, be recharged by adding
 additional substrate, luciferase or other exhausted component.
 a. Bottle-type single chamber container/bladder apparatus
 One example of a fluid packaging apparatus, contemplated for use herein, is
 a bottle shaped device having a bladder within it that contains at least
 one of the bioluminescent system components. A piercing pin or other means
 for rupturing the bladder is also located within the bottle. When the
 bladder is ruptured, within the bottle, its contents mix with the contents
 of the bottle and the resulting mixture becomes illuminated or glows upon
 contact with an activator, such as air.
 Because the bioluminescent system components are mixed within the entire
 bottle, those contents must be used shortly after mixing. Thus, this type
 of packaging is particularly suitable for use with bioluminescence systems
 that are consumed in a single use or activity such as bubble-blowing.
 b. Dual chambered bottle type container/bladder apparatus for use with
 foods and beverages
 Another example of a fluid packaging apparatus provided herein is a single
 use, dual chambered bottle. This apparatus is configured with a membrane
 between the two chambers. One chamber is designed to readily collapse
 against the other chamber thereby rupturing the membrane which divides the
 chambers. The contents of the two chambers then mix, resulting in
 illumination of the fluids. Alternatively, instead of a membrane
 separation means, a one-way valve may be situated between the two
 chambers. Such a single use, dual chamber apparatus is particularly
 suitable for use with bubble-making compositions, beverages, single use
 amounts of shampoos, soaps, creams or lotions, or similar substances.
 c. Can type container/bladder apparatus for use with foods and beverages
 Another example of a fluid packaging apparatus, which is amenable to use
 with bioluminescent food or beverage, is a container/bladder combination.
 In one embodiment, the container is configured like a pop-top can, such as
 a soda can. A bladder, containing at least one of the bioluminescent
 system components, is positioned under the top of the can. Within the can
 is a beverage that contains the remaining bioluminescent system
 components. Upon opening the can, the bladder is punctured and its
 contents mixed with the rest of the contents of the can; thereby
 illuminating the beverage. Preferably, the container is clear, so that the
 illumination will be almost immediately visible. Other pop top cans that
 can be modified for use herein are known [see, e.g., U.S. Pat. No.
 5,397,014].
 Alternative configurations of the container/bladder apparatus are likewise
 contemplated. For example, the container may be in any shape and
 configured with a removable cap to which the bladder is attached. To cause
 the beverage to glow, the bladder is punctured or otherwise compromised
 and its contents added to the container; thereby causing illumination of
 the food or beverage. The contents of the container need not be a food or
 beverage, any fluid or semi-solid may be used and is herein contemplated.
 7. Cap Apparatus for use a single chamber vessel
 Another example of a packaging apparatus contemplated herein is a cap
 apparatus for use with a vessel. In this embodiment, one or more of the
 bioluminescent system components, up to all but one component, is [are]
 within the cap of the vessel and the remaining components are contained in
 the vessel. Upon operation of the cap apparatus, the bioluminescent system
 components are added to the composition in the vessel and the composition
 glows. Preferably the vessel is translucent to the bioluminescence;
 however, the glowing composition may be dispensed from the vessel.
 Generally, the cap is configured with a pocket within it which opens to the
 bottom of the cap. For example, the bottom of the cap can be U-shaped,
 curving into the cap and thereby forming the pocket. The cap apparatus
 contains a capsule or similar package, containing one or more, up to all
 but one, of the bioluminescent system components, within the pocket in the
 cap. Means for deploying the bioluminescent system components into the
 vessel are attached to the cap. Such deployment means can be, for example,
 a plunger assembly. The cap apparatus is operated by depressing the
 plunger, thereby forcing the packaged components into the composition
 within the vessel or breaking the packaging, releasing its contents into
 the composition within the vessel. The package should be dissolvable in
 the composition or amenable to diffusion of the components contained
 therein or readily rupturable upon contact with the plunger assembly.
 Alternatively, the packaging within the cap apparatus can comprise a
 membrane or series of membranes separating the bioluminescent system
 components from the composition within the vessel or from the composition
 within the vessel and from each other. In this alternative, the plunger
 can rupture the membrane(s) thereby permitting the bioluminescent system
 components contained therein to be released into the composition contained
 in the vessel. Again, upon mixture of the components with the composition,
 illumination ensues.
 The bioluminescent system components contained within the cap apparatus may
 be in a composition, such as a solution, a powder or a suspension of
 particles or other form amenable to packaging within the cap apparatus
 that can be mixed with the composition contained within the vessel. The
 cap apparatus also may be adapted with a screen or filter attached to the
 bottom of the cap to prevent membrane fragments from entering the vessel.
 The cap apparatus, as all the apparatus described herein that are in
 contact with a bioluminescent system component, should be non-reactive
 with the components and is preferably non-toxic, particularly if used with
 a composition intended for human consumption. The cap can be constructed
 of cork, for example, and situated in a wine or champagne bottle.
 Alternatively, the cap can be a screw-top type cap, having a plunger
 integral thereto, such that tightening of the screw-cap onto the top of
 the vessel forces the plunger against the packaged bioluminescent system
 components either rupturing the packaging or pushing it into the vessel.
 E. Combinations of articles of manufacture and bioluminescence
 Combinations of articles of manufacture and bioluminescence are provided
 herein. By virtue of the bioluminescence the combinations are novelty
 items because the bioluminescence provides entertainment, amusement or
 recreation. Any such combination of an article of manufacture with
 bioluminescence that produces a novelty item [i.e., provides
 entertainment, amusement, or recreation] is intended herein. The
 combination is formed by contacting the article of manufacture or
 materials in the manufacture with a bioluminescence generating system or
 an apparatus therefore. The components of the bioluminescence generating
 system is manufactured as part of the item, coated thereon, impregnated
 therein, or added after manufacture. Alternatively, the article of
 manufacture is combined with an apparatus that contains or to which
 components of the bioluminescence generating system are added, and that
 produces the bioluminescence.
 The bioluminescent systems provided herein are contemplated for use with
 various substances to glow the substance. For example, as discussed below,
 the bioluminescent system components may be used to produce glowing
 aqueous mixtures housed in transparent portions of articles of
 manufacture, thereby illuminating that portion of the article of
 manufacture. Additionally, the bioluminescent system components may be
 used to produce glowing food or beverage products, textiles, creams,
 lotions, gels, soaps, bubbles, papers, powders or water. Following are
 brief examples of combinations of bioluminescence systems with articles of
 manufacture and the resulting novelty items contemplated herein.
 1. Personal care products, including bath powders, bubble baths, products
 for use on the nails, hair, skin, lips and elsewhere
 Personal care products can be in the form of powders, pressed powders,
 sprays, foams, aerosols, lotions, gels, ointments and other suitable
 formulations. The common element will be the combination of such items
 with bioluminescence generating reagents, so that before use or upon
 application to the body or when used the product will glow. These items
 include, body powders, lotions, gels, solutions, nail polishes, make-up,
 body paints, dentifrices. As described herein, the items are combined with
 one or more components of a bioluminescence generating system, and, when a
 glow is desired, the remaining components are added or combined with the
 other components.
 a. Bath powders
 Numerous bath powders exemplified herein, are suitable for use in
 combination with the bioluminescent generating systems herein. Such bath
 powders are preferably non-detergent with a pH close to neutral. The
 selected bioluminescence generating system must be selected to be active
 at the resulting pH. In addition, capsular delivery vehicles, such as
 liposomes or time release delivery vehicles, preferably microcapsules,
 that contain a luciferase and luciferin, such as the Renilla, Vargula, or
 Aequorin system, and that are pH, temperature sensitive, or that dissolve
 in water or that are otherwise released are preferred for use herein. In
 certain embodiments, there will be two types of capsules, one type
 containing up to all but one of the components required for the
 bioluminescence reaction, and the other containing the remaining
 components [except, if desired, for those components that will be present
 in the bath water, such as Ca.sup.2+ ]. Such capsules may be components of
 the bath powder or may be added to a bath to give it a glow. Upon contact
 with the warm water or with water of a particular pH the contents of the
 capsule or pellet will be released, preferably over time, and will glow.
 In other embodiments, there will be one type of capsule that contains the
 luciferase and other components. The luciferin may be included in the bath
 powder or added separately. Other ways in which the components may be
 combined will, in light of the disclosure herein, be apparent to those of
 skill in the art. The bath powders and bioluminescence generating
 reactions will be provided as a combination or in a kit.
 Suitable bath powders and bubble baths and other bubble compositions for
 use in these combinations are well known to those of skill in the art
 [see, eg., U.S. Pat. Nos.: 5,478,501 4,565,647; 5,478,490; 5,412,118;
 5,401,773; and many other examples]. These may be modified by adding the
 bioluminescence generating system components.
 b. Glowing dust or powder
 Another embodiment of the combination described herein is as a glowing dust
 or powder substance, or a vapor, such as for use in the theatrical
 productions. In this embodiment, lyophilized or desiccated forms,
 micronized powdered forms, or, a suitable composition, of up to all but
 one of the bioluminescence generating system components are encapsulated
 in readily rupturable or time release or temperature or pH or light
 sensitive microspheres or capsules, as described above. Preferable
 encapsulating agents are light or temperature sensitive so that upon
 exposure to the environment, the contents are released from the capsules.
 Moisture or oxygen in the air or a spray of water on the skin with
 dissolved oxygen in the vicinity of the "dust" will produce a glow. The
 dust can be added to another powder, such as body powder, provided it is
 stored in an airtight container. Once the powder contacts the moisture in
 the air and on the wearer's skin, it glows.
 Alternatively, micronized particles of lyophilized powders are packaged
 such in manner so that the powder remains dry. Upon exposure to moist air
 or to air with water droplets [such as a fog], the micronized powders will
 glow.
 c. Lotions, gels and other topical application formulations
 For application to the skin, the macro or microparticles or the luciferase,
 luciferin or mixture thereof, may be added to cosmetic compositions. The
 compositions may be provided in the form of gels, creams, lotions, solids,
 and other compositions, such as solutions and suspensions, aerosols or
 solid base or vehicle known in the art to be non-toxic and
 dermatologically acceptable to which sufficient number of such particles
 are added under conditions in which the contents are released into the
 gels, creams, lotions, solids, solutions or suspensions, or aerosols,
 which contain either molecular oxygen and/or Ca.sup.2+ to react with the
 contents of particles. Upon application to the skin the gels, creams,
 lotions, solids, solutions or suspensions, or aerosols glow.
 (1) Lotions
 The lotions contain an effective concentration of less than all reagents
 for one or more bioluminescent systems. Preferably, the reagents are
 encapsulated in a vehicle that releases its contents upon exposure to
 light or temperature, such that as the contents of the vehicle are
 released they react with oxygen or Ca.sup.2+ in the lotion and/or on the
 skin. Prior to use the skin can be sprayed with a mist of water, buffer or
 other composition containing the requisite ions. The effective
 concentration is that sufficient to produce a visible glow when contacting
 the skin. Any emollients, as long as they do not inactivate the
 bioluminescent reaction, known to those of skill in the art as suitable
 for application to human skin may be used. These include, but are not
 limited to, the following:
 (a) Hydrocarbon oils and waxes, including mineral oil, petrolatum,
 paraffin, ceresin, ozokerite, microcrystalline wax, polyethylene, and
 perhydrosqualene. p1 (b) Silicone oils, including dimethylpolysiloxanes,
 methylphenylpolysiloxanes, water-soluble and alcohol-soluble
 silicone-glycol copolymers. p1 (c) Triglyceride fats and oils, including
 those derived from vegetable, animal and marine sources. Examples include,
 but are not limited to, castor oil, safflower oil, cotton seed oil, corn
 oil, olive oil, cod liver oil, almond oil, avocado oil, palm oil, sesame
 oil, and soybean oil.
 (d) Acetoglyceride esters, such as acetylated monoglycerides.
 (e) Ethoxylated glycerides, such as ethoxylated glyceryl monstearate.
 (f) Alkyl esters of fatty acids having 10 to 20 carbon atoms. Methyl,
 isopropyl and butyl esters of fatty acids are useful herein. Examples
 include, but are not limited to, hexyl laurate, isohexyl laurate, isohexyl
 palmitate, isopropyl palmitate, isopropyl myristate, decyl oleate,
 isodecyl oleate, hexadecyl stearate, decyl stearate, isopropyl
 isostearate, diisopropyl adipate, diisohexyl adipate, dihexyldecyl
 adipate, diisopropyl sebacate, lauryl lactate, myristyl lactate, and cetyl
 lactate.
 (g) Alkenyl esters of fatty acids having 10 to 20 carbon atoms. Examples
 thereof include, but are not limited to, oleyl myristate, oleyl stearate,
 and oleyl oleate.
 (h) Fatty acids having 9 to 22 carbon atoms. Suitable examples include, but
 are not limited to, pelargonic, lauric, myristic, palmitic, stearic,
 isostearic, hydroxystearic, oleic, linoleic, ricinoleic, arachidonic,
 behenic, and erucic acids. p1 (i) Fatty alcohols having 10 to 22 carbon
 atoms, such as, but not limited to, lauryl, myristyl, cetyl, hexadecyl,
 stearyl, isostearyl, hydroxystearyl, oleyl, ricinoleyl, behenyl, erucyl,
 and 2-octyl dodecyl alcohols.
 (j) Fatty alcohol ethers, including, but not limited to ethoxylated fatty
 alcohols of 10 to 20 carbon atoms, such as, but are not limited to, the
 lauryl, cetyl, stearyl, isostearyl, oleyl, and cholesterol alcohols having
 attached thereto from 1 to 50 ethylene oxide groups or 1 to 50 propylene
 oxide groups or mixtures thereof.
 (k) Ether-esters, such as fatty acid esters of ethoxylated fatty alcohols.
 (l) Lanolin and derivatives, including, but not limited to, lanolin,
 lanolin oil, lanolin wax, lanolin alcohols, lanolin fatty acids, isopropyl
 lanolate, ethoxylated lanolin, ethoxylated lanolin alcohols, ethoxylated
 cholesterol, propoxylated lanolin alcohols, acetylated lanolin, acetylated
 lanolin alcohols, lanolin alcohols linoleate, lanolin alcohols
 ricinoleate, acetate of lanolin alcohols ricinoleate, acetate of
 ethoxylated alcohols-esters, hydrogenolysis of lanolin, ethoxylated
 hydrogenated lanolin, ethoxylated sorbitol lanolin, and liquid and
 semisolid lanolin absorption bases.
 (m) Polyhydric alcohols and polyether derivatives, including, but not
 limited to, propylene glycol, dipropylene glycol, polypropylene glycol
 [M.W. 2000-4000], polyoxyethylene polyoxypropylene glycols,
 polyoxypropylene polyoxyethylene glycols, glycerol, ethoxylated glycerol,
 propoxylated glycerol, sorbitol, ethoxylated sorbitol, hydroxypropyl
 sorbitol, polyethylene glycol [M.W. 200-6000], methoxy polyethylene
 glycols 350, 550, 750, 2000, 5000, poly(ethylene oxide) homopolymers [M.W.
 100,000-5,000,000], polyalkylene glycols and derivatives, hexylene glycol
 (2-methyl-2,4-pentanediol), 1,3-butylene glycol, 1,2,6,-hexanetriol,
 ethohexadiol USP (2-ethyl-1,3-hexanediol), C.sub.15 -C.sub.18 vicinal
 glycol and polyoxypropylene derivatives of trimethylolpropane.
 (n) Polyhydric alcohol esters, including, but not limited to, ethylene
 glycol mono- and di-fatty acid esters, diethylene glycol mono- and
 di-fatty acid esters, polyethylene glycol [M.W. 200-6000], mono- and
 di-fatty esters, propylene glycol mono- and di-fatty acid esters,
 polypropylene glycol 2000 monooleate, polypropylene glycol 2000
 monostearate, ethoxylated propylene glycol monostearate, glyceryl mono-
 and di-fatty acid esters, polyglycerol poly-fatty acid esters, ethoxylated
 glyceryl monostearate, 1,3-butylene glycol monostearate, 1,3-butylene
 glycol distearate, polyoxyethylene polyol fatty acid ester, sorbitan fatty
 acid esters, and polyoxyethylene sorbitan fatty acid esters.
 (o) Wax esters, including, but not limited to, beeswax, spermaceti,
 myristyl myristate, and stearyl stearate and beeswax derivatives,
 including, but not limited to, polyoxyethylene sorbitol beeswax, which are
 reaction products of beeswax with ethoxylated sorbitol of varying ethylene
 oxide content that form a mixture of ether-esters.
 (p) Vegetable waxes, including, but not limited to, carnauba and candelilla
 waxes.
 (q) Phospholipids, such as lecithin and derivatives.
 (r) Sterols, including, but not limited to, cholesterol and cholesterol
 fatty acid esters.
 (s) Amides, such as fatty acid amides, ceramides, ethoxylated fatty acid
 amides, and solid fatty acid alkanolamides.
 The lotions further preferably contain [by weight] from 1% to 10%, more
 preferably from 2% to 5%, of an emulsifier. The emulsifiers can be
 nonionic, anionic or cationic. Examples of satisfactory nonionic
 emulsifiers include, but are not limited to, fatty alcohols having 10 to
 20 carbon atoms, fatty alcohols having 10 to 20 carbon atoms condensed
 with 2 to 20 moles of ethylene oxide or propylene oxide, alkyl phenols
 with 6 to 12 carbon atoms in the alkyl chain condensed with 2 to 20 moles
 of ethylene oxide, mono- and di-fatty acid esters of ethylene oxide, mono-
 and di-fatty acid esters of ethylene glycol wherein the fatty acid moiety
 contains from 10 to 20 carbon atoms, diethylene glycol, polyethylene
 glycols of molecular weight 200 to 6000, propylene glycols of molecular
 weight 200 to 3000, glycerol, sorbitol, sorbitan, polyoxyethylene
 sorbitol, polyoxyethylene sorbitan and hydrophilic wax esters. Suitable
 anionic emulsifiers include, but are not limited to, the fatty acid soaps,
 e.g. sodium, potassium and triethanolamine soaps, wherein the fatty acid
 moiety contains from 10 to 20 carbon atoms. Other suitable anionic
 emulsifiers include, but are not limited to, the alkali metal, ammonium or
 substituted ammonium alkyl sulfates, alkyl arylsulfonates, and alkyl
 ethoxy ether sulfonates having 10 to 30 carbon atoms in the alkyl moiety.
 The alkyl ethoxy ether sulfonates contain from 1 to 50 ethylene oxide
 units. Among satisfactory cationic emulsifiers are quaternary ammonium,
 morpholinium and pyridinium compounds. Certain of the emollients described
 in preceding paragraphs also have emulsifying properties. When a lotion is
 formulated containing such an emollient, an additional emulsifier is not
 needed, though it can be included in the composition.
 Other conventional components of such lotions may be included. One such
 additive is a thickening agent at a level from 1% to 10% by weight of the
 composition. Examples of suitable thickening agents include, but are not
 limited to: cross-linked carboxypolymethylene polymers, ethyl cellulose,
 polyethylene glycols, gum tragacanth, gum kharaya, xanthan gums and
 bentonite, hydroxyethyl cellulose, and hydroxypropyl cellulose.
 The balance of the lotion is water or a C.sub.2 or C.sub.3 alcohol, or a
 mixture of water and the alcohol. The lotions are formulated by simply
 admixing all of the components together. Preferably bioluminescence
 generating system reagents are suspended or otherwise uniformly dispersed
 in the mixture.
 In certain embodiments the components may be mixed just prior to use.
 Devices for effecting such mixture are known to those of skill in the art
 or are exemplified herein.
 Kits containing the lotion and powders, capsular vehicles and, optionally,
 buffer compositions containing ATP, Ca.sup.2+ and other ingredients
 required for the bioluminescence reaction are also provided.
 (2) Creams
 The creams are similarly formulated to contain an effective concentration
 typically at between about 0.1%, preferably at greater than 1% up to and
 greater than 50%, preferably between about 3% and 50%, more preferably
 between about 5% and 15% [by weight] of one ore more the bioluminescent
 systems provided herein. The creams also contain from 5% to 50%,
 preferably from 10% to 25%, of an emollient and the remainder is water or
 other suitable non-toxic carrier, such as an isotonic buffer. The
 emollients, as described above for the lotions, can also be used in the
 cream compositions. The cream may also contain a suitable emulsifier, as
 described above. The emulsifier is included is in the composition at a
 level from 3% to 50%, preferably from 5% to 20%.
 (3) Solutions and suspensions for topical application
 These compositions are formulated to contain an amount sufficient to
 produce a visible glow, typically at a concentration of between about
 0.1-10 mg/l preferably between 1 and 5 mg/l of the luciferase. The amount
 of luciferin is similarly between about 0.1 and 10 mg/l, although the
 amount can be selected based on the desired duration of the glow. The
 balance is water, a suitable organic solvent or other suitable solvent or
 buffer. Suitable organic materials useful as the solvent or a part of a
 solvent system are as follows: propylene glycol, polyethylene glycol [M.W.
 200-600], polypropylene glycol [M.W. 425-2025], glycerine, sorbitol
 esters, 1,2,6-hexanetriol, ethanol, isopropanol, diethyl tartrate,
 butanediol, and mixtures thereof. Such solvent systems can also contain
 water.
 Solutions or suspensions used for topical application can include any of
 the following components: a diluent, such as water saline solution, fixed
 oil, polyethylene glycol, glycerine, propylene glycol or other synthetic
 solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens;
 antioxidants, such as ascorbic acid and sodium bisulfite; chelating
 agents, such as ethylenediaminetetraacetic acid [EDTA]; buffers, such as
 acetates, citrates and phosphates; and agents for the adjustment of
 tonicity such as sodium chloride or dextrose. Liquid preparations can be
 enclosed in ampules, disposable syringes or multiple dose vials made of
 glass, plastic or other suitable material. Suitable carriers may include
 physiological saline or phosphate buffered saline [PBS], and the
 suspensions and solutions may contain thickening and solubilizing agents,
 such as glucose, polyethylene glycol, and polypropylene glycol and
 mixtures thereof. Liposomal suspensions, may also be suitable as
 pharmaceutically acceptable carriers. These may be prepared according to
 methods known to those skilled in the art.
 These compositions that are formulated as solutions or suspensions may be
 applied to the skin, or, may be formulated as an aerosol or foam and
 applied to the skin as a spray-on. The aerosol compositions typically
 contain [by weight] from 25% to 80%, preferably from 30% to 50%, of a
 suitable propellant. Examples of such propellants are the chlorinated,
 fluorinated and chlorofluorinated lower molecular weight hydrocarbons.
 Nitrous oxide, carbon dioxide, butane, and propane are also used as
 propellant gases. These propellants are used as understood in the art in a
 quantity and under a pressure suitable to expel the contents of the
 container.
 Solutions, may be formulated as 0.01%-10% isotonic solutions, pH about 5-8,
 with appropriate salts, and preferably containing one or more of the
 compounds herein at a concentration of about 0.1%, preferably greater than
 1%, up to 50% or more. Suitable mild solutions are known [see, eg., U.S.
 Pat. No. 5,116,868, which describes typical compositions of ophthalmic
 irrigation solutions and solutions for topical application]. Such
 solutions, which have a pH adjusted to about 7.4, contain, for example,
 90-100 mM sodium chloride, 4-6 mM dibasic potassium phosphate, 4-6 mM
 dibasic sodium phosphate, 8-12 mM sodium citrate, 0.5-1.5 mM magnesium
 chloride, 1.5-2.5 mM calcium chloride, 15-25 mM sodium acetate, 10-20 mM
 D.L.-sodium .beta.-hydroxybutyrate and 5-5.5 mM glucose.
 The active materials can also be mixed with other active materials, that do
 not impair the desired action, or with materials that supplement the
 desired action.
 (4) Gels
 Gel compositions can be formulated by simply admixing a suitable thickening
 agent to the previously described [(3)] solution or suspension
 compositions. Examples of suitable thickening agents have been previously
 described with respect to the lotions.
 The gelled compositions contain an effective amount of one or more an
 anti-hyperalgesic amount, typically at a concentration of between about
 0.1 mg/l-10 mg/l or more of one or more of systems provided herein, from
 0% to 75%, from 0.5% to 20%, preferably from 1% to 10% of the thickening
 agent; the balance being water or other aqueous carrier.
 (5) Solids
 Compositions of solid forms may be formulated as stick-type compositions
 intended for application to the lips or other parts of the body. Such
 compositions contain an effective amount of one or more of the compounds
 provided herein. The amount is typically an amount effective to glow when
 contacted with moist skin, such as lips, typically at a concentration of
 between about 0.1 mg/l-10 mg/l or more of one or more of the systems
 provided herein. The solids also contain from about 40% to 98%, preferably
 from about 50% to 90%, of the previously described emollients. This
 composition can further contain from 1% to 20%, preferably from 5% to 15%,
 of a suitable thickening agent, and, if desired or needed, emulsifiers and
 water or buffers. Thickening agents previously described with respect to
 lotions are suitably employed in the compositions in solid form.
 Other ingredients, such as preservatives, including methyl-paraben or
 ethyl-paraben, perfumes, dyes or the like, that are known in the art to
 provide desirable stability, fragrance or color, or other desirable
 properties, such as shielding from actinic rays from the sun, to
 compositions for application to the skin may also be employed in a
 composition for such topical application.
 2. Glowing toys and other items
 Examples of uses of the bioluminescent systems in toys include illumination
 of dolls, toy vehicles, hoolahoops, yo-yos, balloons and any other toy
 amenable to having a generally translucent covering defining a space for
 containment of the bioluminescent system and addition of the final
 ingredients necessary for the illumination reaction. Also contemplated
 herein are toys that eject or spew a fluid. For example, toy or game
 projectiles are contemplated that contain a luciferase and bioluminescence
 substrate in an oxygen-free environment. The projectiles rupture upon
 impact with a hard surface thereby exposing the contents to moisture in
 the air that contains dissolved oxygen, the bioluminescence activator, and
 causing reaction.
 Dolls and dummies containing one or two of the bioluminescent system
 components within a transparent or translucent portion of their bodies are
 contemplated herein. Addition of the remaining bioluminescent system
 components results in illumination of that body part or area. For example,
 a doll can have a visible, translucent digestive system containing a
 luciferase and substrate in a water-free environment. Upon "ingestion" of
 water by the doll, that is addition of water through its mouth, for
 example, the digestive system glows or is illuminated.
 Other examples of uses of the bioluminescent systems in toys include
 illumination hoolahoops, yo-yos, slimy play materials based on sodium
 alginate and glycerine [U.S. Pat. No. 5,310,421], such as those sold by
 MATTEL.RTM. as FLOAM.RTM., GAK.RTM., and SMUD.RTM.. The biolumine-scence
 generating components can be incorporated into the slime material during
 manufacture, as liposomes, or linked to the material. These slime
 materials are fabricated from self crosslinking sodium alginate, a
 glycerin solution [concentration over 90%], water and preservatives. The
 play material contains 2.5%-4.0 by weight, 3.33 weight %, of a
 self-crosslinking sodium alginate; 1.0-3.5 weight % of a glycerin and
 water solution in excess of 90% glycerin; a preservative; 4.0 weight %
 NaCl; and water, and can include 0.04-0.08 weight % of a colorant. It will
 also include up to all but one component of a bioluminescence generating
 system, such as a luciferase, such as Renilla or Vargula or a firefly
 luciferase, or a luciferin and luciferase, such as the Aequorin
 photoprotein and EDTA. A second mixture of the slime material will contain
 the remaining components or a water solution. Prior to use the remaining
 components, such as Ca.sup.2+, in a mixture of the same composition as the
 slime will be mixed with the other mixture. The second mixture could also
 contain a different colorant, so that upon mixing not only will the
 material glow, it will change color. The concentrations of luciferase
 components, such as luciferase will be those sufficient to generate a
 visible glow. The concentrations of luciferase can be empirically
 determined, but generally will be between about 0.1 and 1 mg per liter of
 material. The amount of luciferin generally will be in excess. The
 luciferases and luciferin and other components can also be provided as
 time release vehicles in the material or provided separately for
 subsequent addition. This slime material can be packaged as a kit or
 article of manufacture containing a first slime composition containing all
 but at least one bioluminescence generating reagent, and a second slime
 composition containing the remaining components. The kit will include
 instructions for mixing the two compositions to produce a glowing
 composition. The kit can also contain additional compositions or vehicles
 or dried powders of bioluminescence generating reagents so that they can
 be added prior to use so that the material can be reused. In other
 embodiments, the slime is provided without bioluminescence generating
 reagents and the bioluminescence generating reagents are provided as
 separate compositions, in time release vehicles or other delivery vehicles
 and are mixed into the material prior to use.
 Other toys, games, novelty items, clothes, accessories, foods, beverages,
 fountains, water dispensing apparatus, soaps, creams, cosmetics and
 sporting equipment amenable to bioluminescence are further embodiments of
 the presently disclosed combination. Thus, any article of manufacture or
 substance capable of modification to allow bioluminescence thereof is
 contemplated herein.
 Articles of manufacture that are amenable to use with the bioluminescent
 systems provided herein are well known [see, e.g., U.S. Pat. Nos.
 5,415,151, 5,018,449, 3,539,794, 5,171,081, 4,687,663, 5,038,963,
 4,765,510, 4,282,678, 5,366,108, 5,398,827, 5,397,014, 5,219,096,
 5,305,919, 5,184,755, 5,029,732, 4,214,674, 4,750,641, 4,676,406], which
 describe devices useful as toy water guns or vessels for beverages or
 creams and lotions. To be amenable to use in the embodiments described
 herein, each may require some modification, such as, for example, addition
 of a mixing chamber.
 In light of the disclosure herein, such modification will be apparent. Some
 of the patents describe other toy devices, training mock weapon devices,
 dolls, and beverage containers and dentifrice containers [i.e., toothpaste
 tubes]. In the simplest modification, powdered or capsular vehicles
 containing bioluminescence generating systems may be added to the
 water-holding chambers of the toy gun or other water spewing toy. As the
 powder dissolves or the vehicle releases its contents, typically luciferin
 and luciferase, contact with the water in the gun will cause the
 bioluminescence reaction to occur.
 As is apparent from the above, toy guns are well known items and materials
 and specifications for manufacture thereof are also well known [see, the
 above list and see, also, U.S. Pat. Nos. 5,029,732, and 5,415,151]. Any
 single chamber squirt gun may used in combination with bioluminescent
 systems herein by mixing the components in the gun chamber. Of course the
 selected system should be one that has sustained illumination.
 Alternatively, pellets of encapsulated bioluminescent components, such as
 the aequorin photoprotein or the Renilla luciferase and luciferin, may be
 added to water in the gun chamber. In the case of the aequorin
 photoprotein and Renilla luciferase, added tap water may be sufficient.
 For the Renilla system the pellets may contain the luciferase and
 luciferin or either. The remaining component will be added to the gun
 chamber. If pellets are used, the pellets will slowly release their
 contents thereby providing for a continuous glow.
 Similar apparatus and designs are also used for any fountain or water
 propelling device. Any such device [see, e.g., U.S. Pat. No. 5,360,142]
 may be modified to include a bioluminescence system to produce a glowing
 stream.
 In all of these devices, the water is can be tap water or a selected
 buffer, particularly phosphate buffered saline. The items may packaged as
 kits with the packaged luciferin, luciferase, and including the water.
 a. Single chamber toy guns and other toy weapons that shoot pellets or
 liquid
 Numerous toy guns and other toy weapons that shoot pellets or liquid, in
 addition to those exemplified herein, are suitable for use in combination
 with the bioluminescent generating systems herein. The toy weapons may be
 loaded with a solution containing microspheres of luciferin and/or
 luciferase, or with lyophilized luciferin/luciferin, or other mixtures as
 described herein. Suitable toy weapons and devices that shoot jets or
 sprays of water are described in the following sampling of U.S. Pat. No.
 5,462,469 [toy gun that shoots bubbles]; U.S. Pat. No. 5,448,984 [toy gun
 that shoots balls and water and can be modified to shoot light or
 temperature sensitive pellets, which should be stored under appropriate
 conditions or appropriately packaged, that release luciferin/luciferase
 when exposed to light]; U.S. Pat. Nos. 5,439,139; 5,427,320; 5,419,458;
 5,381,928; 5,377,656; 5,373,975; 5,373,833 and 5,373,832 [which describe
 toy guns that rely upon a pressurizable bladder for release of air
 pressure to shoot a projectile, which can be modified to shoot projectiles
 of encapsulated luciferin/luciferase]; U.S. Pat. No. 5,370,278 [which
 describes liquid from a port mounted to a headband]; U.S. Pat. No.
 5,366,108; 5,360,142 [which describes a supply and delivery assembly for
 use in combination with a pump type water gun or other water propelling
 device]; U.S. Pat. Nos. 5,346,418; 5,343,850 [which describes a projectile
 launcher for use in combination with the pellets provided herein]; U.S.
 Pat. Nos. 5,343,849; 5,339,987 [which describes water guns that have at
 least one pressurizable air/water storage tank, a pressurizing mechanism,
 a channel of release for shooting water and a release mechanism]; U.S.
 Pat. Nos. 5,326,303; 5,322,191; 5,305,919; 5,303,847 [which describes a
 device worn on a user's hand with sheaths for the tips of the fingers that
 includes a housing for a water reservoir, a water pump and electrical
 motor and a battery pack to be secured to the user's body]; U.S. Pat. Nos.
 5,292,032; 5,284,274 [which describes an action to system including a
 capsule for containing water, which will herein contain components of a
 bioluminescence generating system, having an orifice and a plunger and a
 spring loaded mechanism for driving the water from the orifice. The action
 toy may be configured as a shotgun accepting a plurality of prefilled
 shell capsules into its breechblock for firing through its barrel, as a
 missile launcher in which the capsules are mounted to the front of the
 launcher and the water is ejected directly from the capsule against the
 target, or as a crossbow with the bow loading the spring-loaded mechanism
 and a water stream obtained on release of the bow]; U.S. Pat. No.
 5,284,272 [which describes a bottle and cap combination for spewing
 liquid]; U.S. Pat. Nos. 5,256,099; 5,244,153; 5,241,944; 5,238,149;
 5,234,129; 5,224,625; 5,213,335; 4,854,480; 5,213,089; 5,184,755;
 5,174,477; 5,150,819; 5,141,467; 5,141,462; 5,088,950; 5,071,387 [which
 describes a figurine-shaped water squirting toy]; U.S. Pat. No. 5,064,095
 [which describes a water cannon apparatus]; U.S. Pat. Nos. 5,029,732;
 5,004,444; 4,892,228; 4,867,208 [which describes an apparatus for storing
 and dispensing fluid under pressure]; U.S. Pat. Nos. 4,808,143; 4,784,293,
 4,768,681; 4,733,799; 4,615,488 and many others. U.S. Pat. No. 5,415,151
 describes a toy gun that launches projectiles that can be adapted for
 shooting the pellets, such as light sensitive pellets that are degraded
 upon exposure to light, provided herein.
 b. Bubble-making toys
 Soap bubbles are blown from water solutions comprising soap or another
 surfactant. A great variety of bubble solution formulations are available,
 including those that feature special effects in bubble making. There are
 solutions for making large bubbles, "long lasting" bubbles, split bubbles,
 self-healing bubbles, multiple bubbles, vanishing bubbles, flaking
 bubbles, bursting bubbles, high and/or far-flying bubbles, sinking bubbles
 etc. In general, many anionic, non-ionic or amphoteric aqueous solutions
 with low surface tension lend themselves to bubble or foam-making when air
 or other gases are blown into such compositions.
 Such compositions, preferably those that have near neutral pH, can be
 combined with the components of the bioluminescence generating systems
 provided herein. In particular, a mixture of luciferase and luciferin,
 such as the Renilla system or firefly system or Cypridina system,
 preferably in the form of pellets or microspheres, such as liposomes or
 other time release capsule, can be added to the bubble mixture. When used,
 the air added to the mixture will cause a glow, or a glow will be produced
 as the contents of the pellets are released into the composition.
 Kits containing such soap compositions, with preferably a moderate pH
 [between 5 and 8] and bioluminescence generating reagents, including
 luciferase and luciferin are provided herein. These kits can be used with
 bubble-blowing or producing toy.
 Toys that produce bubbles include bubbles with wand for blowing, bicycles,
 flying toys, dolls, swords, toy musical instruments, bubble beards, and
 numerous other toys are well known [see, e.g., U.S. Pat. No. RE 32,973,
 which describes a toy bubble-blowing lawn mower;, U.S. Pat. No. 4,511,497,
 which describes a non-toxic non-irritating bubble composition for toys,
 U.S. Pat. Nos. 2,579,714; 5,480,334; 5,041,042; 5,478,267; 5,462,469;
 5,419,728; 5,393,256; 5,366,402; 5,348,507; 5,322,464; 5,304,085;
 5,269,715; 5,224,893; 5,183,428; 5,181,875; 5,156,564; 5,135,422;
 5,080,623; 5,078,636; 4,957,464; 4,955,840; 4,943,255; 4,923,426,
 4,867,724; 4,861,303; 4,840,597 4,808,138; 4,804,346; 4,764,141;
 4,700,965; 4,556,392 4,334,383; 4,292,754; 4,246,717; and many others].
 3. Glowing textiles and paper products
 The bioluminescent systems described herein are also contemplated for use
 with textiles and paper. One or two of the bioluminescent system
 components are applied to the textile or paper and the remaining
 components are added when illumination is desired. For example, the
 luciferase in association with the bioluminescence substrate may be
 applied to the textile or paper, through covalent or non-covalent
 interaction. When water, or other appropriate activator, is applied to the
 material, illumination ensues. Examples of uses for the textile include
 the fabric portion of an umbrella, clothing, towels, the fabric portion of
 artificial plants or flowers, toys having a fabric component or any item
 susceptible to manufacture from textile material.
 With respect to paper, the luciferase may be applied to the paper in
 association with the bioluminescence substrate. The paper glows upon
 addition of the bioluminescence activator to the paper. Thus, if the
 bioluminescence activator is water, addition of water to the paper, for
 example as an aerosol, produce a glow on the paper. The paper may also be
 illuminated by "writing" upon it with one or two of the bioluminescent
 system components then "writing" or spraying over those components with
 the remaining component(s). As with the other systems disclosed herein,
 the critical aspect to operation is maintaining at least one of the
 bioluminescent system components separate from the other components until
 illumination is desired. The paper may be in almost any form or of almost
 any type, such as writing paper, wrapping paper, boxes, poster paper,
 books, paper jewelry, paper towels, napkins or other paper products.
 4. Foods and beverages, including ice cubes
 Examples of beverages and foodstuffs amenable to combination with
 bioluminescence systems include, but are not limited to, alcoholic
 beverages, as well as sodas and juices, and such foods as applesauce and
 mashed potatoes. Further, bioluminescent systems can be chosen and adapted
 for use in such foodstuffs as cakes and ice creams or almost any other
 edible item. Considerations in combining bioluminescence systems with food
 and/or beverages are primarily the stability of the system throughout
 processing of the food or beverage, unless the system is added subsequent
 to any such processing; the ability to contact the system with its finally
 required ingredients to produce bioluminescence; and taste of the
 components of the system with the foodstuffs to which they are added.
 Bioluminescent food products are also contemplated herein. Such products,
 amenable to illumination using the bioluminescent systems described
 herein, include those that may be stored between about 0.degree. C. and
 35.degree. C. Generally, once the luciferase or bioluminescence substrate
 is added to the food product, it cannot be heated above about 100.degree.
 C. Thus, food products requiring cooking prior to consumption also can be
 cooked prior to addition of either the luciferase or bioluminescence
 substrate.
 Examples of food products amenable to illumination using the bioluminescent
 systems described herein include, icings and other toppings or sauces,
 cookies, biscuits, and similar prepared foods. Bioluminescent icings, for
 example, may be prepared by including the luciferase and bioluminescence
 substrate in a dehydrated icing mixture. Addition of water, just prior to
 use causes the mixture to glow. Alternatively, the bioluminescence
 activator and either the luciferase or bioluminescence substrate may be
 included in the prepared icing mixture and the absent bioluminescent
 system component stirred into the icing just prior to use.
 a. Beverages
 Beverage products are likewise contemplated for use in combination with the
 bioluminescent systems described herein. As with other embodiments, at
 least one of the bioluminescent system components is excluded from the
 beverage until bioluminescence is desired. For example, a
 container/bladder apparatus, as described generally above and in detail
 below, maintains the luciferase and bioluminescence substrate separate
 from the beverage. Upon opening of the container, the luciferase and
 substrate are added to the beverage causing it to glow.
 Alternatively, the beverage may be produced and packaged already containing
 one or two of the bioluminescent system components, such that addition of
 the remaining components causes illumination. An example of such a
 beverage is bioluminescent beer, wine, champagne or a soft drink. In this
 embodiment, the yeast used to produce the alcohol component of the beer
 are genetically transformed to contain, for example, a gene encoding a
 luciferase and the complementary genes necessary to direct the yeast to
 manufacture and secrete the luciferase. Assuming O.sub.2 or air is the
 bioluminescence activator, then when illumination is desired, the
 bioluminescence substrate is simply added to the beer.
 Another example of a bioluminescent beverage contemplated herein is a soft
 drink containing two of the three bioluminescent system components. When
 luminescence is desired, the third bioluminescent system component is
 added. If the bioluminescent system is, for example, the Aequorin system
 or Renilla system, then the Aequorin luciferase with bound luciferin or
 the Renilla luciferase and the luciferin may be included in the soft drink
 and the bioluminescence activator, Ca.sup.2+ [for the aequorin system] or
 dissolved O.sub.2, added to the or in the beverage will result in cause
 illumination. Suitable vessels for such beverages are provided herein
 [see, EXAMPLES] and also are known to those of skill in the art [see,
 e.g., U.S. Pat. No. 5,398,827].
 b. Ice cubes
 Ice cubes containing bioluminescent components, such as lyophilized
 components or encapsulated components are contemplated herein. Upon
 addition to a beverage containing any remaining components, the contents
 of the cubes will be released as they melt into the beverage to produce a
 glow in the beverage.
 The components may also be combined with dry ice, which as it sublimes and
 releases the components contact with moisture condensing in the air will
 cause a glow.
 5. Jewelry, Clothing and Other Items of Manufacture
 The bioluminescence generating systems can be used in combination with
 articles of manufacture that include jewelry, clothing, figurines and
 other such items. In particular, these items may be manufactured from
 matrix materials or from mixtures of the matrix material and other
 materials. Alternatively, the matrix material may be coated on or
 impregnated in such articles. Bioluminescence generating reagents,
 particularly, luciferases can be linked to the matrix material. When a
 glow is desired the article can be contacted with composition containing
 the remaining components.
 In addition, articles, such as clothing, particularly, T-shirts and sports
 gear, and paper items may be sprayed with two compositions, the first
 containing less than all of the necessary reagents and the second
 containing the remaining reagents.
 In other embodiments, the article may be made of two vessels separated by a
 removable separating means, so that when desired components contained
 therein communicate and react resulting in bioluminescence.
 6. Fountains
 Numerous fountains and other water spraying apparatus and devices for use
 in such apparatus, in addition to those exemplified herein, are suitable
 for use in combination with the bioluminescent generating systems herein
 [see, e.g., U.S. Pat. Nos. 5,480,094; 5,472,140; 5,439,170; 5,402,836;
 5,388,285; 5,381,956; 5,337,956; 5,288,018; 5,167,368; 4,852,801;
 3,894,689; 3,889,880; 3,838,816; 3,820,715; 3,773,258; 3,749,311]. For use
 herein, the fountains will be modified or adapted [see, e.g., EXAMPLES] so
 that jets of liquid containing bioluminescent will spew.
 Fountains can be recharged, for example, by adding additional substrate and
 other activators. Spent substrate should be removed, such us by passing
 the water through an affinity matrix specific for the oxidized substrate.
 The following examples are included for illustrative purposes only and are
 not intended to limit the scope of the invention.
 EXAMPLE 1
 Dual Chamber Fluid Dispensing Apparatus--Toy Water Gun
 A preferred embodiment of the dual chamber fluid dispensing apparatus is a
 toy water gun as illustrated in FIGS. 1 through 3. The following
 description of that preferred embodiment is made with reference to those
 figures. The toy water gun includes two housings [or chambers] 10, 12 that
 conveniently may be constructed of injection-molded plastic or other
 suitable material. The two housings 10, 12 are affixed to one another,
 such as glued, heat sealed or by other such means, along a median seam 46
 to form the body of the water gun. See especially FIGS. 2 and 3.
 In operation, one housing 10 contains a mixture having less than all the
 components necessary for generating bioluminescence and the other housing
 12 contains a mixture having the remaining components or the remaining
 components, save the bioluminescent activator. Depression of the trigger
 14 pushes the pistons 26, 36 into their respective cylinders 38, 48
 compressing the trigger springs 28, 43 and pushing the contents of the
 cylinder through the second check-valve 34, into the mixing chamber 20 and
 out the nozzle orifice 22. As the trigger 14 is released, the trigger
 springs 28, 43 return to their relaxed state pushing the pistons 26, 36
 out of the cylinders 38, 48 creating a vacuum therein which pulls the
 contents of the housings 10, 12 past the first check-valves 33, 32,
 respectively and into the cylinders 38, 48 respectively. Pumping the
 trigger, that is repeatedly depressing and releasing it, moves the
 mixtures contained in the housings through the gun and out the nozzle
 orifice 22.
 As the mixtures leave the cylinders 38, 48, they enter the mixing chamber
 20, via the conduit means 44 and second check-valve 34. Luminescence
 begins either upon mixing of the components or as the mixed composition
 contacts the air upon expulsion from the toy gun. The mixtures may be
 powdered, such as those produced by lyophilization, or they may be liquid.
 If powdered, water can be added prior to use.
 The housings 10, 12 may be filled and refilled through the filling caps 17,
 19, respectively, located at the top of each housing. A trigger 14 is
 attached to a trigger guide 13 which serves to guide the trigger 14
 towards two piston assemblies 25. Depression of the trigger 14 activates
 the two piston assemblies 25. This causes a portion of the composition
 located in each housing 10, 12 to move through the water gun into a mixing
 chamber 20 and out a nozzle orifice 22. The preferred embodiment
 illustrated has a trigger guard 15 which helps prevent accidental
 discharge of the gun and makes the gun appear more realistic. The sighting
 aids 21, 23 aid in aiming the toy gun and also serve to make the gun
 appear realistic.
 Only one of the two piston assemblies 25 is completely illustrated, and it
 is visible in FIG. 1. The other piston assembly is adjacent and, in this
 preferred embodiment, identical to the one illustrated. Both assemblies
 operate by identical means and are activated by depression of the single
 trigger 14. The piston assembly 25 includes a piston 26 which passes
 through a sealing o-ring 30, is connected to a trigger spring 28 and moves
 within a cylinder 38. The piston assembly also includes a spring retainer
 40 that secures one end of the trigger spring 28 to the end wall of the
 cylinder. The cylinder 38 is in communication with one end of a pick-up
 tube 18 and lies about perpendicular to the pick-up tube 18. The cylinder
 38 also communicates with the mixing chamber 20 via conduit means 44.
 In the sectional views of the water gun, illustrated in FIGS. 2 and 3,
 portions of the second, adjacent piston assembly are visible. Namely, the
 second trigger spring retainer 42 and trigger spring 43 are visible in
 FIG. 2, and the second piston 36 is visible in FIG. 3.
 Referring to the piston assembly 25 illustrated in FIG. 1, the piston 26
 passes into the water gun through the sealing o-ring 30 and into the
 cylinder 38. The trigger spring 28 is attached by one end to the piston
 and by its other end to the spring retainer 40 located at the opposite end
 of the cylinder from the piston. As the trigger 14 is depressed, the
 piston 26 moves into the cylinder 38 and through the sealing o-ring 30.
 This compresses the trigger spring 28 within the cylinder 38. As the
 trigger 14 is released, the trigger spring 28 expands, returning it and
 the piston 26 to a resting position.
 Because the piston 26 is sealed within the cylinder 38 by the sealing
 o-ring 30, its repeated movement causes the air within the cylinder to be
 displaced thereby creating a vacuum within the pick-up tube 18 of the
 water gun. The composition located in the housing 12 is then drawn into
 the pick-up tube 18, past a first check valve 32, past the trigger spring
 28, past a second check valve 34, into the mixing chamber 20 and out the
 nozzle orifice 22 via an outlet tube 24. The second check valve 34 is
 illustrated with a spring mechanism 35 which serves to maintain the check
 valve 34 in a closed position isolating the piston assembly cylinders 28
 and conduit means 44 from the mixing chamber 20, allowing a vacuum to form
 within the gun during operation.
 The same mechanism operates to simultaneously withdraw composition from the
 complementary housing 10 into the mixing chamber 20 via a pick-up tube 16.
 Thus, referring to FIGS. 2 and 3, the action of the piston 36 within its
 cylinder compresses the trigger spring 43 against the spring retainer 42
 creating a vacuum within the pick-up tube 16 and moving some of the
 composition located in the housing 10 through the pick-up tube 16 into the
 mixing chamber 20 and out the nozzle orifice 22.
 As illustrated in FIG. 2, the two pick-up tubes 16 and 18 originate in the
 housings 10 and 12, respectively. Each pick-up tube 16, 18 includes a
 check valve 32 and 33, respectively. The first check valves 32, 33 serve
 to prevent fluid flow from the piston assembly cylinders 38, 48 back into
 the housings 10, 12. The single second check valve 34 prevents the mixed
 compositions from flowing out of the mixing chamber 20 back into the
 piston assembly cylinders 38, 48.
 Thus, repeated depression of the trigger 14 increases the pressure within
 the gun, thereby filling the mixing chamber 20 with a combination of the
 compositions located in the two housings 10, 12, then forcing the mixed
 compositions through the outlet-tube 24 and out the nozzle orifice 22.
 EXAMPLE 2
 Dual Chamber Fluid Dispensing Apparatus--Gas-Charged Toy Water Gun
 In contrast to the above-described toy water gun, the gas-charged toy water
 gun operates using pressurized gas, rather than the piston assembly, to
 move the bioluminescent mixtures through the system. A preferred
 embodiment of this device is illustrated in FIGS. 4 and 5. In this
 embodiment the butt of the water gun 86 houses the two chambers 64, 74
 that contain the bioluminescent system components. Further, the butt 86 is
 detachable and thus readily replaced.
 To pressurize the gun for operation, a CO.sub.2 or air [or other suitable
 gas or mixtures thereof] canister 50 is inserted into a gas chamber 56 as
 shown. A screw cap 52, located at the base of the gas chamber, secures the
 canister 50 into the chamber 56. As the screw cap 52 is tightened, the
 CO.sub.2 or air canister is forced against a piercing pin 54, thereby
 releasing CO.sub.2 or air into the gas chamber 56 and charging the water
 gun for use.
 Depression of a trigger 58 aligns a gas cock 60 with each of two gas
 conduits 62 and 72 and the gas chamber 56. With the gas cock 60
 so-aligned, CO.sub.2 gas or air enters the gas conduits 62 and 72 and
 passes into the two chambers 64 and 74. The pressure of the gas forces
 some of each mixture out of the chambers 64, 74, via composition pick-up
 tubes 66, 76. The composition pick-up tubes 66, 76 are connected to outlet
 conduits 78 and 80 through which the mixtures pass into a mixing chamber
 68, and are combined. The continued pressure of the CO.sub.2 gas or air
 forces the combined mixture from the mixing chamber 68 and out a nozzle
 orifice 70.
 The gas conduits 62, 72 and outlet conduits 78, 80 are housed within the
 main body of the water gun and extend beyond it in the region where the
 butt 86 of the gun is attached to the main body. The composition pick-up
 tubes 66, 76 are completely within the butt of the water gun 86. In order
 to obtain a leak-free assembly of the butt of the gun to the main body,
 the gas conduits 62, 72 and outlet conduits 78, 80 each pass through a
 leak seal 88 located within the butt of the gun 86. The leak seals 88 may
 be constructed of rubber or similar soft sealing material and should be
 covered, either with a removable cap or with a material susceptible to
 piercing, to prevent spillage of the compositions contained therein.
 In attaching the butt of the gun 86 to the main body, the gas conduits 62,
 72 and outlet conduits 78, 80 pass through the leak seals 88 forming a
 tight seal between the tubes and the butt of the gun. Also, as can be seen
 in FIG. 4, the delivery tubes 78, 80 set within the composition pick-up
 tubes 66, 76 at the point where they enter the butt of the gun. This
 permits fluid communication between the composition pick-up tubes 66, 76
 and the outlet conduits 78, 80.
 Additional features of the preferred embodiment, as illustrated in FIGS. 4
 and 5 include retaining hooks or latches 90, 92 and 94 positioned on the
 main body of the water gun and used to secure the butt of the gun to the
 main body. Additionally, the two chambers 64 and 74 can be configured with
 filler caps 82 and 84, as illustrated, thereby allowing them to be
 refilled as an alternative to replacement.
 It will be appreciated that the gas used to operate the gas-charged fluid
 dispensing apparatus described herein may be other than carbon dioxide.
 Any gas or mixture of gases, such as air or mixtures of O.sub.2 and
 CO.sub.2, that operates in the same manner may be used.
 EXAMPLE 3
 Dual Chamber Fluid Dispensing Apparatus--Gas-Charged
 FIGS. 6, 7 and 8 illustrate a preferred embodiment of a gas-charged fluid
 dispensing apparatus as provided herein. This embodiment may be adapted
 for particular uses; for example, it may be housed within a decorative
 sculpture, thereby functioning as a decorative water fountain. Alternative
 embodiments incorporating this embodiment are illustrated in FIGS. 4 and 5
 [EXAMPLE 2] and FIGS. 9 and 10 [EXAMPLE 4].
 Referring to FIGS. 6 and 7, the gas-charged dual chamber dispensing
 apparatus has two chambers 100 and 102. In a preferred embodiment as
 illustrated, the two chambers 100 and 102 are refillable via filler caps
 104 and 106 located on the upper end of the chambers. A gas chamber 108 is
 situated about equidistant from the two chambers and communicates with
 each of them via gas conduits 117. The gas conduits 117 end at gas inlets
 118 that communicate with the two chambers 100, 102. The gas inlets 118
 are positioned near the upper end of the chambers 100 and 102. While one
 gas inlet 118 is depicted, it is understood that each chamber 100, 102 has
 such an inlet.
 A gas canister 112 fits into the gas chamber 108, being secured therein by
 a screw cap 110. Screwing the screw cap 110 tightly into place forces the
 top of the gas canister 112 against a piercing needle 114, thereby
 releasing gas into the gas chamber 108. A gas control valve 116 is used to
 control the flow of the gas from the gas chamber 108 into the gas conduits
 118.
 A mixing chamber 124 is also situated about equidistant from the two
 chambers 100 and 102 and communicates with them via outlet conduit means
 122, such as fluid ports. The outlet conduits [fluid ports] 122 are
 located sufficiently near the bottom of the chambers 100 and 102 to permit
 the chamber contents to empty. Near the lower end of the two chambers 100,
 102 are fluid outlets that connect to the fluid ports 122. Blow-out plugs
 120 prevent the compositions contained herein from leaving the chambers
 and entering the fluid ports before activation of the device. One-way
 valves or similar devices can be substituted for the blow-out plugs 120.
 The mixing chamber 124, having bottom inlets and a top outlet, is
 associated with a nozzle 126, which may be attached or integral to the
 mixing chamber. Optionally, the nozzle 126 has a closure cap 132 distal to
 the mixing chamber 124.
 In a preferred embodiment, illustrated in FIGS. 6, 7 and 8, an upper
 support 130 is shown. This upper support 130 spans the upper ends of both
 chambers 100 and 102 and over the top end of the gas chamber 108. The gas
 conduits 118 and inlets 117 are within the upper support 130. The nozzle
 126 passes through the upper support 130 and is supported thereby.
 Also illustrated in this preferred embodiment, is a base support 123 that
 spans across the lower ends of the chambers 100 and 102 and that is
 integral to the mixing chamber 124. The fluid ports 122 connecting the
 chambers 100 and 102 with the mixing chamber 124 are contained within the
 base support 123 [see, FIGS. 6 and 7].
 To operate the basic dual chamber gas-charged fluid dispensing apparatus, a
 gas canister 112 containing gas under pressure, for example pressurized
 CO.sub.2, is inserted into the gas chamber 108. The screw cap 110 is
 tightened, forcing the gas canister against the piercing needle 114. As
 gas escapes from the canister, it fills the gas chamber. The gas control
 valve 116 is opened, permitting the gas to enter the gas conduits 117 and
 pass into the chambers 100 and 102 through the gas inlets 118.
 The pressure of the gas in the chambers pushes the mixtures therein against
 the blow-out plugs 120, or through the one-way valves, out the fluid
 outlets, into the fluid ports 122 or other fluid conduit means, and into
 the mixing chamber 124 via the bottom inlets. In the mixing chamber 124,
 the mixtures combine with one another while the continued pressure from
 the gas propels the combined mixtures through the nozzle 126 and out the
 nozzle orifice 128.
 EXAMPLE 4
 Dual Chamber Fluid Dispensing Apparatus--Volcano-Shaped Gas-Charged
 Apparatus
 FIGS. 9 and 10 illustrate a preferred embodiment of the gas-charged fluid
 dispensing apparatus illustrated in FIGS. 6, 7 and 8 and described above.
 In this embodiment, each chamber has a generally half-conical shape, or
 other suitable shape [depending upon the intended use], such that, when
 attached to one another they form, in this embodiment, a volcano-shaped
 apparatus. The gas chamber 160 and gas conduit 162 are defined by the
 inner walls 176, 178 of the chambers 150, 152, respectively. Similarly,
 the mixing chamber 170 and nozzle 172 are defined by the inner walls 176,
 178 of the chambers 150, 152, respectively.
 As in the apparatus, FIGS. 6, 7 and 8, a gas canister 154 is housed in the
 gas chamber 160 and is activated by tightening a gas screw-cap 156 which
 forces the gas canister 154 against a piercing needle 158 thereby
 releasing the gas into the gas chamber 160. The gas enters the gas
 conduits 162, forces out the blow-out plugs 164 and passes into the
 chambers 150, 152 via the gas inlets 166. Alternatively, a control valve,
 or other suitable control means, is situated between the gas chamber and
 gas conduits or within the gas conduit means and used to control the flow
 of gas into the gas chambers.
 Within the two chambers 150, 152, one containing, for example, up to all
 except one component necessary for the bioluminescence generating reaction
 and the other the remaining component(s), the gas forces the
 bioluminescence generating mixtures into the mixing chamber 170. Blow-out
 plugs 168, situated between the chambers 150, 152 and mixing chamber 170,
 prevent the bioluminescence mixtures from entering the mixing chamber 170
 until the apparatus is activated. The continued pressure of the gas forces
 the combined mixtures from the mixing chamber 170 through the nozzle 172
 and out the nozzle orifice 174.
 This apparatus is particularly designed for use as "fireworks" configured
 in the shape of a volcano. As the combined bioluminescent mixtures are
 forced from the apparatus into the air, they glow in a similar manner to
 traditional fireworks.
 Alternatives to the specific embodiment described herein are likewise
 contemplated. For example, blow-out plugs may be replaced by one-way or
 control valves. Manually operated valves may be replaced by electronically
 or mechanically controlled valves. The apparatus does not have to be in
 the shape of a volcano, but may be formed into any shape, such as animals,
 humans, plants or abstract forms.
 EXAMPLE 5
 Compressible Dispensing Apparatus--Lotion/Cream container
 FIG. 11 illustrates a preferred embodiment of a compressible dispensing
 apparatus particularly useful for dispensing waxy, pasty or semi-solid
 compositions such as body lotions or finger paints. In this embodiment,
 the container, preferably a tube, has two chambers 200, 202. In certain
 embodiments, within one chamber are all, except for one or more,
 components of the bioluminescence generating system, and in the other
 chamber are the remaining components. The composition, such as body lotion
 or cream is in one or, preferably both chambers. The container is
 preferably constructed of a pliable collapsible or compressible material,
 such as plastic, plastic/metal laminate or similar collapsible composite,
 which can be squeezed by hand. Numerous such tubes are known to those of
 skill in this art are used to dispense products such as finger paints,
 toothpaste, gels, lotions and other such items.
 A membrane seal 204 at the top end [dispensing end] of the container
 prevents the contents of the chambers from mixing. The cap apparatus 206
 of the container has a dispensing cap at the top 210 and is configured
 such that a space 208 exists between the membrane seal 204 and the
 dispensing cap 210, which space acts as a mixing chamber 208.
 Thus, to operate the lotion/cream container, the membrane seal 204 is
 punctured, or otherwise opened, and a portion of the contents of the two
 chambers 200, 202 are simultaneously squeezed into the mixing chamber 208
 by applying pressure to the container. The dispensing cap 210 is removed
 and the contents of the mixing chamber 208 are squeezed out the dispensing
 orifice 212. The mixed composition may be dispensed by squeezing the
 container or by squeezing the cap apparatus 206. Alternatively, a
 plunger/syringe device [not illustrated] may be attached to the dispensing
 orifice and the mixed cream composition thereby withdrawn from the mixing
 chamber 208.
 The membrane seal, 204 situated between the chambers 200, 202 and the
 mixing chamber 208, functions to prevent the contents of the mixing
 chamber 208 from returning into either of the chambers 200, 202. It may be
 constructed, for example, of a thin layer of rubber, plastic, or other
 suitable porous material, having a small hole or holes through which the
 contents pass. As the sides of the container are compressed, portions of
 the contents of the chambers are forced through the holes in the membrane
 and into the mixing chamber, with the membrane returning to its "sealed"
 state once the pressure is relieved. A one-way valve or similar device may
 be substituted for the membrane seal 204, provided it too prevents the
 contents of the mixing chamber 208 from flowing back into either of the
 chambers 200, 202.
 EXAMPLE 6
 Bottle/Bladder Apparatus--Bubble Composition Bottle
 FIGS. 12 and 13 illustrate a preferred embodiment of the bottle/bladder
 apparatus adapted for use with bioluminescent bubble compositions. This
 bubble composition bottle has a bladder 300 positioned within it and held
 in place, in the neck 302 of the bottle, by friction. A collar 304 is
 positioned on the neck of the bottle 302, preventing the cap 306 from
 being screwed completely onto the top of the bottle. The cap 306 contains
 a plunger 308 which operates to push the bladder 300 into the body of the
 bottle when the collar 304 is removed and the cap 306 is screwed down
 tightly. Upon entering the body of the bottle, the bladder is pierced by a
 piercing pin 310 located on the bottom of the bottle; thereby releasing
 the contents of the bladder into the bottle. FIG. 13 shows the bottle with
 the collar 304 removed, the cap 306 screwed on tightly, and the bladder
 300 collapsed within it.
 Component(s) [less than all] of the bioluminescence generating reaction are
 contained in the bladder. The components may be in the form of a solution,
 suspension, suspended particles, or particles. Prior to use the bottle may
 be gently agitated. The particles may be time release capsules that
 release their contents upon exposure to the bubble composition or from
 which the contents diffuse upon mixing of the contents of the bladder with
 the bubble composition. The remaining component(s), such as Ca.sup.2+ or
 ATP, are contained in the bubble composition 314, which is preferably a
 mild bubble forming composition. Selection of the bioluminescence
 generating composition depends upon the selected bubble composition and
 also the desired action. In other embodiments, remaining components, such
 as ATP, FMN, a flavin reductase or other component that may be somewhat
 sensitive to the bubble composition, of the bioluminescence generating
 system may be added to the bubble composition just prior to use.
 The collar 304 of the bottle is adapted with a bubble blowing ring 312,
 with arm, integral thereto. Thus, the collar 304 is removed, the bladder
 300 pierced within the bottle as described and the bubble blowing ring 312
 dipped into the mixed composition, withdrawn and bioluminescent bubbles
 blown. A standard bubble blowing wand [arm with ring] may be used and/or
 provided in place of that depicted in FIG. 12.
 The bladder 300 should be constructed of a material that can be pierced by
 a piercing means, such as a needle or pin, made for example of thin
 plastic or other polymeric film. Preferably the distance from the base of
 the neck of the bottle to the tip of the piercing needle is less than the
 length of the bladder, so that the bladder will be pierced by the needle
 before its top edge clears the base of the neck of the bottle.
 The bottle 316 may be fabricated of any material ordinarily used for
 dispensing bubbles. It may be transparent or translucent to the
 bioluminescent light so that any glow in the bottle can be seen.
 EXAMPLE 7
 Container/Bladder Apparatus--Beverage Can
 A preferred embodiment of the container/bladder apparatus, illustrated in
 FIG. 14, is a beverage can or bottle. It is configured similarly to a
 pop-top aluminum drink can but has a bladder 400 under the top which is
 pierced by the pop-top 402 when the can is opened. The bladder may be
 centered under the top of the can, as illustrated, may be off-center or
 may be attached to the top and side of the can. Positioning of the bladder
 is chosen such that it may be readily pierced and its contents mixed with
 the contents of the container 404. Thus, the bladder should be
 sufficiently thin that the pop-top 402 is able to pierce it allowing its
 contents to mix with the contents of the beverage can.
 The can is preferably fabricated of translucent or transparent material
 such that the glowing beverage can be observed.
 An alternative embodiment includes a beverage container with two pop-tops,
 in which one is designed, such as including by having a point at the end,
 to puncture the bladder and the other can be a typical pop-top that is
 used for emptying the contents of the can, such as by pouring into a glass
 or into a person's mouth. Since the novelty of these items resides in the
 resulting glow in the beverage, the beverage should be poured into a
 glass, or the container should be transparent or translucent to the
 bioluminescent light.
 Another alternative contemplated herein includes a mesh filter surrounding
 the bladder and functioning to prevent small pieces of the ruptured
 bladder from mixing with the contents of the can. The contents of the
 bladder are in aqueous composition; thus, the density of the mesh of the
 filter that is permeable to the luciferase and other bioluminescence
 generating components.
 Similarly, embodiments employing other opening types are contemplated
 herein. For example, the bladder and corresponding container opening may
 be pierced with a point-ended straw, or other sharp device. Likewise, the
 dispensing opening [which may be the same as the bladder-associated
 opening] may be covered with a thin aluminum pull tab. Critical to the
 operation of the can/bladder combination is that the bladder preclude
 mixing of the contents of the bladder and the can until the consumer takes
 action to rupture the bladder.
 The bladder may be constructed of any material which is amenable to being
 pierced as described and is preferably constructed of a material which
 will rarely if ever break into small pieces when pierced. For example,
 aluminum foil with a thin plastic coating, when pierced with a point-ended
 straw in particular, will rarely break into small pieces. The body of the
 can may be constructed of aluminum, plastic or similar material and is
 preferably constructed of a translucent material such as plastic.
 The bladder includes up to all except for one component of the
 bioluminescent generating system, and the beverage includes the remaining
 component. For example, the bladder includes the aequorin photoprotein
 [typically 0.1 to 1 mg or more] in a composition containing a chelator to
 prevent activation of the photoprotein, and the beverage contains
 Ca.sup.2+.
 EXAMPLE 8
 Single Use, Dual Chamber Fluid Packaging Apparatus
 FIG. 15 illustrates a preferred embodiment of the single use, dual chamber
 fluid packaging apparatus or bottle described generally above, and the
 following description is with reference to that FIGURE. The bottle has a
 first chamber 500 which contains a composition including one or more, up
 to all but one, of the bioluminescent system components. Below the first
 chamber and operatively attached thereto, is a second chamber 502,
 containing the remaining bioluminescent system components in composition.
 In the preferred embodiment illustrated, the first chamber is seated in
 the first chamber along a side seam 506 and a separation membrane 504.
 The second chamber 502 is constructed of pliable material, such as plastic,
 that is convoluted 508 such that it can be readily collapsed against the
 bottom of the first chamber in the direction of the illustrated arrow.
 When collapsed in this way, the force of the composition contained within
 the second chamber ruptures the separation membrane 504A, permitting the
 compositions to mix. Once mixed, the compositions begin to illuminate.
 This apparatus, as illustrated, is adapted for use with bubble-blowing
 compositions in that the cap of the bottle 510 has a bubble-blowing wand
 512 attached to it. Alternatively, the apparatus may be used with a
 beverage and, if so used, would not have the illustrated bubble-blowing
 wand 512.
 Another embodiment of this apparatus, not illustrated, but contemplated
 herein, is the bottle wherein the second chamber may be secured to the
 first chamber or to itself in a collapsed position. For example, the
 second chamber can be adapted with a hooking mechanism on its exterior
 such that it can be hooked to itself when collapsed.
 EXAMPLE 9
 Cap Apparatus for Use with Composition Vessels
 FIGS. 16, 17 and 18 & 19 illustrate three preferred embodiments of the cap
 apparatus for use with composition vessels.
 A. Cork Cap Apparatus
 Referring to FIG. 16, a cork 600, situated within the neck 602 of a bottle
 and having a rupturable capsule 604 housed within it, is illustrated. In
 this embodiment, the bottom edge of the cork 600 is about U-shaped such
 that a pocket is formed. Contained within the pocket is the capsule which
 is in communication with the screen 608 which is permanently attached to
 the bottom of the cork. The capsule contains one or more, up to all but
 one, of the bioluminescent system components. A plunger assembly 606 is
 positioned, partially within the cork, such that depression of the plunger
 assembly 606 results in rupture of the capsule and release of its contents
 into the composition within the bottle. The screen 608 or other filtering
 device prevents fragments of the ruptured capsule from entering the
 vessel.
 The plunger assembly 606, illustrated in FIG. 16, has a top portion 610
 integral to the stem portion 612. Pressing on the top portion 610 forces
 the stem 612 to move within the cork 600 and against the capsule 604,
 thereby rupturing the capsule and releasing its contents into the vessel.
 FIG. 17 illustrates an alternative embodiment of the cork cap apparatus. In
 this embodiment, the cork 700 is illustrated as being about flush with the
 top of the neck 702 of the bottle. The plunger apparatus 704 is adapted
 with a finger ring 706 for ease in handling. The stem 708, which may be
 pointed or blunt or any combination thereof, is threaded 710. In
 operation, the plunger assembly 704 is screwed into the cork 700 where it
 contacts a capsule 712, rupturing it and releasing its contents against
 the screen 714 or filter. The capsule will preferably contain powdered
 bioluminescence generating components.
 It will be appreciated that the cork cap alone, with encapsulated
 compositions encased within and screen or filter attached thereto, is an
 alternative embodiment of the two illustrated cork cap apparatus. In this
 embodiment a corkscrew may be employed to rupture the capsule and to
 remove the cork cap.
 B. Screw-top Cap Apparatus
 FIGS. 18 and 19 illustrate another preferred embodiment of the cap
 apparatus for use with composition vessels. FIG. 18 shows the cap
 apparatus before activation or engagement. This is particularly adapted
 for use with a wine or champagne bottle, and includes encapsulated
 bioluminescence generating system components.
 This preferred embodiment generally comprises a bottle-shaped vessel with a
 collar 802 situated about the neck 804 of the bottle and a cap 800
 attached to the top of the bottle just above the collar 802. The neck of
 the bottle 804 is threaded to receive the screw-on cap 800. The collar 802
 is situated such that a lower portion of the threads on the neck of the
 bottle 804 are covered thereby preventing the screw-on cap 800 from being
 completely attached to the bottle. Enough threads remain exposed on the
 top of the bottle such that the screw-on cap 800 is securely, though not
 completely, attached to the top of the bottle.
 The screw-on cap 800 has a plunger 806 integral thereto which extends into
 the bottle neck 804. A screen or filter assembly 812 is attached to the
 interior of the bottle within the bottle neck 804. A membrane system 808,
 810 or capsule or similar composition packaging is situated between the
 plunger 806 of the screw-on cap 800 and the screen/filter assembly 812. In
 operation, the collar 802 is removed, for example by removing the
 screw-cap 800 and lifting off or screwing off the collar 802 or by tearing
 off the collar 802, and the screw-on cap 800 is tightened against the top
 of the bottle. This forces the plunger 806 through the membranes 808, 810,
 rupturing them and releasing the composition(s) contained therein. The
 composition(s) pass through the screen assembly 812 and are mixed with the
 contents of the bottle. FIG. 19 illustrates the cap apparatus fully
 engaged with the membrane system ruptured.
 In the preferred embodiment illustrated, the screen assembly 812 is
 attached along the interior of the neck of the bottle 804 as well as
 across the interior of the neck, thereby forming a basket within which the
 membrane system 808, 810 sits. Alternatively, the screen assembly can be
 attached around the circumference of the bottle neck only and not along
 its sides to the top of the bottle, as illustrated.
 The precise height of the collar 802 will be determined by the length of
 the plunger 806 and location of the membrane system 808, 810. The height
 will be sufficient to prevent the plunger 806 from being engaged through
 the membrane system 808, 810 prior to activation by the user, while
 permitting the screw-on cap 800 to be secured to the top of the bottle.
 The membrane system 808, 812 contains one or more, up to all but one, of
 the bioluminescent system components. Typically the those components will
 consist of the luciferase and luciferin in lyophilized form.
 The illustrated preferred embodiment is shown and described as attached to
 a bottle. It will be appreciated, however, that the vessel to which the
 cap apparatus is attached may be a can, tube or any other container.
 Additionally, the preferred embodiment is described and illustrated with
 reference to the neck of the bottle. It is not necessary that the vessel
 have a "neck" for the cap apparatus to function. For example, if the
 vessel does not have a neck, other means may be employed to hold the
 collar in place below the screw-on cap, such as, a lip formed on the
 container, below the threads, to stop the collar at an approriate point.
 With respect to all three preferred embodiments of the cap apparatus
 adapted for use with composition vessels, the stem of the plunger assembly
 is short enough not to pierce the screen or filter device, yet long enough
 to effectively rupture the capsule, membrane or other packaging once
 engaged. The bioluminescent system component(s) contained within the cap
 apparatus may be powdered or in composition or in any form amenable to
 addition to the composition contained within the vessel. Additionally, the
 components may be contained in more than one capsule, membrane or other
 packaging. In this case, the component packages are positioned adjacent
 one another, such that each is ruptured by engagement of the plunger.
 Preferably, the remaining components required for completion of the
 bioluminescent reaction are contained within in the vessel with in any
 compostion. These preferred embodiments are particularly adapted to use
 with wine or champagne or other beverage.
 Since modifications will be apparent to those of skill in this art, it is
 intended that this invention be limited only by the scope of the appended
 claims.
 Summary of Sequences of Representative luciferases and the reductase set
 forth in the Sequence Listing
 1. SEQ ID NO. 1 Renilla reinformis Luciferase [U.S. Pat. No. 5,418,155]
 2. SEQ ID NO. 2 Cypridina hilgendorfii luciferase [EP 0 387 355]
 3. SEQ ID NO. 3 Modified Luciola Cruciata Luciferase [firefly; U.S. Pat.
 No. 4,968,613]
 4. SEQ ID NO. 4 Vargula (Cypridina) luciferase [Thompson et al. (1989)
 Proc. Natl. Acad. Sci. U.S.A. 86:6567-6571 and from JP 3-30678 Osaka
 5. SEQ ID NO. 5 Apoaequorin-encoding gene [U.S. Pat. No. 5,093,240, pAQ440]
 6. SEQ ID NO. 6 Recombinant Aequorin AEQ1 [Prasher et al. (1987) "Sequence
 Comparisons of cDNAs Encoding for Aequorin Isotypes," Biochemistry
 26:1326-1332]
 7. SEQ ID NO. 7 Recombinant Aequorin AEQ2 [Prasher et al. (1987)]
 8. SEQ ID NO. 8 Recombinant Aequorin AEQ3 [Prasher et al. (1987)]
 9. SEQ ID NO. 9 Aequorin photoprotein [Charbonneau et al. (1985) "Amino
 Acid Sequence of the Calcium-Dependent Photoprotein Aequorin,"
 Biochemistry 24:6762-6771]
 10. SEQ ID NO. 10 Aequorin mutant with increased bioluminescence activity
 [U.S. Pat. No. 5,360,728; Asp 124 changed to Ser]
 11. SEQ ID NO. 11 Aequorin mutant with increased bioluminescence activity
 [U.S. Pat. No. 5,360,728; Glu 135 changed to Ser]
 12. SEQ ID NO. 12 Aequorin mutant with increased bioluminescence activity
 [U.S. Pat. No. 5,360,728 Gly 129 changed to Ala]
 13. SEQ ID NO. 13 Recombinant apoaequorin [sold by Sealite, Sciences,
 Bogart, Ga. as AQUALITE.RTM., when reconstituted to form aequorin]
 14. SEQ ID NO. 14 Vibrio Fisheri Flavin reductase [U.S. Pat. No. 5,484,723]

SEQUENCE LISTING
 (1) GENERAL INFORMATION:
 (iii) NUMBER OF SEQUENCES: 14
 (2) INFORMATION FOR SEQ ID NO:1:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 1196 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (vi) ORIGINAL SOURCE:
 (ix) FEATURE:
 (A) NAME/KEY: Coding Sequence
 (B) LOCATION: 1...942
 (D) OTHER INFORMATION: Renilla Reinformis Luciferase
 (x) PUBLICATION INFORMATION:
 (H) DOCUMENT NUMBER: 5,418,155
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
 AGC TTA AAG ATG ACT TCG AAA GTT TAT GAT CCA GAA CAA AGG AAA CGG 48
 Ser Leu Lys Met Thr Ser Lys Val Tyr Asp Pro Glu Gln Arg Lys Arg
 1 5 10 15
 ATG ATA ACT GGT CCG CAG TGG TGG GCC AGA TGT AAA CAA ATG AAT GTT 96
 Met Ile Thr Gly Pro Gln Trp Trp Ala Arg Cys Lys Gln Met Asn Val
 20 25 30
 CTT GAT TCA TTT ATT AAT TAT TAT GAT TCA GAA AAA CAT GCA GAA AAT 144
 Leu Asp Ser Phe Ile Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn
 35 40 45
 GCT GTT ATT TTT TTA CAT GGT AAC GCG GCC TCT TCT TAT TTA TGG CGA 192
 Ala Val Ile Phe Leu His Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg
 50 55 60
 CAT GTT GTG CCA CAT ATT GAG CCA GTA GCG CGG TGT ATT ATA CCA GAT 240
 His Val Val Pro His Ile Glu Pro Val Ala Arg Cys Ile Ile Pro Asp
 65 70 75 80
 CTT ATT GGT ATG GGC AAA TCA GGC AAA TCT GGT AAT GGT TCT TAT AGG 288
 Leu Ile Gly Met Gly Lys Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg
 85 90 95
 TTA CTT GAT CAT TAC AAA TAT CTT ACT GCA TGG TTG AAC TTC TTA ATT 336
 Leu Leu Asp His Tyr Lys Tyr Leu Thr Ala Trp Leu Asn Phe Leu Ile
 100 105 110
 TAC CAA AGA AGA TCA TTT TTT GTC GGC CAT GAT TGG GGT GCT TGT TTG 384
 Tyr Gln Arg Arg Ser Phe Phe Val Gly His Asp Trp Gly Ala Cys Leu
 115 120 125
 GCA TTT CAT TAT AGC TAT GAG CAT CAA GAT AAG ATC AAA GCA ATA GTT 432
 Ala Phe His Tyr Ser Tyr Glu His Gln Asp Lys Ile Lys Ala Ile Val
 130 135 140
 CAC GCT GAA AGT GTA GTA GAT GTG ATT GAA TCA TGG GAT GAA TGG CCT 480
 His Ala Glu Ser Val Val Asp Val Ile Glu Ser Trp Asp Glu Trp Pro
 145 150 155 160
 GAT ATT GAA GAA GAT ATT GCG TTG ATC AAA TCT GAA GAA GGA GAA AAA 528
 Asp Ile Glu Glu Asp Ile Ala Leu Ile Lys Ser Glu Glu Gly Glu Lys
 165 170 175
 ATG GTT TTG GAG AAT AAC TTC TTC GTG GAA ACC ATG TTG CCA TCA AAA 576
 Met Val Leu Glu Asn Asn Phe Phe Val Glu Thr Met Leu Pro Ser Lys
 180 185 190
 ATC ATG AGA AAG TTA GAA CCA GAA GAA TTT GCA GCA TAT CTT GAA CCA 624
 Ile Met Arg Lys Leu Glu Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro
 195 200 205
 TTC AAA GAG AAA GGT GAA GTT CGT CGT CCA ACA TTA TCA TGG CCT CGT 672
 Phe Lys Glu Lys Gly Glu Val Arg Arg Pro Thr Leu Ser Trp Pro Arg
 210 215 220
 GAA ATC CCG TTA GTA AAA GGT GGT AAA CCT GAC GTT GTA CAA ATT GTT 720
 Glu Ile Pro Leu Val Lys Gly Gly Lys Pro Asp Val Val Gln Ile Val
 225 230 235 240
 AGG AAT TAT AAT GCT TAT CTA CGT GCA AGT GAT GAT TTA CCA AAA ATG 768
 Arg Asn Tyr Asn Ala Tyr Leu Arg Ala Ser Asp Asp Leu Pro Lys Met
 245 250 255
 TTT ATT GAA TCG GAT CCA GGA TTC TTT TCC AAT GCT ATT GTT GAA GGC 816
 Phe Ile Glu Ser Asp Pro Gly Phe Phe Ser Asn Ala Ile Val Glu Gly
 260 265 270
 GCC AAG AAG TTT CCT AAT ACT GAA TTT GTC AAA GTA AAA GGT CTT CAT 864
 Ala Lys Lys Phe Pro Asn Thr Glu Phe Val Lys Val Lys Gly Leu His
 275 280 285
 TTT TCG CAA GAA GAT GCA CCT GAT GAA ATG GGA AAA TAT ATC AAA TCG 912
 Phe Ser Gln Glu Asp Ala Pro Asp Glu Met Gly Lys Tyr Ile Lys Ser
 290 295 300
 TTC GTT GAG CGA GTT CTC AAA AAT GAA CAA TAA TTACTTTGGT TTTTTATTTA 965
 Phe Val Glu Arg Val Leu Lys Asn Glu Gln
 305 310
 CATTTTTCCC GGGTTTAATA ATATAAATGT CATTTTCAAC AATTTTATTT TAACTGAATA 1025
 TTTCACAGGG AACATTCATA TATGTTGATT AATTTAGCTC GAACTTTACT CTGTCATATC 1085
 ATTTTGGAAT ATTACCTCTT TCAATGAAAC TTTATAAACA GTGGTTCAAT TAATTAATAT 1145
 ATATTATAAT TACATTTGTT ATGTAATAAA CTCGGTTTTA TTATAAAAAA A 1196
 (2) INFORMATION FOR SEQ ID NO:2:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 1822 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (ix) FEATURE:
 (A) NAME/KEY: Coding Sequence
 (B) LOCATION: 1...1665
 (D) OTHER INFORMATION: Cypridina hilgendorfii luciferase
 (x) PUBLICATION INFORMATION:
 (H) DOCUMENT NUMBER: EP 0 387 355 TORAY
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
 ATG AAG CTA ATA ATT CTG TCT ATT ATA TTG GCC TAC TGT GTC ACA GTC 48
 Met Lys Leu Ile Ile Leu Ser Ile Ile Leu Ala Tyr Cys Val Thr Val
 1 5 10 15
 AAC TGC CAG GAT GCA TGT CCT GTA GAA GCT GAA GCA CCG TCA AGT ACA 96
 Asn Cys Gln Asp Ala Cys Pro Val Glu Ala Glu Ala Pro Ser Ser Thr
 20 25 30
 CCA ACA GTC CCA ACA TCT TGT GAA GCT AAA GAA GGA GAA TGT ATC GAT 144
 Pro Thr Val Pro Thr Ser Cys Glu Ala Lys Glu Gly Glu Cys Ile Asp
 35 40 45
 ACC AGA TGC GCA ACA TGT AAA CGA GAC ATA CTA TCA GAC GGA CTG TGT 192
 Thr Arg Cys Ala Thr Cys Lys Arg Asp Ile Leu Ser Asp Gly Leu Cys
 50 55 60
 GAA AAT AAA CCA GGG AAG ACA TGC TGT AGA ATG TGC CAG TAT GTA ATT 240
 Glu Asn Lys Pro Gly Lys Thr Cys Cys Arg Met Cys Gln Tyr Val Ile
 65 70 75 80
 GAA TCC AGA GTA GAA GCT GCT GGA TAT TTT AGA ACG TTT TAC GCC AAA 288
 Glu Ser Arg Val Glu Ala Ala Gly Tyr Phe Arg Thr Phe Tyr Ala Lys
 85 90 95
 AGA TTT AAT TTT CAG GAA CCT GGT AAA TAT GTG CTG GCT CGA GGA ACC 336
 Arg Phe Asn Phe Gln Glu Pro Gly Lys Tyr Val Leu Ala Arg Gly Thr
 100 105 110
 AAG GGT GGC GAC TGG TCT GTA ACC CTC ACC ATG GAG AAT CTA GAT GGA 384
 Lys Gly Gly Asp Trp Ser Val Thr Leu Thr Met Glu Asn Leu Asp Gly
 115 120 125
 CAG AAG GGA GCT GTA CTG ACT AAG ACA ACA CTG GAG GTA GTA GGA GAC 432
 Gln Lys Gly Ala Val Leu Thr Lys Thr Thr Leu Glu Val Val Gly Asp
 130 135 140
 GTA ATA GAC ATT ACT CAA GCT ACT GCA GAT CCT ATC ACA GTT AAC GGA 480
 Val Ile Asp Ile Thr Gln Ala Thr Ala Asp Pro Ile Thr Val Asn Gly
 145 150 155 160
 GGA GCT GAC CCA GTT ATC GCT AAC CCG TTC ACA ATT GGT GAG GTG ACC 528
 Gly Ala Asp Pro Val Ile Ala Asn Pro Phe Thr Ile Gly Glu Val Thr
 165 170 175
 ATT GCT GTT GTC GAA ATA CCC GGC TTC AAT ATT ACA GTC ATC GAA TTC 576
 Ile Ala Val Val Glu Ile Pro Gly Phe Asn Ile Thr Val Ile Glu Phe
 180 185 190
 TTT AAA CTA ATC GTG ATA GAT ATT CTG GGA GGA AGA TCT GTG AGA ATT 624
 Phe Lys Leu Ile Val Ile Asp Ile Leu Gly Gly Arg Ser Val Arg Ile
 195 200 205
 GCT CCA GAC ACA GCA AAC AAA GGA CTG ATA TCT GGT ATC TGT GGT AAT 672
 Ala Pro Asp Thr Ala Asn Lys Gly Leu Ile Ser Gly Ile Cys Gly Asn
 210 215 220
 CTG GAG ATG AAT GAC GCT GAT GAC TTT ACT ACA GAC GCA GAT CAG CTG 720
 Leu Glu Met Asn Asp Ala Asp Asp Phe Thr Thr Asp Ala Asp Gln Leu
 225 230 235 240
 GCG ATC CAA CCC AAC ATA AAC AAA GAG TTC GAC GGC TGC CCA TTC TAC 768
 Ala Ile Gln Pro Asn Ile Asn Lys Glu Phe Asp Gly Cys Pro Phe Tyr
 245 250 255
 GGG AAT CCT TCT GAT ATC GAA TAC TGC AAA GGT CTC ATG GAG CCA TAC 816
 Gly Asn Pro Ser Asp Ile Glu Tyr Cys Lys Gly Leu Met Glu Pro Tyr
 260 265 270
 AGA GCT GTA TGT CGT AAC AAT ATC AAC TTC TAC TAT TAC ACT CTG TCC 864
 Arg Ala Val Cys Arg Asn Asn Ile Asn Phe Tyr Tyr Tyr Thr Leu Ser
 275 280 285
 TGC GCC TTC GCT TAC TGT ATG GGA GGA GAA GAA AGA GCT AAA CAC GTC 912
 Cys Ala Phe Ala Tyr Cys Met Gly Gly Glu Glu Arg Ala Lys His Val
 290 295 300
 CTT TTC GAC TAT GTT GAG ACA TGC GCT GCA CCG GAA ACG AGA GGA ACG 960
 Leu Phe Asp Tyr Val Glu Thr Cys Ala Ala Pro Glu Thr Arg Gly Thr
 305 310 315 320
 TGT GTT TTA TCA GGA CAT ACT TTC TAT GAC ACA TTC GAC AAA GCC AGA 1008
 Cys Val Leu Ser Gly His Thr Phe Tyr Asp Thr Phe Asp Lys Ala Arg
 325 330 335
 TAT CAA TTC CAG GGC CCA TGC AAA GAG CTT CTG ATG GCC GCA GAC TGT 1056
 Tyr Gln Phe Gln Gly Pro Cys Lys Glu Leu Leu Met Ala Ala Asp Cys
 340 345 350
 TAC TGG AAC ACA TGG GAT GTA AAG GTT TCA CAT AGA GAT GTT GAG TCA 1104
 Tyr Trp Asn Thr Trp Asp Val Lys Val Ser His Arg Asp Val Glu Ser
 355 360 365
 TAC ACT GAG GTA GAG AAA GTA ACA ATC AGG AAA CAG TCA ACT GTA GTA 1152
 Tyr Thr Glu Val Glu Lys Val Thr Ile Arg Lys Gln Ser Thr Val Val
 370 375 380
 GAT TTG ATT GTG GAT GGC AAG CAG GTC AAG GTT GGA GGA GTG GAT GTA 1200
 Asp Leu Ile Val Asp Gly Lys Gln Val Lys Val Gly Gly Val Asp Val
 385 390 395 400
 TCT ATC CCG TAC AGT TCT GAG AAC ACA TCC ATA TAC TGG CAG GAT GGA 1248
 Ser Ile Pro Tyr Ser Ser Glu Asn Thr Ser Ile Tyr Trp Gln Asp Gly
 405 410 415
 GAC ATC CTG ACG ACG GCC ATC CTA CCT GAA GCT CTT GTC GTT AAG TTC 1296
 Asp Ile Leu Thr Thr Ala Ile Leu Pro Glu Ala Leu Val Val Lys Phe
 420 425 430
 AAC TTT AAG CAG CTC CTT GTA GTT CAT ATC AGA GAT CCA TTC GAT GGA 1344
 Asn Phe Lys Gln Leu Leu Val Val His Ile Arg Asp Pro Phe Asp Gly
 435 440 445
 AAG ACA TGC GGC ATA TGT GGT AAC TAT AAT CAA GAT TCA ACT GAT GAT 1392
 Lys Thr Cys Gly Ile Cys Gly Asn Tyr Asn Gln Asp Ser Thr Asp Asp
 450 455 460
 TTC TTT GAC GCA GAA GGA GCA TGC GCT CTG ACC CCC AAT CCC CCA GGA 1440
 Phe Phe Asp Ala Glu Gly Ala Cys Ala Leu Thr Pro Asn Pro Pro Gly
 465 470 475 480
 TGT ACA GAG GAG CAG AAA CCA GAA GCT GAG CGA CTC TGC AAT AGT CTA 1488
 Cys Thr Glu Glu Gln Lys Pro Glu Ala Glu Arg Leu Cys Asn Ser Leu
 485 490 495
 TTT GAT AGT TCT ATC GAC GAG AAA TGT AAT GTC TGC TAC AAG CCT GAC 1536
 Phe Asp Ser Ser Ile Asp Glu Lys Cys Asn Val Cys Tyr Lys Pro Asp
 500 505 510
 CGT ATT GCA CGA TGT ATG TAC GAG TAT TGC CTG AGG GGA CAG CAA GGA 1584
 Arg Ile Ala Arg Cys Met Tyr Glu Tyr Cys Leu Arg Gly Gln Gln Gly
 515 520 525
 TTC TGT GAC CAT GCT TGG GAG TTC AAA AAA GAA TGC TAC ATA AAG CAT 1632
 Phe Cys Asp His Ala Trp Glu Phe Lys Lys Glu Cys Tyr Ile Lys His
 530 535 540
 GGA GAC ACT CTA GAA GTA CCA CCT GAA TGC CAA TAA ATGAACAAAG 1678
 Gly Asp Thr Leu Glu Val Pro Pro Glu Cys Gln
 545 550 555
 ATACAGAAGC TAAGACTACT ACAGCAGAAG ATAAAAGAGA AGCTGTAGTT CTTCAAAAAC 1738
 AGTATATTTT GATGTACTCA TTGTTTACTT ACATAAAAAT AAATTGTTAT TATCATAACG 1798
 TAAAGAAAAA AAAAAAAAAA AAAA 1822
 (2) INFORMATION FOR SEQ ID NO:3:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 1644 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (ix) FEATURE:
 (A) NAME/KEY: Coding Sequence
 (B) LOCATION: 1...1644
 (D) OTHER INFORMATION: Luciola Cruciata Luciferase (Firefly)
 (x) PUBLICATION INFORMATION:
 (H) DOCUMENT NUMBER: 4,968,613
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
 ATG GAA AAC ATG GAA AAC GAT GAA AAT ATT GTA GTT GGA CCT AAA CCG 48
 Met Glu Asn Met Glu Asn Asp Glu Asn Ile Val Val Gly Pro Lys Pro
 1 5 10 15
 TTT TAC CCT ATC GAA GAG GGA TCT GCT GGA ACA CAA TTA CGC AAA TAC 96
 Phe Tyr Pro Ile Glu Glu Gly Ser Ala Gly Thr Gln Leu Arg Lys Tyr
 20 25 30
 ATG GAG CGA TAT GCA AAA CTT GGC GCA ATT GCT TTT ACA AAT GCA GTT 144
 Met Glu Arg Tyr Ala Lys Leu Gly Ala Ile Ala Phe Thr Asn Ala Val
 35 40 45
 ACT GGT GTT GAT TAT TCT TAC GCC GAA TAC TTG GAG AAA TCA TGT TGT 192
 Thr Gly Val Asp Tyr Ser Tyr Ala Glu Tyr Leu Glu Lys Ser Cys Cys
 50 55 60
 CTA GGA AAA GCT TTG CAA AAT TAT GGT TTG GTT GTT GAT GGC AGA ATT 240
 Leu Gly Lys Ala Leu Gln Asn Tyr Gly Leu Val Val Asp Gly Arg Ile
 65 70 75 80
 GCG TTA TGC AGT GAA AAC TGT GAA GAA TTT TTT ATT CCT GTA ATA GCC 288
 Ala Leu Cys Ser Glu Asn Cys Glu Glu Phe Phe Ile Pro Val Ile Ala
 85 90 95
 GGA CTG TTT ATA GGT GTA GGT GTT GCA CCC ACT AAT GAG ATT TAC ACT 336
 Gly Leu Phe Ile Gly Val Gly Val Ala Pro Thr Asn Glu Ile Tyr Thr
 100 105 110
 TTA CGT GAA CTG GTT CAC AGT TTA GGT ATC TCT AAA CCA ACA ATT GTA 384
 Leu Arg Glu Leu Val His Ser Leu Gly Ile Ser Lys Pro Thr Ile Val
 115 120 125
 TTT AGT TCT AAA AAA GGC TTA GAT AAA GTT ATA ACA GTA CAG AAA ACA 432
 Phe Ser Ser Lys Lys Gly Leu Asp Lys Val Ile Thr Val Gln Lys Thr
 130 135 140
 GTA ACT ACT ATT AAA ACC ATT GTT ATA CTA GAT AGC AAA GTT GAT TAT 480
 Val Thr Thr Ile Lys Thr Ile Val Ile Leu Asp Ser Lys Val Asp Tyr
 145 150 155 160
 CGA GGA TAT CAA TGT CTG GAC ACC TTT ATA AAA AGA AAC ACT CCA CCA 528
 Arg Gly Tyr Gln Cys Leu Asp Thr Phe Ile Lys Arg Asn Thr Pro Pro
 165 170 175
 GGT TTT CAA GCA TCC AGT TTC AAA ACT GTG GAA GTT GAC CGT AAA GAA 576
 Gly Phe Gln Ala Ser Ser Phe Lys Thr Val Glu Val Asp Arg Lys Glu
 180 185 190
 CAA GTT GCT CTT ATA ATG AAC TCT TCG GGT TCT ACC GGT TTG CCA AAA 624
 Gln Val Ala Leu Ile Met Asn Ser Ser Gly Ser Thr Gly Leu Pro Lys
 195 200 205
 GGC GTA CAA CTT ACT CAC GAA AAT ACA GTC ACT AGA TTT TCT CAT GCT 672
 Gly Val Gln Leu Thr His Glu Asn Thr Val Thr Arg Phe Ser His Ala
 210 215 220
 AGA GAT CCG ATT TAT GGT AAC CAA GTT TCA CCA GGC ACC GCT GTT TTA 720
 Arg Asp Pro Ile Tyr Gly Asn Gln Val Ser Pro Gly Thr Ala Val Leu
 225 230 235 240
 ACT GTC GTT CCA TTC CAT CAT GGT TTT GGT ATG TTC ACT ACT CTA GGG 768
 Thr Val Val Pro Phe His His Gly Phe Gly Met Phe Thr Thr Leu Gly
 245 250 255
 TAT TTA ATT TGT GGT TTT CGT GTT GTA ATG TTA ACA AAA TTC GAT GAA 816
 Tyr Leu Ile Cys Gly Phe Arg Val Val Met Leu Thr Lys Phe Asp Glu
 260 265 270
 GAA ACA TTT TTA AAA ACT CTA CAA GAT TAT AAA TGT ACA AGT GTT ATT 864
 Glu Thr Phe Leu Lys Thr Leu Gln Asp Tyr Lys Cys Thr Ser Val Ile
 275 280 285
 CTT GTA CCG ACC TTG TTT GCA ATT CTC AAC AAA AGT GAA TTA CTC AAT 912
 Leu Val Pro Thr Leu Phe Ala Ile Leu Asn Lys Ser Glu Leu Leu Asn
 290 295 300
 AAA TAC GAT TTG TCA AAT TTA GTT GAG ATT GCA TCT GGC GGA GCA CCT 960
 Lys Tyr Asp Leu Ser Asn Leu Val Glu Ile Ala Ser Gly Gly Ala Pro
 305 310 315 320
 TTA TCA AAA GAA GTT GGT GAA GCT GTT GCT AGA CGC TTT AAT CTT CCC 1008
 Leu Ser Lys Glu Val Gly Glu Ala Val Ala Arg Arg Phe Asn Leu Pro
 325 330 335
 GGT GTT CGT CAA GGT TAT GGT TTA ACA GAA ACA ACA TCT GCC ATT ATT 1056
 Gly Val Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Ile
 340 345 350
 ATT ACA CCA GAA GGA GAC GAT AAA CCA GGA GCT TCT GGA AAA GTC GTG 1104
 Ile Thr Pro Glu Gly Asp Asp Lys Pro Gly Ala Ser Gly Lys Val Val
 355 360 365
 CCG TTG TTT AAA GCA AAA GTT ATT GAT CTT GAT ACC AAA AAA TCT TTA 1152
 Pro Leu Phe Lys Ala Lys Val Ile Asp Leu Asp Thr Lys Lys Ser Leu
 370 375 380
 GGT CCT AAC AGA CGT GGA GAA GTT TGT GTT AAA GGA CCT ATG CTT ATG 1200
 Gly Pro Asn Arg Arg Gly Glu Val Cys Val Lys Gly Pro Met Leu Met
 385 390 395 400
 AAA GGT TAT GTA AAT AAT CCA GAA GCA ACA AAA GAA CTT ATT GAC GAA 1248
 Lys Gly Tyr Val Asn Asn Pro Glu Ala Thr Lys Glu Leu Ile Asp Glu
 405 410 415
 GAA GGT TGG CTG CAC ACC GGA GAT ATT GGA TAT TAT GAT GAA GAA AAA 1296
 Glu Gly Trp Leu His Thr Gly Asp Ile Gly Tyr Tyr Asp Glu Glu Lys
 420 425 430
 CAT TTC TTT ATT GTC GAT CGT TTG AAG TCT TTA ATC AAA TAC AAA GGA 1344
 His Phe Phe Ile Val Asp Arg Leu Lys Ser Leu Ile Lys Tyr Lys Gly
 435 440 445
 TAC CAA GTA CCA CCT GCC GAA TTA GAA TCC GTT CTT TTG CAA CAT CCA 1392
 Tyr Gln Val Pro Pro Ala Glu Leu Glu Ser Val Leu Leu Gln His Pro
 450 455 460
 TCT ATC TTT GAT GCT GGT GTT GCC GGC GTT CCT GAT CCT GTA GCT GGC 1440
 Ser Ile Phe Asp Ala Gly Val Ala Gly Val Pro Asp Pro Val Ala Gly
 465 470 475 480
 GAG CTT CCA GGA GCC GTT GTT GTA CTG GAA AGC GGA AAA AAT ATG ACC 1488
 Glu Leu Pro GLy Ala Val Val Val Leu Glu Ser Gly Lys Asn Met Thr
 485 490 495
 GAA AAA GAA GTA ATG GAT TAT GTT GCA AGT CAA GTT TCA AAT GCA AAA 1536
 Glu Lys Glu Val Met Asp Tyr Val Als Ser Gln Val Ser Asn Ala Lys
 500 505 510
 CGT TTA CGT GGT GGT GTT CGT TTT GTG GAT GAA GTA CCT AAA GGT CTT 1584
 Arg Leu Arg Gly Gly Val Arg Phe Val Asp Glu Val Pro Lys Gly Leu
 515 520 525
 ACT GGA AAA ATT GAC GGC AGA GCA ATT AGA GAA ATC CTT AAG AAA CCA 1632
 Thr Gly Lys Ile Asp Gly Arg Ala Ile Arg Glu Ile Leu Lys Lys Pro
 530 535 540
 GTT GCT AAG ATG 1644
 Val Ala Lys Met
 545
 (2) INFORMATION FOR SEQ ID NO:4:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 1820 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (ix) FEATURE:
 (A) NAME/KEY: Coding Sequence
 (B) LOCATION: 1...1664
 (D) OTHER INFORMATION: Vargula (cypridina) luciferase
 (x) PUBLICATION INFORMATION:
 (A) AUTHORS: Thompson et al.
 (C) JOURNAL: Proc. Natl. Acad. Sci. U.S.A.
 (D) VOLUME: 86
 (F) PAGES: 6567-6571
 (G) DATE: (1989)
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
 ATG AAG ATA ATA ATT CTG TCT GTT ATA TTG GCC TAC TGT GTC ACC GAC 48
 Met Lys Ile Ile Ile Leu Ser Val Ile Leu Ala Tyr Cys Val Thr Asp
 1 5 10 15
 AAC TGT CAA GAT GCA TGT CCT GTA GAA GCG GAA CCG CCA TCA AGT ACA 96
 Asn Cys Gln Asp Ala Cys Pro Val Glu Ala Glu Pro Pro Ser Ser Thr
 20 25 30
 CCA ACA GTT CCA ACT TCT TGT GAA GCT AAA GAA GGA GAA TGT ATA GAT 144
 Pro Thr Val Pro Thr Ser Cys Glu Ala Lys Glu Gly Glu Cys Ile Asp
 35 40 45
 ACC AGA TGC GCA ACA TGT AAA CGA GAT ATA CTA TCA GAT GGA CTG TGT 192
 Thr Arg Cys Ala Thr Cys Lys Arg Asp Ile Leu Ser Asp Gly Leu Cys
 50 55 60
 GAA AAT AAA CCA GGG AAG ACA TGC TGT AGA ATG TGC CAG TAT GTG ATT 240
 Glu Asn Lys Pro Gly Lys Thr Cys Cys Arg Met Cys Gln Tyr Val Ile
 65 70 75 80
 GAA TGC AGA GTA GAA GCA GCT GGT TAT TTT AGA ACG TTT TAC GGC AAA 288
 Glu Cys Arg Val Glu Ala Ala Gly Tyr Phe Arg Thr Phe Tyr Gly Lys
 85 90 95
 AGA TTT AAT TTT CAG GAA CCT GGT AAA TAT GTG CTG GCT AGG GGA ACC 336
 Arg Phe Asn Phe Gln Glu Pro Gly Lys Tyr Val Leu Ala Arg Gly Thr
 100 105 110
 AAG GGT GGC GAT TGG TCT GTA ACC CTC ACC ATG GAG AAT CTA GAT GGA 384
 Lys Gly Gly Asp Trp Ser Val Thr Leu Thr Met Glu Asn Leu Asp Gly
 115 120 125
 CAG AAG GGA GCT GTG CTG ACT AAG ACA ACA CTG GAG GTT GCA GGA GAC 432
 Gln Lys Gly Ala Val Leu Thr Lys Thr Thr Leu Glu Val Ala Gly Asp
 130 135 140
 GTA ATA GAC ATT ACT CAA GCT ACT GCA GAT CCT ATC ACA GTT AAC GGA 480
 Val Ile Asp Ile Thr Gln Ala Thr Ala Asp Pro Ile Thr Val Asn Gly
 145 150 155 160
 GGA GCT GAC CCA GTT ATC GCT AAC CCG TTC ACA ATT GGT GAG GTG ACC 528
 Gly Ala Asp Pro Val Ile Ala Asn Pro Phe Thr Ile Gly Glu Val Thr
 165 170 175
 ATT GCT GTT GTT GAA ATA CCG GGC TTC AAT ATC ACA GTC ATC GAA TTC 576
 Ile Ala Val Val Glu Ile Pro Gly Phe Asn Ile Thr Val Ile Glu Phe
 180 185 190
 TTT AAA CTA ATC GTG ATT GAT ATT CTG GGA GGA AGA TCT GTC AGA ATT 624
 Phe Lys Leu Ile Val Ile Asp Ile Leu Gly Gly Arg Ser Val Arg Ile
 195 200 205
 GCT CCA GAC ACA GCA AAC AAA GGA CTG ATA TCT GGT ATC TGT GGT AAT 672
 Ala Pro Asp Thr Ala Asn Lys Gly Leu Ile Ser Gly Ile Cys Gly Asn
 210 215 220
 CTG GAG ATG AAT GAC GCT GAT GAC TTT ACT ACA GAT GCA GAT CAG CTG 720
 Leu Glu Met Asn Asp Ala Asp Asp Phe Thr Thr Asp Ala Asp Gln Leu
 225 230 235 240
 GCG ATC CAA CCC AAC ATA AAC AAA GAG TTC GAC GGC TGC CCA TTC TAT 768
 Ala Ile Gln Pro Asn Ile Asn Lys Glu Phe Asp Gly Cys Pro Phe Tyr
 245 250 255
 GGC AAT CCT TCT GAT ATC GAA TAC TGC AAA GGT CTG ATG GAG CCA TAC 816
 Gly Asn Pro Ser Asp Ile Glu Tyr Cys Lys Gly Leu Met Glu Pro Tyr
 260 265 270
 AGA GCT GTA TGT CGT AAC AAT ATC AAC TTC TAC TAT TAC ACT CTA TCC 864
 Arg Ala Val Cys Arg Asn Asn Ile Asn Phe Tyr Tyr Tyr Thr Leu Ser
 275 280 285
 TGT GCC TTC GCT TAC TGT ATG GGA GGA GAA GAA AGA GCT AAA CAC GTC 912
 Cys Ala Phe Ala Tyr Cys Met Gly Gly Glu Glu Arg Ala Lys His Val
 290 295 300
 CTT TTC GAC TAT GTT GAG ACA TGC GCT GCG CCG GAA ACG AGA GGA ACG 960
 Leu Phe Asp Tyr Val Glu Thr Cys Ala Ala Pro Glu Thr Arg Gly Thr
 305 310 315 320
 TGT GTT TTA TCA GGA CAT ACT TTC TAT GAC ACA TTC GAC AAA GCA AGA 1008
 Cys Val Leu Ser Gly His Thr Phe Tyr Asp Thr Phe Asp Lys Ala Arg
 325 330 335
 TAT CAA TTC CAG GGC CCA TGC AAG GAG ATT CTG ATG GCC GCA GAC TGT 1056
 Tyr Gln Phe Gln Gly Pro Cys Lys Glu Ile Leu Met Ala Ala Asp Cys
 340 345 350
 TAC TGG AAC ACA TGG GAT GTA AAG GTT TCA CAT AGA GAC GTC GAA TCA 1104
 Tyr Trp Asn Thr Trp Asp Val Lys Val Ser His Arg Asp Val Glu Ser
 355 360 365
 TAC ACT GAG GTA GAG AAA GTA ACA ATC AGG AAA CAG TCA ACT GTA GTA 1152
 Tyr Thr Glu Val Glu Lys Val Thr Ile Arg Lys Gln Ser Thr Val Val
 370 375 380
 GAT CTC ATT GTG GAT GGC AAG CAG GTC AAG GTT GGA GGA GTG GAT GTA 1200
 Asp Leu Ile Val Asp Gly Lys Gln Val Lys Val Gly Gly Val Asp Val
 385 390 395 400
 TCT ATC CCG TAC AGC TCT GAG AAC ACT TCC ATA TAC TGG CAG GAT GGA 1248
 Ser Ile Pro Tyr Ser Ser Glu Asn Thr Ser Ile Tyr Trp Gln Asp Gly
 405 410 415
 GAC ATC CTG ACG ACG GCC ATC CTA CCT GAA GCT CTT GTC GTT AAG TTC 1296
 Asp Ile Leu Thr Thr Ala Ile Leu Pro Glu Ala Leu Val Val Lys Phe
 420 425 430
 AAC TTT AAG CAG CTC CTT GTA GTT CAT ATC AGA GAT CCA TTC GAT GCA 1344
 Asn Phe Lys Gln Leu Leu Val Val His Ile Arg Asp Pro Phe Asp Ala
 435 440 445
 AAG ACA TGC GGC ATA TGT GGT AAC TAT AAT CAA GAT TCA ACT GAT GAT 1392
 Lys Thr Cys Gly Ile Cys Gly Asn Tyr Asn Gln Asp Ser Thr Asp Asp
 450 455 460
 TTC TTT GAC GCA GAA GGA GCA TGC GCT CTA ACC CCC AAC CCC CCA GGA 1440
 Phe Phe Asp Ala Glu Gly Ala Cys Ala Leu Thr Pro Asn Pro Pro Gly
 465 470 475 480
 TGT ACA GAG GAA CAG AAA CCA GAA GCT GAG CGA CTT TGC AAT AAT CTC 1488
 Cys Thr Glu Glu Gln Lys Pro Glu Ala Glu Arg Leu Cys Asn Asn Leu
 485 490 495
 TTT GAT TCT TCT ATC GAC GAG AAA TGT AAT GTC TGC TAC AAG CCT GAC 1536
 Phe Asp Ser Ser Ile Asp Glu Lys Cys Asn Val Cys Tyr Lys Pro Asp
 500 505 510
 CGG ATT GCC CGA TGT ATG TAC GAG TAT TGC CTG AGG GGA CAA CAA GGA 1584
 Arg Ile Ala Arg Cys Met Tyr Glu Tyr Cys Leu Arg Gly Gln Gln Gly
 515 520 525
 TTT TGT GAC CAT GCT TGG GAG TTC AAG AAA GAA TGC TAC ATA AAA CAT 1632
 Phe Cys Asp His Ala Trp Glu Phe Lys Lys Glu Cys Tyr Ile Lys His
 530 535 540
 GGA GAC ACT CTA GAA GTA CCA CCT GAA TGT CAA TAA ACGTACAAAG 1678
 Gly Asp Thr Leu Glu Val Pro Pro Glu Cys Gln
 545 550 555
 ATACAGAAGC TAAGGCTACT ACAGCAGAAG ATAAAAAAGA AACTGTAGTT CCTTCAAAAA 1738
 CCGTGTATTT TATGTACTCA TTGTTTAATT AGAGCAAAAT AAATTGTTAT TATCATAACT 1798
 TAAACTAAAA AAAAAAAAAA AA 1820
 (2) INFORMATION FOR SEQ ID NO:5:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 958 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (iii) HYPOTHETICAL: NO
 (iv) ANTI-SENSE: NO
 (v) FRAGMENT TYPE:
 (vi) ORIGINAL SOURCE:
 (ix) FEATURE:
 (A) NAME/KEY: Coding Sequence
 (B) LOCATION: 115...702
 (D) OTHER INFORMATION: apoaequorin-encoding gene
 (x) PUBLICATION INFORMATION:
 (H) DOCUMENT NUMBER: 5,093,240
 (A) AUTHORS: Inouye et al.
 (C) JOURNAL: Proc. Natl. Acad. Sci. U.S.A.
 (D) VOLUME: 82
 (F) PAGES: 3154-3158
 (G) DATE: (1985)
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
 GGGGGGGGGG GGGGGGGGGG GGGGGGGGGG GGGAATGCAA TTCATCTTTG CATCAAAGAA 60
 TTACATCAAA TCTCTAGTTG ATCAACTAAA TTGTCTCGAC AACAACAAGC AAAC ATG 117
 Met
 1
 ACA AGC AAA CAA TAC TCA GTC AAG CTT ACA TCA GAC TTC GAC AAC CCA 165
 Thr Ser Lys Gln Tyr Ser Val Lys Leu Thr Ser Asp Phe Asp Asn Pro
 5 10 15
 AGA TGG ATT GGA CGA CAC AAG CAT ATG TTC AAT TTC CTT GAT GTC AAC 213
 Arg Trp Ile Gly Arg His Lys His Met Phe Asn Phe Leu Asp Val Asn
 20 25 30
 CAC AAT GGA AAA ATC TCT CTT GAC GAG ATG GTC TAC AAG GCA TCT GAT 261
 His Asn Gly Lys Ile Ser Leu Asp Glu Met Val Tyr Lys Ala Ser Asp
 35 40 45
 ATT GTC ATC AAT AAC CTT GGA GCA ACA CCT GAG CAA GCC AAA CGA CAC 309
 Ile Val Ile Asn Asn Leu Gly Ala Thr Pro Glu Gln Ala Lys Arg His
 50 55 60 65
 AAA GAT GCT GTA GAA GCC TTC TTC GGA GGA GCT GGA ATG AAA TAT GGT 357
 Lys Asp Ala Val Glu Ala Phe Phe Gly Gly Ala Gly Met Lys Tyr Gly
 70 75 80
 GTG GAA ACT GAT TGG CCT GCA TAT ATT GAA GGA TGG AAA AAA TTG GCT 405
 Val Glu Thr Asp Trp Pro Ala Tyr Ile Glu Gly Trp Lys Lys Leu Ala
 85 90 95
 ACT GAT GAA TTG GAG AAA TAC GCC AAA AAC GAA CCA ACG CTC ATC CGT 453
 Thr Asp Glu Leu Glu Lys Tyr Ala Lys Asn Glu Pro Thr Leu Ile Arg
 100 105 110
 ATA TGG GGT GAT GCT TTG TTT GAT ATC GTT GAC AAA GAT CAA AAT GGA 501
 Ile Trp Gly Asp Ala Leu Phe Asp Ile Val Asp Lys Asp Gln Asn Gly
 115 120 125
 GCC ATT ACA CTG GAT GAA TGG AAA GCA TAC ACC AAA GCT GCT GGT ATC 549
 Ala Ile Thr Leu Asp Glu Trp Lys Ala Tyr Thr Lys Ala Ala Gly Ile
 130 135 140 145
 ATC CAA TCA TCA GAA GAT TGC GAG GAA ACA TTC AGA GTG TGC GAT ATT 597
 Ile Gln Ser Ser Glu Asp Cys Glu Glu Thr Phe Arg Val Cys Asp Ile
 150 155 160
 GAT GAA AGT GGA CAA CTC GAT GTT GAT GAG ATG ACA AGA CAA CAT TTA 645
 Asp Glu Ser Gly Gln Leu Asp Val Asp Glu Met Thr Arg Gln His Leu
 165 170 175
 GGA TTT TGG TAC ACC ATG GAT CCT GCT TGC GAA AAG CTC TAC GGT GGA 693
 Gly Phe Trp Tyr Thr Met Asp Pro Ala Cys Glu Lys Leu Tyr Gly Gly
 180 185 190
 GCT GTC CCC TAAGAAGCTC TACGGTGGTG ATGCACCCTA GGAAGATGAT GTGATTTTGA 752
 Ala Val Pro
 195
 ATAAAACACT GATGAATTCA ATCAAAATTT TCCAAATTTT TGAACGATTT CAATCGTTTG 812
 TGTTGATTTT TGTAATTAGG AACAGATTAA ATCGAATGAT TAGTTGTTTT TTTAATCAAC 872
 AGAACTTACA AATCGAAAAA GTAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 932
 AAAAAAAAAA AAAAAAAAAA AAAAAA 958
 (2) INFORMATION FOR SEQ ID NO:6:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 591 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (iii) HYPOTHETICAL: NO
 (iv) ANTI-SENSE: NO
 (v) FRAGMENT TYPE:
 (vi) ORIGINAL SOURCE:
 (ix) FEATURE:
 (A) NAME/KEY: Coding Sequence
 (B) LOCATION: 1...588
 (D) OTHER INFORMATION: Recombinant Aequorin AEQ1
 (x) PUBLICATION INFORMATION:
 (A) AUTHORS: Prasher et al.
 (B) TITLE: Sequence Comparisons of Complementary
 DNAs Encoding Aequorin Isotypes
 (C) JOURNAL: Biochemistry
 (D) VOLUME: 26
 (F) PAGES: 1326-1332
 (G) DATE: 1987
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
 ATG ACC AGC GAA CAA TAC TCA GTC AAG CTT ACA CCA GAC TTC GAC AAC 48
 Met Thr Ser Glu Gln Tyr Ser Val Lys Leu Thr Pro Asp Phe Asp Asn
 1 5 10 15
 CCA AAA TGG ATT GGA CGA CAC AAG CAC ATG TTT AAT TTT CTT GAT GTC 96
 Pro Lys Trp Ile Gly Arg His Lys His Met Phe Asn Phe Leu Asp Val
 20 25 30
 AAC CAC AAT GGA AGG ATC TCT CTT GAC GAG ATG GTC TAC AAG GCG TCC 144
 Asn His Asn Gly Arg Ile Ser Leu Asp Glu Met Val Tyr Lys Ala Ser
 35 40 45
 GAT ATT GTT ATA AAC AAT CTT GGA GCA ACA CCT GAA CAA GCC AAA CGT 192
 Asp Ile Val Ile Asn Asn Leu Gly Ala Thr Pro Glu Gln Ala Lys Arg
 50 55 60
 CAC AAA GAT GCT GTA GAA GCC TTC TTC GGA GGA GCT GGA ATG AAA TAT 240
 His Lys Asp Ala Val Glu Ala Phe Phe Gly Gly Ala Gly Met Lys Tyr
 65 70 75 80
 GGT GTA GAA ACT GAA TGG CCT GAA TAC ATC GAA GGA TGG AAA AGA CTG 288
 Gly Val Glu Thr Glu Trp Pro Glu Tyr Ile Glu Gly Trp Lys Arg Leu
 85 90 95
 GCT TCC GAG GAA TTG AAA AGG TAT TCA AAA AAC CAA ATC ACA CTT ATT 336
 Ala Ser Glu Glu Leu Lys Arg Tyr Ser Lys Asn Gln Ile Thr Leu Ile
 100 105 110
 CGT TTA TGG GGT GAT GCA TTG TTC GAT ATC ATT GAC AAA GAC CAA AAT 384
 Arg Leu Trp Gly Asp Ala Leu Phe Asp Ile Ile Asp Lys Asp Gln Asn
 115 120 125
 GGA GCT ATT TCA CTG GAT GAA TGG AAA GCA TAC ACC AAA TCT GAT GGC 432
 Gly Ala Ile Ser Leu Asp Glu Trp Lys Ala Tyr Thr Lys Ser Asp Gly
 130 135 140
 ATC ATC CAA TCG TCA GAA GAT TGC GAG GAA ACA TTC AGA GTG TGC GAT 480
 Ile Ile Gln Ser Ser Glu Asp Cys Glu Glu Thr Phe Arg Val Cys Asp
 145 150 155 160
 ATT GAT GAA AGT GGA CAG CTC GAT GTT GAT GAG ATG ACA AGA CAA CAT 528
 Ile Asp Glu Ser Gly Gln Leu Asp Val Asp Glu Met Thr Arg Gln His
 165 170 175
 TTA GGA TTT TGG TAC ACC ATG GAT CCT GCT TGC GAA AAG CTC TAC GGT 576
 Leu Gly Phe Trp Tyr Thr Met Asp Pro Ala Cys Glu Lys Leu Tyr Gly
 180 185 190
 GGA GCT GTC CCC TAA 591
 Gly Ala Val Pro *
 195
 (2) INFORMATION FOR SEQ ID NO:7:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 591 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (iii) HYPOTHETICAL: NO
 (iv) ANTI-SENSE: NO
 (v) FRAGMENT TYPE:
 (vi) ORIGINAL SOURCE:
 (ix) FEATURE:
 (A) NAME/KEY: Coding Sequence
 (B) LOCATION: 1...588
 (D) OTHER INFORMATION: Recombinant Aequorin AEQ2
 (x) PUBLICATION INFORMATION:
 (A) AUTHORS: Prasher et al.
 (B) TITLE: Sequence Comparisons of Complementary
 DNAs Encoding Aequorin Isotypes
 (C) JOURNAL: Biochemistry
 (D) VOLUME: 26
 (F) PAGES: 1326-1332
 (G) DATE: 1987
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
 ATG ACC AGC GAA CAA TAC TCA GTC AAG CTT ACA TCA GAC TTC GAC AAC 48
 Met Thr Ser Glu Gln Tyr Ser Val Lys Leu Thr Ser Asp Phe Asp Asn
 1 5 10 15
 CCA AGA TGG ATT GGA CGA CAC AAG CAT ATG TTC AAT TTC CTT GAT GTC 96
 Pro Arg Trp Ile Gly Arg His Lys His Met Phe Asn Phe Leu Asp Val
 20 25 30
 AAC CAC AAT GGA AAA ATC TCT CTT GAC GAG ATG GTC TAC AAG GCA TCT 144
 Asn His Asn Gly Lys Ile Ser Leu Asp Glu Met Val Tyr Lys Ala Ser
 35 40 45
 GAT ATT GTC ATC AAT AAC CTT GGA GCA ACA CCT GAG CAA GCC AAA CGA 192
 Asp Ile Val Ile Asn Asn Leu Gly Ala Thr Pro Glu Gln Ala Lys Arg
 50 55 60
 CAC AAA GAT GCT GTA GAA GCC TTC TTC GGA GGA GCT GGA ATG AAA TAT 240
 His Lys Asp Ala Val Glu Ala Phe Phe Gly Gly Ala Gly Met Lys Tyr
 65 70 75 80
 GGT GTG GAA ACT GAT TGG CCT GCA TAT ATT GAA GGA TGG AAA AAA TTG 288
 Gly Val Glu Thr Asp Trp Pro Ala Tyr Ile Glu Gly Trp Lys Lys Leu
 85 90 95
 GCT ACT GAT GAA TTG GAG AAA TAC GCC AAA AAC GAA CCA ACG CTC ATC 336
 Ala Thr Asp Glu Leu Glu Lys Tyr Ala Lys Asn Glu Pro Thr Leu Ile
 100 105 110
 CGT ATA TGG GGT GAT GCT TTG TTC GAT ATC GTT GAC AAA GAT CAA AAT 384
 Arg Ile Trp Gly Asp Ala Leu Phe Asp Ile Val Asp Lys Asp Gln Asn
 115 120 125
 GGA GCC ATT ACA CTG GAT GAA TGG AAA GCA TAC ACC AAA GCT GCT GGT 432
 Gly Ala Ile Thr Leu Asp Glu Trp Lys Ala Tyr Thr Lys Ala Ala Gly
 130 135 140
 ATC ATC CAA TCA TCA GAA GAT TGC GAG GAA ACA TTC AGA GTG TGC GAT 480
 Ile Ile Gln Ser Ser Glu Asp Cys Glu Glu Thr Phe Arg Val Cys Asp
 145 150 155 160
 ATT GAT GAA AGT GGA CAA CTC GAT GTT GAT GAG ATG ACA AGA CAA CAT 528
 Ile Asp Glu Ser Gly Gln Leu Asp Val Asp Glu Met Thr Arg Gln His
 165 170 175
 TTA GGA TTT TGG TAC ACC ATG GAT CCT GCT TGC GAA AAG CTC TAC GGT 576
 Leu Gly Phe Trp Tyr Thr Met Asp Pro Ala Cys Glu Lys Leu Tyr Gly
 180 185 190
 GGA GCT GTC CCC TAA 591
 Gly Ala Val Pro *
 195
 (2) INFORMATION FOR SEQ ID NO:8:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 591 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (iii) HYPOTHETICAL: NO
 (iv) ANTI-SENSE: NO
 (v) FRAGMENT TYPE:
 (vi) ORIGINAL SOURCE:
 (ix) FEATURE:
 (A) NAME/KEY: Coding Sequence
 (B) LOCATION: 1...588
 (D) OTHER INFORMATION: Recombinant Aequorin AEQ3
 (x) PUBLICATION INFORMATION:
 (A) AUTHORS: Prasher et al.
 (B) TITLE: Sequence Comparisons of Complementary
 DNAs Encoding Aequorin Isotypes
 (C) JOURNAL: Biochemistry
 (D) VOLUME: 26
 (F) PAGES: 1326-1332
 (G) DATE: 1987
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
 ATG ACC AGC GAA CAA TAC TCA GTC AAG CTT ACA TCA GAC TTC GAC AAC 48
 Met Thr Ser Glu Gln Tyr Ser Val Lys Leu Thr Ser Asp Phe Asp Asn
 1 5 10 15
 CCA AGA TGG ATT GGA CGA CAC AAG CAT ATG TTC AAT TTC CTT GAT GTC 96
 Pro Arg Trp Ile Gly Arg His Lys His Met Phe Asn Phe Leu Asp Val
 20 25 30
 AAC CAC AAT GGA AAA ATC TCT CTT GAC GAG ATG GTC TAC AAG GCA TCT 144
 Asn His Asn Gly Lys Ile Ser Leu Asp Glu Met Val Tyr Lys Ala Ser
 35 40 45
 GAT ATT GTC ATC AAT AAC CTT GGA GCA ACA CCT GAG CAA GCC AAA CGA 192
 Asp Ile Val Ile Asn Asn Leu Gly Ala Thr Pro Glu Gln Ala Lys Arg
 50 55 60
 CAC AAA GAT GCT GTA GGA GAC TTC TTC GGA GGA GCT GGA ATG AAA TAT 240
 His Lys Asp Ala Val Gly Asp Phe Phe Gly Gly Ala Gly Met Lys Tyr
 65 70 75 80
 GGT GTG GAA ACT GAT TGG CCT GCA TAC ATT GAA GGA TGG AAA AAA TTG 288
 Gly Val Glu Thr Asp Trp Pro Ala Tyr Ile Glu Gly Trp Lys Lys Leu
 85 90 95
 GCT ACT GAT GAA TTG GAG AAA TAC GCC AAA AAC GAA CCA ACG CTC ATC 336
 Ala Thr Asp Glu Leu Glu Lys Tyr Ala Lys Asn Glu Pro Thr Leu Ile
 100 105 110
 CGT ATA TGG GGT GAT GCT TTG TTC GAT ATC GTT GAC AAA GAT CAA AAT 384
 Arg Ile Trp Gly Asp Ala Leu Phe Asp Ile Val Asp Lys Asp Gln Asn
 115 120 125
 GGA GCC ATT ACA CTG GAT GAA TGG AAA GCA TAC ACC AAA GCT GCT GGT 432
 Gly Ala Ile Thr Leu Asp Glu Trp Lys Ala Tyr Thr Lys Ala Ala Gly
 130 135 140
 ATC ATC CAA TCA TCA GAA GAT TGC GAG GAA ACA TTC AGA GTG TGC GAT 480
 Ile Ile Gln Ser Ser Glu Asp Cys Glu Glu Thr Phe Arg Val Cys Asp
 145 150 155 160
 ATT GAT GAA AAT GGA CAA CTC GAT GTT GAT GAG ATG ACA AGA CAA CAT 528
 Ile Asp Glu Asn Gly Gln Leu Asp Val Asp Glu Met Thr Arg Gln His
 165 170 175
 TTA GGA TTT TGG TAC ACC ATG GAT CCT GCT TGC GAA AAG CTC TAC GGT 576
 Leu Gly Phe Trp Tyr Thr Met Asp Pro Ala Cys Glu Lys Leu Tyr Gly
 180 185 190
 GGA GCT GTC CCC TAA 591
 Gly Ala Val Pro *
 195
 (2) INFORMATION FOR SEQ ID NO:9:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 567 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (iii) HYPOTHETICAL: NO
 (iv) ANTI-SENSE: NO
 (v) FRAGMENT TYPE:
 (vi) ORIGINAL SOURCE:
 (ix) FEATURE:
 (A) NAME/KEY: Coding Sequence
 (B) LOCATION: 1...567
 (D) OTHER INFORMATION: Aequorin photoprotein
 (x) PUBLICATION INFORMATION:
 (A) AUTHORS: Charbonneau et al.
 (B) TITLE: Amino acid sequence of the calcium-dependent
 photoprotein aequorin
 (C) JOURNAL: Am. Chem. Soc.
 (D) VOLUME: 24
 (E) ISSUE: 24
 (F) PAGES: 6762-6771
 (G) DATE: 1985
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
 GTC AAG CTT ACA CCA GAC TTC GAC AAC CCA AAA TGG ATT GGA CGA CAC 48
 Val Lys Leu Thr Pro Asp Phe Asp Asn Pro Lys Trp Ile Gly Arg His
 1 5 10 15
 AAG CAC ATG TTT AAT TTT CTT GAT GTC AAC CAC AAT GGA AGG ATC TCT 96
 Lys His Met Phe Asn Phe Leu Asp Val Asn His Asn Gly Arg Ile Ser
 20 25 30
 CTT GAC GAG ATG GTC TAC AAG GCG TCC GAT ATT GTT ATA AAC AAT CTT 144
 Leu Asp Glu Met Val Tyr Lys Ala Ser Asp Ile Val Ile Asn Asn Leu
 35 40 45
 GGA GCA ACA CCT GAA CAA GCC AAA CGT CAC AAA GAT GCT GTA GAA GCC 192
 Gly Ala Thr Pro Glu Gln Ala Lys Arg His Lys Asp Ala Val Glu Ala
 50 55 60
 TTC TTC GGA GGA GCT GCA ATG AAA TAT GGT GTA GAA ACT GAA TGG CCT 240
 Phe Phe Gly Gly Ala Ala Met Lys Tyr Gly Val Glu Thr Glu Trp Pro
 65 70 75 80
 GAA TAC ATC GAA GGA TGG AAA AGA CTG GCT TCC GAG GAA TTG AAA AGG 288
 Glu Tyr Ile Glu Gly Trp Lys Arg Leu Ala Ser Glu Glu Leu Lys Arg
 85 90 95
 TAT TCA AAA AAC CAA ATC ACA CTT ATT CGT TTA TGG GGT GAT GCA TTG 336
 Tyr Ser Lys Asn Gln Ile Thr Leu Ile Arg Leu Trp Gly Asp Ala Leu
 100 105 110
 TTC GAT ATC ATT GAC AAA GAC CAA AAT GGA GCT ATT TCA CTG GAT GAA 384
 Phe Asp Ile Ile Asp Lys Asp Gln Asn Gly Ala Ile Ser Leu Asp Glu
 115 120 125
 TGG AAA GCA TAC ACC AAA TCT GCT GGC ATC ATC CAA TCG TCA GAA GAT 432
 Trp Lys Ala Tyr Thr Lys Ser Ala Gly Ile Ile Gln Ser Ser Glu Asp
 130 135 140
 TGC GAG GAA ACA TTC AGA GTG TGC GAT ATT GAT GAA AGT GGA CAG CTC 480
 Cys Glu Glu Thr Phe Arg Val Cys Asp Ile Asp Glu Ser Gly Gln Leu
 145 150 155 160
 GAT GTT GAT GAG ATG ACA AGA CAA CAT TTA GGA TTT TGG TAC ACC ATG 528
 Asp Val Asp Glu Met Thr Arg Gln His Leu Gly Phe Trp Tyr Thr Met
 165 170 175
 GAT CCT GCT TGC GAA AAG CTC TAC GGT GGA GCT GTC CCC 567
 Asp Pro Ala Cys Glu Lys Leu Tyr Gly Gly Ala Val Pro
 180 185
 (2) INFORMATION FOR SEQ ID NO:10:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 588 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (iii) HYPOTHETICAL: NO
 (iv) ANTI-SENSE: NO
 (v) FRAGMENT TYPE:
 (vi) ORIGINAL SOURCE:
 (ix) FEATURE:
 (A) NAME/KEY: Coding Sequence
 (B) LOCATION: 1...588
 (D) OTHER INFORMATION: Aequorin mutant w/increased
 bioluminescence activity
 (x) PUBLICATION INFORMATION:
 (H) DOCUMENT NUMBER: 5,360,728
 (K) RELEVANT RESIDUES IN SEQ ID NO: 10:
 Asp 124 changed to Ser
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
 ATG ACC AGC GAA CAA TAC TCA GTC AAG CTT ACA CCA GAC TTC GAC AAC 48
 Met Thr Ser Glu Gln Tyr Ser Val Lys Leu Thr Pro Asp Phe Asp Asn
 1 5 10 15
 CCA AAA TGG ATT GGA CGA CAC AAG CAC ATG TTT AAT TTT CTT GAT GTC 96
 Pro Lys Trp Ile Gly Arg His Lys His Met Phe Asn Phe Leu Asp Val
 20 25 30
 AAC CAC AAT GGA AGG ATC TCT CTT GAC GAG ATG GTC TAC AAG GCG TCC 144
 Asn His Asn Gly Arg Ile Ser Leu Asp Glu Met Val Tyr Lys Ala Ser
 35 40 45
 GAT ATT GTT ATA AAC AAT CTT GGA GCA ACA CCT GAA CAA GCC AAA CGT 192
 Asp Ile Val Ile Asn Asn Leu Gly Ala Thr Pro Glu Gln Ala Lys Arg
 50 55 60
 CAC AAA GAT GCT GTA GAA GCC TTC TTC GGA GGA GCT GCA ATG AAA TAT 240
 His Lys Asp Ala Val Glu Ala Phe Phe Gly Gly Ala Ala Met Lys Tyr
 65 70 75 80
 GGT GTA GAA ACT GAA TGG CCT GAA TAC ATC GAA GGA TGG AAA AGA CTG 288
 Gly Val Glu Thr Glu Trp Pro Glu Tyr Ile Glu Gly Trp Lys Arg Leu
 85 90 95
 GCT TCC GAG GAA TTG AAA AGG TAT TCA AAA AAC CAA ATC ACA CTT ATT 336
 Ala Ser Glu Glu Leu Lys Arg Tyr Ser Lys Asn Gln Ile Thr Leu Ile
 100 105 110
 CGT TTA TGG GGT GAT GCA TTG TTC GAT ATC ATT TCC AAA GAC CAA AAT 384
 Arg Leu Trp Gly Asp Ala Leu Phe Asp Ile Ile Ser Lys Asp Gln Asn
 115 120 125
 GGA GCT ATT TCA CTG GAT GAA TGG AAA GCA TAC ACC AAA TCT GCT GGC 432
 Gly Ala Ile Ser Leu Asp Glu Trp Lys Ala Tyr Thr Lys Ser Ala Gly
 130 135 140
 ATC ATC CAA TCG TCA GAA GAT TGC GAG GAA ACA TTC AGA GTG TGC GAT 480
 Ile Ile Gln Ser Ser Glu Asp Cys Glu Glu Thr Phe Arg Val Cys Asp
 145 150 155 160
 ATT GAT GAA AGT GGA CAG CTC GAT GTT GAT GAG ATG ACA AGA CAA CAT 528
 Ile Asp Glu Ser Gly Gln Leu Asp Val Asp Glu Met Thr Arg Gln His
 165 170 175
 TTA GGA TTT TGG TAC ACC ATG GAT CCT GCT TGC GAA AAG CTC TAC GGT 576
 Leu Gly Phe Trp Tyr Thr Met Asp Pro Ala Cys Glu Lys Leu Tyr Gly
 180 185 190
 GGA GCT GTC CCC 588
 Gly Ala Val Pro
 195
 (2) INFORMATION FOR SEQ ID NO:11:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 588 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (iii) HYPOTHETICAL: NO
 (iv) ANTI-SENSE: NO
 (v) FRAGMENT TYPE:
 (vi) ORIGINAL SOURCE:
 (ix) FEATURE:
 (A) NAME/KEY: Coding Sequence
 (B) LOCATION: 1...588
 (D) OTHER INFORMATION: Recombinant site-directed Aequorin
 mutant w/increased biolum. activity
 (x) PUBLICATION INFORMATION:
 (H) DOCUMENT NUMBER: 5,360,728
 (K) RELEVANT RESIDUES IN SEQ ID NO: 11:
 Glu 135 changed to Ser
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
 ATG ACC AGC GAA CAA TAC TCA GTC AAG CTT ACA CCA GAC TTC GAC AAC 48
 Met Thr Ser Glu Gln Tyr Ser Val Lys Leu Thr Pro Asp Phe Asp Asn
 1 5 10 15
 CCA AAA TGG ATT GGA CGA CAC AAG CAC ATG TTT AAT TTT CTT GAT GTC 96
 Pro Lys Trp Ile Gly Arg His Lys His Met Phe Asn Phe Leu Asp Val
 20 25 30
 AAC CAC AAT GGA AGG ATC TCT CTT GAC GAG ATG GTC TAC AAG GCG TCC 144
 Asn His Asn Gly Arg Ile Ser Leu Asp Glu Met Val Tyr Lys Ala Ser
 35 40 45
 GAT ATT GTT ATA AAC AAT CTT GGA GCA ACA CCT GAA CAA GCC AAA CGT 192
 Asp Ile Val Ile Asn Asn Leu Gly Ala Thr Pro Glu Gln Ala Lys Arg
 50 55 60
 CAC AAA GAT GCT GTA GAA GCC TTC TTC GGA GGA GCT GCA ATG AAA TAT 240
 His Lys Asp Ala Val Glu Ala Phe Phe Gly Gly Ala Ala Met Lys Tyr
 65 70 75 80
 GGT GTA GAA ACT GAA TGG CCT GAA TAC ATC GAA GGA TGG AAA AGA CTG 288
 Gly Val Glu Thr Glu Trp Pro Glu Tyr Ile Glu Gly Trp Lys Arg Leu
 85 90 95
 GCT TCC GAG GAA TTG AAA AGG TAT TCA AAA AAC CAA ATC ACA CTT ATT 336
 Ala Ser Glu Glu Leu Lys Arg Tyr Ser Lys Asn Gln Ile Thr Leu Ile
 100 105 110
 CGT TTA TGG GGT GAT GCA TTG TTC GAT ATC ATT TCC AAA GAC CAA AAT 384
 Arg Leu Trp Gly Asp Ala Leu Phe Asp Ile Ile Ser Lys Asp Gln Asn
 115 120 125
 GGA GCT ATT TCA CTG GAT TCA TGG AAA GCA TAC ACC AAA TCT GCT GGC 432
 Gly Ala Ile Ser Leu Asp Ser Trp Lys Ala Tyr Thr Lys Ser Ala Gly
 130 135 140
 ATC ATC CAA TCG TCA GAA GAT TGC GAG GAA ACA TTC AGA GTG TGC GAT 480
 Ile Ile Gln Ser Ser Glu Asp Cys Glu Glu Thr Phe Arg Val Cys Asp
 145 150 155 160
 ATT GAT GAA AGT GGA CAG CTC GAT GTT GAT GAG ATG ACA AGA CAA CAT 528
 Ile Asp Glu Ser Gly Gln Leu Asp Val Asp Glu Met Thr Arg Gln His
 165 170 175
 TTA GGA TTT TGG TAC ACC ATG GAT CCT GCT TGC GAA AAG CTC TAC GGT 576
 Leu Gly Phe Trp Tyr Thr Met Asp Pro Ala Cys Glu Lys Leu Tyr Gly
 180 185 190
 GGA GCT GTC CCC 588
 Gly Ala Val Pro
 195
 (2) INFORMATION FOR SEQ ID NO:12:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 588 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (iii) HYPOTHETICAL: NO
 (iv) ANTI-SENSE: NO
 (v) FRAGMENT TYPE:
 (vi) ORIGINAL SOURCE:
 (ix) FEATURE:
 (A) NAME/KEY: Coding Sequence
 (B) LOCATION: 1...588
 (D) OTHER INFORMATION: Recombinant site-directed Aequorin
 mutant w/increased biolum. activity
 (x) PUBLICATION INFORMATION:
 (H) DOCUMENT NUMBER: 5,360,728
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
 ATG ACC AGC GAA CAA TAC TCA GTC AAG CTT ACA CCA GAC TTC GAC AAC 48
 Met Thr Ser Glu Gln Tyr Ser Val Lys Leu Thr Pro Asp Phe Asp Asn
 1 5 10 15
 CCA AAA TGG ATT GGA CGA CAC AAG CAC ATG TTT AAT TTT CTT GAT GTC 96
 Pro Lys Trp Ile Gly Arg His Lys His Met Phe Asn Phe Leu Asp Val
 20 25 30
 AAC CAC AAT GGA AGG ATC TCT CTT GAC GAG ATG GTC TAC AAG GCG TCC 144
 Asn His Asn Gly Arg Ile Ser Leu Asp Glu Met Val Tyr Lys Ala Ser
 35 40 45
 GAT ATT GTT ATA AAC AAT CTT GGA GCA ACA CCT GAA CAA GCC AAA CGT 192
 Asp Ile Val Ile Asn Asn Leu Gly Ala Thr Pro Glu Gln Ala Lys Arg
 50 55 60
 CAC AAA GAT GCT GTA GAA GCC TTC TTC GGA GGA GCT GCA ATG AAA TAT 240
 His Lys Asp Ala Val Glu Ala Phe Phe Gly Gly Ala Ala Met Lys Tyr
 65 70 75 80
 GGT GTA GAA ACT GAA TGG CCT GAA TAC ATC GAA GGA TGG AAA AGA CTG 288
 Gly Val Glu Thr Glu Trp Pro Glu Tyr Ile Glu Gly Trp Lys Arg Leu
 85 90 95
 GCT TCC GAG GAA TTG AAA AGG TAT TCA AAA AAC CAA ATC ACA CTT ATT 336
 Ala Ser Glu Glu Leu Lys Arg Tyr Ser Lys Asn Gln Ile Thr Leu Ile
 100 105 110
 CGT TTA TGG GGT GAT GCA TTG TTC GAT ATC ATT TCC AAA GAC CAA AAT 384
 Arg Leu Trp Gly Asp Ala Leu Phe Asp Ile Ile Ser Lys Asp Gln Asn
 115 120 125
 GCA GCT ATT TCA CTG GAT GAA TGG AAA GCA TAC ACC AAA TCT GCT GGC 432
 Ala Ala Ile Ser Leu Asp Glu Trp Lys Ala Tyr Thr Lys Ser Ala Gly
 130 135 140
 ATC ATC CAA TCG TCA GAA GAT TGC GAG GAA ACA TTC AGA GTG TGC GAT 480
 Ile Ile Gln Ser Ser Glu Asp Cys Glu Glu Thr Phe Arg Val Cys Asp
 145 150 155 160
 ATT GAT GAA AGT GGA CAG CTC GAT GTT GAT GAG ATG ACA AGA CAA CAT 528
 Ile Asp Glu Ser Gly Gln Leu Asp Val Asp Glu Met Thr Arg Gln His
 165 170 175
 TTA GGA TTT TGG TAC ACC ATG GAT CCT GCT TGC GAA AAG CTC TAC GGT 576
 Leu Gly Phe Trp Tyr Thr Met Asp Pro Ala Cys Glu Lys Leu Tyr Gly
 180 185 190
 GGA GCT GTC CCC 588
 Gly Ala Val Pro
 195
 (2) INFORMATION FOR SEQ ID NO:13:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 567 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (ix) FEATURE:
 (A) NAME/KEY: Coding Sequence
 (B) LOCATION: 1...567
 (D) OTHER INFORMATION: Recombinant apoaequorin (AQUALITE )
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
 GTC AAG CTT ACA CCA GAC TTC GAC AAC CCA AAA TGG ATT GGA CGA CAC 48
 Val Lys Leu Thr Pro Asp Phe Asp Asn Pro Lys Trp Ile Gly Arg His
 1 5 10 15
 AAG CAC ATG TTT AAT TTT CTT GAT GTC AAC CAC AAT GGA AGG ATC TCT 96
 Lys His Met Phe Asn Phe Leu Asp Val Asn His Asn Gly Arg Ile Ser
 20 25 30
 CTT GAC GAG ATG GTC TAC AAG GCG TCC GAT ATT GTT ATA AAC AAT CTT 144
 Leu Asp Glu Met Val Tyr Lys Ala Ser Asp Ile Val Ile Asn Asn Leu
 35 40 45
 GGA GCA ACA CCT GAA CAA GCC AAA CGT CAC AAA GAT GCT GTA GAA GCC 192
 Gly Ala Thr Pro Glu Gln Ala Lys Arg His Lys Asp Ala Val Glu Ala
 50 55 60
 TTC TTC GGA GGA GCT GGA ATG AAA TAT GGT GTA GAA ACT GAA TGG CCT 240
 Phe Phe Gly Gly Ala Gly Met Lys Tyr Gly Val Glu Thr Glu Trp Pro
 65 70 75 80
 GAA TAC ATC GAA GGA TGG AAA AAA CTG GCT TCC GAG GAA TTG AAA AGG 288
 Glu Tyr Ile Glu Gly Trp Lys Lys Leu Ala Ser Glu Glu Leu Lys Arg
 85 90 95
 TAT TCA AAA AAC CAA ATC ACA CTT ATT CGT TTA TGG GGT GAT GCA TTG 336
 Tyr Ser Lys Asn Gln Ile Thr Leu Ile Arg Leu Trp Gly Asp Ala Leu
 100 105 110
 TTC GAT ATC ATT GAC AAA GAC CAA AAT GGA GCT ATT CTG TCA GAT GAA 384
 Phe Asp Ile Ile Asp Lys Asp Gln Asn Gly Ala Ile Leu Ser Asp Glu
 115 120 125
 TGG AAA GCA TAC ACC AAA TCT GAT GGC ATC ATC CAA TCG TCA GAA GAT 432
 Trp Lys Ala Tyr Thr Lys Ser Asp Gly Ile Ile Gln Ser Ser Glu Asp
 130 135 140
 TGC GAG GAA ACA TTC AGA GTG TGC GAT ATT GAT GAA AGT GGA CAG CTC 480
 Cys Glu Glu Thr Phe Arg Val Cys Asp Ile Asp Glu Ser Gly Gln Leu
 145 150 155 160
 GAT GTT GAT GAG ATG ACA AGA CAA CAT TTA GGA TTT TGG TAC ACC ATG 528
 Asp Val Asp Glu Met Thr Arg Gln His Leu Gly Phe Trp Tyr Thr Met
 165 170 175
 GAT CCT GCT TGC GAA AAG CTC TAC GGT GGA GCT GTC CCC 567
 Asp Pro Ala Cys Glu Lys Leu Tyr Gly Gly Ala Val Pro
 180 185
 (2) INFORMATION FOR SEQ ID NO:14:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 236 amino acids
 (B) TYPE: amino acid
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (ix) FEATURE:
 (D) OTHER INFORMATION: Vibrio fisheri Flavin reductase
 (x) PUBLICATION INFORMATION:
 (H) DOCUMENT NUMBER: 5,484,723
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
 Met Pro Ile Asn Cys Lys Val Lys Ser Ile Glu Pro Leu Ala Cys Asn
 1 5 10 15
 Thr Phe Arg Ile Leu Leu His Pro Glu Gln Pro Val Ala Phe Lys Ala
 20 25 30
 Gly Gln Tyr Leu Thr Val Val Met Gly Glu Lys Asp Lys Arg Pro Phe
 35 40 45
 Ser Ile Ala Ser Ser Pro Cys Arg His Glu Gly Glu Ile Glu Leu His
 50 55 60
 Ile Gly Ala Ala Glu His Asn Ala Tyr Ala Gly Glu Val Val Glu Ser
 65 70 75 80
 Met Lys Ser Ala Leu Glu Thr Gly Gly Asp Ile Leu Ile Asp Ala Pro
 85 90 95
 His Gly Glu Ala Trp Ile Arg Glu Asp Ser Asp Arg Ser Met Leu Leu
 100 105 110
 Ile Ala Gly Gly Thr Gly Phe Ser Tyr Val Arg Ser Ile Leu Asp His
 115 120 125
 Cys Ile Ser Gln Gln Ile Gln Lys Pro Ile Tyr Leu Tyr Trp Gly Gly
 130 135 140
 Arg Asp Glu Cys Gln Leu Tyr Ala Lys Ala Glu Leu Glu Ser Ile Ala
 145 150 155 160
 Gln Ala His Ser His Ile Thr Phe Val Pro Val Val Glu Lys Ser Glu
 165 170 175
 Gly Trp Thr Gly Lys Thr Gly Asn Val Leu Glu Ala Val Lys Ala Asp
 180 185 190
 Phe Asn Ser Leu Ala Asp Met Asp Ile Tyr Ile Ala Gly Arg Phe Glu
 195 200 205
 Met Ala Gly Ala Ala Arg Glu Gln Phe Thr Thr Glu Lys Gln Ala Lys
 210 215 220
 Lys Glu Gln Leu Phe Gly Asp Ala Phe Ala Phe Ile
 225 230 235