Methods for monitoring multiple gene expression

Tag : nitrocellulose membrane


Abstract
The present invention relates to methods for monitoringdifferential expression of a plurality of genes in a firstfilamentous fungal cell relative to expression of the same genes inone or more second filamentous fungal cells using microarrayscontaining Trichoderma reesei ESTs or SSH clones, or a combinationthereof. The present invention also relates to computer readablemedia and substrates containing such array features for monitoringexpression of a plurality of genes in filamentous fungal cells.
Description of the Invention
SUMMARY OF THE INVENTION

The present invention relates to methods for monitoringdifferential expression of a plurality of genes in a firstfilamentous fungal cell relative to expression of the same genes inone or more second filamentous fungal cells, comprising:

(a) adding a mixture of detection reporter-labeled nucleic acidsisolated from the filamentous fungal cells to a substratecontaining an array of Trichoderma reesei ESTs or SSH clones, or acombination thereof, selected from the group consisting of SEQ IDNOs. 1-1188, nucleic acid fragments of SEQ ID NOs. 1-1188, andnucleic acid sequences having at least 90% homology to SEQ ID NOs.1-1188, under conditions where the nucleic acids hybridize tocomplementary sequences of the ESTs or SSH clones, or a combinationthereof, in the array, wherein the nucleic acids from the firstfilamentous fungal cell and the one or more second filamentousfungal cells are labeled with a first detection reporter and one ormore different second detection reporters, respectively; and

(b) examining the array under conditions wherein the relativeexpression of the genes in the filamentous fungal cells isdetermined by the observed detection signal of each spot in thearray in which (i) the Trichoderma reesei ESTs or SSH clones, or acombination thereof, in the array that hybridize to the nucleicacids obtained from either the first or the one or more secondfilamentous fungal cells produce a distinct first detection signalor one or more second detection signals, respectively, and (ii) theTrichoderma reesei ESTs or SSH clones, or a combination thereof, inthe array that hybridize to the nucleic acids obtained from boththe first and one or more second filamentous fungal cells produce adistinct combined detection signal.

The present invention also relates to computer readable media andsubstrates containing an array of such Trichoderma reesei ESTs orSSH clones, or a combination thereof, for monitoring differentialexpression of a plurality of genes in a first filamentous fungalcell relative to expression of the same genes in one or more secondfilamentous fungal cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for monitoringdifferential expression of a plurality of genes in a firstfilamentous fungal cell relative to expression of the same genes inone or more second filamentous fungal cells. The methods comprise(a) adding a mixture of detection reporter-labeled nucleic acidsisolated from the two or more filamentous fungal cells withdifferent detection reporters for each cell's nucleic acids to asubstrate containing an array of Trichoderma reesei ESTs or SSHclones, or a combination thereof, under conditions where thenucleic acids hybridize to complementary sequences of the ESTs orSSH clones, or a combination thereof, in the array; and (b)examining the array under conditions wherein the relativeexpression of the genes in the two or more cells is determined bythe observed detection signal of each spot in the array.

The methods of the present invention may be used to monitor globalexpression of a plurality of genes from a filamentous fungal cell,discover new genes, identify possible functions of unknown openreading frames, and monitor gene copy number variation andstability. For example, the global view of changes in expression ofgenes may be used to provide a picture of the way in whichfilamentous fungal cells adapt to changes in culture conditions,environmental stress, or other physiological provocation. Otherpossibilities for monitoring global expression include sporemorphogenesis, recombination, metabolic or catabolic pathwayengineering. In a preferred embodiment, the methods of the presentinvention are used to identify microbial genes induced when themicroorganism is grown on cellulose or corn stover. In morepreferred embodiment, the microorganism is a Trichoderma strain. Ina most preferred embodiment, the microorganism is a Trichodermareesei strain.

The methods of the present invention are particularly advantageousbecause one spot on an array equals one gene or open reading frame,extensive follow-up characterization is unnecessary since sequenceinformation is available, and EST and/or SSH microarrays can beorganized based on function of the gene products.

Expressed Sequenced Tags and Suppression Subtractive Hybridization(SSH) Clones

The term "array features" is defined herein as arrayelements of ESTs or SSH clones, or a combination thereof.

The term "expressed sequenced tag" or "EST" isdefined herein as a segment of a sequence from a cDNA clone of anexpressed Trichoderma reesei gene. The term "EST" will beunderstood to also include two or more ESTs assembled into acontig. In the methods of the present invention, the Trichodermareesei ESTs described herein preferably represent a plurality ofgenes or homologues thereof present in the two or more filamentousfungal cells to be evaluated.

ESTs are generally generated as follows: Total polyadenylated mRNAis isolated from a filamentous fungal cell and reverse transcribedinto total cDNA. The total cDNA is digested with a restrictionendonuclease, size-selected by agarose gel electrophoresis,isolated, and ligated into a vector, e.g., pZErO-2.1. The ligationmixture is transformed into competent E. coli cells andtransformants are selected under selective pressure, e.g.,kanamycin selection. The cDNA clones isolated from the selectedtransformants are amplified, isolated, and partially sequenced. Thepartial sequences are then compared to sequences in variouspublicly available databases for identification.

Any method known in the art may be used for generating ESTs (see,for example, Adams et al., 1991, Science 252: 1651-1656; Fields,1996, Tibtech 14: 286-289; Weinstock et al., 1994, Current Opinionin Biotechnology 5: 599-603; Matsubara and Okubo, 1993, CurrentOpinions in Biotechnology 4: 672-677; Nelson et al., 1997, FungalGenet. Biol. 21: 348-363; Zhu at al., Genetics 157: 1057-1065).

In a preferred embodiment, the ESTs are SEQ ID NOs: 1-24.

The term "SSH clones" is defined herein as selectivelyamplified target cDNA fragments which are differentially expressed.SSH is used to selectively amplify these target cDNA fragments andsimultaneously suppress nontarget DNA amplification.

Any method known in the art may be used for generating SSH clones(see, for example, Diatchenko et al., 1996, supra, Yang et al.,1999, supra; Porkka and Visakorpi, 2001, supra).

In a preferred embodiment, the SSH clones are SEQ ID NOs: 25-65.

In the methods of the present invention, the Trichoderma reeseiarray features are preferably at least about 50 bp in length, morepreferably at least about 100 bp in length, even more preferably atleast about 150 bp in length, and most preferably at least about200 bp in length. Furthermore, the array features are preferablydirectional ESTs or SSH clones, or a combination thereof. However,nondirectional ESTs or SSH clones, or a combination thereof, mayalso be used. A "directional EST" is defined as a cDNAcloned in the same orientation relative to the vector cloningsites, e.g., 5'.fwdarw.3' or 3'.fwdarw.5'.

In a preferred embodiment, the array features are obtained fromTrichoderma reesei. In a more preferred embodiment, the arrayfeatures are obtained from Trichoderma reesei strain RutC30(Montenecourt and Eveleigh, 1979, Adv. Chem. Ser. 181: 289-301). Ina most preferred embodiment, the Trichoderma reesei array featuresare selected from the group consisting of SEQ ID NOs. 1-1188,nucleic acid fragments of SEQ ID NOs. 1-1188, or nucleic acidsequences having at least 95%, preferably at least 99% and mostpreferably at least 99.9% homology to a sequence of SEQ ID NOs.1-1188.

In another preferred embodiment, the array features obtained fromTrichoderma reesei are ESTs. In another preferred embodiment, thearray features obtained from Trichoderma reesei are SSH clones. Inanother preferred embodiment, the array features obtained fromTrichoderma reesei are a combination of two or more of ESTs and SSHclones.

For purposes of the present invention, the degree of homologybetween two nucleic acid sequences is determined by theWilbur-Lipman method (Wilbur and Lipman, 1983, Proceedings of theNational Academy of Science USA 80: 726-730) using theLASERGENE.TM. MEGALIGN.TM. software (DNASTAR, Inc., Madison, Wis.)with an identity table and the following multiple alignmentparameters: Gap penalty of 10 and gap length penalty of 10.Pairwise alignment parameters are Ktuple=3, gap penalty=3, andwindows=20.

Microarrays

The term "an array of Trichoderma reesei ESTs or SSH clones,or a combination thereof" is defined herein as a linear ortwo-dimensional array of preferably discrete array features, eachhaving a finite area, formed on the surface of a solid support.

The term "microarray" is defined herein as an array offeatures (i.e., ESTs or SSH clones, or a combination thereof)having a density of discrete array elements of at least about100/cm.sup.2, and preferably at least about 1000/cm.sup.2. Theprinted elements in a microarray have typical dimensions, e.g.,diameters, in the range of between about 10 to about 250 .mu.m,preferably in the range of between about 10 to about 200 .mu.m,more preferably in the range of between about 20 to about 150.mu.m, even more preferably in the range of between about 20 toabout 100 .mu.m, most preferably in the range of between about 20to about 75 .mu.m, and even most preferably in the range of betweenabout 25 to about 50 .mu.m, and are separated from other printedelements in the microarray by about the same distance.

Methods and instruments for forming microarrays on the surface of asolid support are well known in the art. See, for example, U.S.Pat. No. 5,807,522; U.S. Pat. No. 5,700,637; and U.S. Pat. No.5,770,151. The instrument may be an automated device such asdescribed in U.S. Pat. No. 5,807,522.

The term "a substrate containing an array of Trichodermareesei ESTs or SSH clones, or a combination thereof," isdefined herein as a solid support having deposited on the surfaceof the support one or more of a plurality of array features, foruse in detecting binding of labeled cDNAs to the array features.

The substrate may, in one aspect, be a glass support (eg., glassslide) having a hydrophilic or hydrophobic coating on the surfaceof the support, and an array of distinct array featureselectrostatically bound non-covalently to the coating, where eachdistinct array features is disposed at a separate, definedposition.

Each microarray in the substrate preferably contains at least10.sup.3 distinct array features in a surface area of less thanabout 1 cm.sup.2. Each distinct array feature (i) is disposed at aseparate, defined position in the array, (ii) has a length of atleast 50 bp, and (iii) is present in a defined amount between about0.1 femtomoles and 100 nanomoles or higher if necessary.

For a hydrophilic coating, the glass slide is coated by placing afilm of a polycationic polymer with a uniform thickness on thesurface of the slide and drying the film to form a dried coating.The amount of polycationic polymer added should be sufficient toform at least a monolayer of polymers on the glass surface. Thepolymer film is bound to the surface via electrostatic bindingbetween negative silyl-OH groups on the surface and chargedcationic groups in the polymers. Such polycationic polymersinclude, but are not limited to, polylysine and polyarginine.

Another coating strategy employs reactive aldehydes to couple DNAto the slides (Schena et al., 1996, Proceedings of the NationalAcademy of Science USA 93: 10614-10619; Heller at al., 1997,Proceedings of the National Academy of Science USA 94: 2150-2155).

Alternatively, the surface may have a relatively hydrophobiccharacter, i.e., one that causes aqueous medium deposited on thesurface to bead. A variety of known hydrophobic polymers, such aspolystyrene, polypropylene, or polyethylene, have desirablehydrophobic properties, as do glass and a variety of lubricant orother hydrophobic films that may be applied to the support surface.A support surface is "hydrophobic" if an aqueous dropletapplied to the surface does not spread out substantially beyond thearea size of the applied droplet, wherein the surface acts toprevent spreading of the droplet applied to the surface byhydrophobic interaction with the droplet.

In another aspect, the substrate may be a multi-cell substratewhere each cell contains a microarray of array features, andpreferably an identical microarray, formed on a porous surface. Forexample, a 96-cell array may typically have array dimensionsbetween about 12 and 244 mm in width and 8 and 400 mm in length,with the cells in the array having width and length dimension of1/12 and 1/8 the array width and length dimensions, respectively,i.e., between about 1 and 20 in width and 1 and 50 mm in length.

The solid support may include a water-impermeable backing such as aglass slide or rigid polymer sheet, or other non-porous material.Formed on the surface of the backing is a water-permeable filmwhich is made of porous material. Such porous materials include,but are not limited to, nitrocellulose membrane nylon,polypropylene, and PVDF polymer. The thickness of the film ispreferably between about 10 and 1000 .mu.m. The film may be appliedto the backing by spraying or coating, or by applying a preformedmembrane to the backing.

The film surface may be partitioned into a desirable array of cellsby water-impermeable grid lines typically at a distance of about100 to 2000 .mu.m above the film surface. The grid lines can beformed on the surface of the film by laying down an uncuredflowable resin or elastomer solution in an array grid, allowing thematerial to infiltrate the porous film down to the backing, andthen curing the grid lines to form the cell-array substrate.

The barrier material of the grid lines may be a flowable silicone,wax-based material, thermoset material (e.g., epoxy), or any otheruseful material. The grid lines may be applied to the solid supportusing a narrow syringe, printing techniques, heat-seal stamping, orany other useful method known in the art.

Each well preferably contains a microarray of distinct arrayfeatures. "Distinct array features" as applied to theESTs or SSH clones, or a combination thereof, forming a microarrayis defined herein as an array feature which is distinct from otherarray features on the basis of a different nucleic add sequence,and/or different concentrations of the same or distinct arrayfeatures, and/or different mixtures of distinct array features ordifferent-concentrations of array features. Thus an array of"distinct array features" may be an array containing, asits components, (i) distinct array features, which may have adefined amount in each component, (ii) different, gradedconcentrations of given-sequence array features, and/or (iii)different-composition mixtures of two or more distinct arrayfeatures.

However, any type of substrate known in the art may be used in themethods of the present invention.

The delivery of a known amount of a selected EST or SSH clone to aspecific position on the support surface is preferably performedwith a dispensing device equipped with one or more tips forinsuring reproducible deposition and location of the array featuresand for preparing multiple arrays. Any dispensing device known inthe art may be used in the methods of the present invention. See,for example, U.S. Pat. No. 5,807,522. The dispensing devicepreferably contains a plurality of tips.

For liquid-dispensing on a hydrophilic surface, the liquid willhave less of a tendency to bead, and the dispensed volume will bemore sensitive to the total dwell time of the dispenser tip in theimmediate vicinity of the support surface.

For liquid-dispensing on a hydrophobic surface, flow of fluid fromthe tip onto the support surface will continue from the dispenseronto the support surface until it forms a liquid bead. At a givenbead size, i.e., volume, the tendency of liquid to flow onto thesurface will be balanced by the hydrophobic surface interaction ofthe bead with the support surface, which acts to limit the totalbead area on the surface, and by the surface tension of thedroplet, which tends toward a given bead curvature. At this point,a given bead volume will have formed, and continued contact of thedispenser tip with the bead, as the dispenser tip is beingwithdrawn, will have little or no effect on bead volume.

The desired deposition volume, i.e., bead volume, formed ispreferably in the range 2 .mu.l (picoliters) to 2 nl (nanoliters),although volumes as high as 100 nl or more may be dispensed. Itwill be appreciated that the selected dispensed volume will dependon (i) the "footprint" of the dispenser tip(s), i.e., thesize of the area spanned by the tip(s), (ii) the hydrophobicity ofthe support surface, and (iii) the time of contact with and rate ofwithdrawal of the tip(s) from the support surface. In addition,bead size may be reduced by increasing the viscosity of the medium,effectively reducing the flow time of liquid from the dispensingdevice onto the support surface. The drop size may be furtherconstrained by depositing the drop in a hydrophilic regionsurrounded by a hydrophobic grid pattern on the support surface.

At a given tip size, bead volume can be reduced in a controlledfashion by increasing surface hydrophobicity, reducing time ofcontact of the tip with the surface, increasing rate of movement ofthe tip away from the surface, and/or increasing the viscosity ofthe medium. Once these parameters are fixed, a selected depositionvolume in the desired pl to nl range can be achieved in arepeatable fashion.

After depositing a liquid droplet of an array feature sample at oneselected location on a support, the tip may be moved to acorresponding position on a second support, the sample is depositedat that position, and this process is repeated until the sample hasbeen deposited at a selected position on a plurality of supports.

This deposition process may then be repeated with another EST orSSH clone sample at another microarray position on each of thesupports.

The diameter of each array feature region is preferably betweenabout 20-200 .mu.m. The spacing between each region and its closest(non-diagonal) neighbor, measured from center-to-center, ispreferably in the range of about 20-400 .mu.m. Thus, for example,an array having a center-to-center spacing of about 250 .mu.mcontains about 40 regions/cm.sup.2 or 1,600 regions/cm.sup.2. Afterformation of the array, the support is treated to evaporate theliquid of the droplet forming each region, to leave a desired arrayof dried, relatively flat array feature regions. This drying may bedone by heating or under vacuum.

Filamentous Fungal Cells

In the methods of the present invention, the two or morefilamentous fungal cells may be any filamentous fungal cell whereone of the cells is used as a reference for identifying differencesin expression of the same or similar complement of genes in theother cell. In one aspect, the two or more cells are the same cell.For example, they may be compared under different growthconditions, e.g., carbon source, oxygen limitation, nutrition,and/or physiology. In another aspect, one or more cells are mutantsof the reference cell. For example, the mutant(s) may have adifferent phenotype. In a further aspect, the two or more cells areof different species (e.g., Trichoderma reesei and Trichodermaviride). In another further aspect, the two or more cells are ofdifferent genera. In an even further aspect, one or more cells aretransformants of the reference cell, wherein the one or moretransformants exhibit a different property. For example, thetransformants may have a different or improved phenotype relativeto the reference cell and/or one of the other transformants. Theterm "phenotype" is defined herein as an observable oroutward characteristic of a cell determined by its genotype andmodulated by its environment. Such different or improved phenotypesmay include, but are not limited to, improved secretion orproduction of a protein or compound, reduced or no secretion orproduction of a protein or compound, improved or reduced expressionof a gene, desirable morphology, an altered growth rate underdesired conditions, relief of over-expression mediated growthinhibition, or tolerance to low oxygen conditions, improvedfilterability or flocculation properties, or altered proteinglycosylation.

In a preferred embodiment, the differential expression of aplurality of genes in a first filamentous fungal cell relative toexpression of the same genes in one or more second filamentousfungal cells is a result of growth of the first filamentous fungalcell on glucose and growth of the one or more second filamentousfungal cells on cellulose, hemicellulose, and/or corn stover toidentify genes that are induced by growth on cellulose,hemicellulose, or corn stover. The corn stover is preferablypre-treated and washed corn stover as described herein.

The filamentous fungal cells may be any filamentous fungal cells,but preferably Acremonium, Aspergillus, Aureobasidium, Bjerkandera,Ceriporiopsis, Coprinus, Coriolus, Cryptococcus, Filibasidium,Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum,Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, orTrichoderma cells, and more preferably Aspergillus awamori,Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina,Ceriporlopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsispannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus,Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum,Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum,Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum,Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicilliumpurpurogenum, Phanerochaete chrysosporium, Phlebia radiata,Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametesversicolor, Trichoderma harzianum, Trichoderma koningii,Trichoderma longibrachiatum, Trichoderma reesei, or Trichodermaviride cells.

In a preferred embodiment, the filamentous fungal cells areTrichoderma cells. In a more preferred embodiment, the Trichodermacells are Trichoderma reesei cells. In a most preferred embodiment,the Trichoderma cells are Trichoderma reesei strain RutC30(Montenecourt and Eveleigh, 1979, supra).

In the methods of the present invention, the cells are cultivatedin a nutrient medium suitable for growth using methods well knownin the art for isolation of the nucleic acids to be used as probes.For example, the cells may be cultivated by shake flaskcultivation, small-scale or large-scale fermentation (includingcontinuous, batch, fed-batch, or solid state fermentations) inlaboratory or industrial fermentors performed in a suitable medium.The cultivation takes place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (eg., in catalogues of the American Type CultureCollection).

Nucleic Acid Probes

The nucleic acid probes from the two or more filamentous fungalcells may be any nucleic acid including genomic DNA, cDNA, and RNA,and may be isolated using standard methods known in the art. Forexample, cDNA probes may be obtained from the total polyadenylatedmRNA isolated from the cells using standard methods and reversetranscribed into total cDNA.

The populations of isolated nucleic acid probes may be labeled withdetection reporters such as colorimetric, radioactive, fluorescentreporters, or other reporters using methods known in the art (Chenet al., 1998, Genomics 51: 313-324; DeRisi et al., 1997, Science278: 680-686; U.S. Pat. No. 5,770,367).

In a preferred embodiment, the probes are labeled with fluorescentreporters. For example, cDNA probes may be labeled during reversetranscription from the respective mRNA pools by incorporation offluorophores as dye-labeled nucleotides (DeRisi et al., 1997,supra), e.g., Cy5-labeled deoxyuridine triphosphate, or theisolated cDNAs may be directly labeled with different fluorescentfunctional groups. Fluorescent-labeled nucleotides include, but arenot limited to, fluorescein conjugated nucleotide analogs (greenfluorescence) and lissamine nucleotide analogs (red fluorescence).Fluorescent functional groups include, but are not limited to, Cy3(a green fluorescent dye) and Cy5 (red fluorescent dye).

Array Hybridization

The labeled nucleic acids from the two or more filamentous fungalcells are then added to a substrate containing an array ofTrichoderma reesei array features under conditions where thenucleic acid pools from the two or more filamentous fungal cellshybridize to complementary sequences of the array features in thearray. For purposes of the present invention, hybridizationindicates that the labeled nucleic acids from the two or more cellshybridize to the array features under very low to very highstringency conditions.

A small volume of the labeled nucleic acids mixture is loaded ontothe substrate. The solution will spread to cover the entiremicroarray. In the case of a multi-cell substrate, one or moresolutions are loaded into each cell which stop at the barrierelements.

The labeled probes are denatured and applied to a microarray slideunder a cover glass, placed in a humidified chamber, and incubatedovernight (15-16 hours) in a water bath at 63.degree. C. Beforescanning, the arrays are washed consecutively in 1.times.SSC with0.03% SDS, 0.2.times.SSC, and 0.05.times.SSC and centrifuged for 2minutes at 500 rpm top remove excess liquid. For further details,see Berka et al., 2003, Proc. Natl. Acad. Sci. USA 100: 5682-5687.

For nucleic acid probes of at least about 100 nucleotides inlength, very low to very high stringency conditions are defined asprehybridization and hybridization at 42.degree. C. in5.times.SSPE, 0.3% SDS, 200 .mu.g/ml sheared and denatured salmonsperm DNA, and either 25% formamide for very low and lowstringencies, 35% formamide for medium and medium-highstringencies, or 50% formamide for high and very high stringencies,following standard Southern blotting procedures for 12 to 24 hoursoptimally.

For nucleic acid probes of at least about 100 nucleotides inlength, the carrier material is finally washed three times each for15 minutes using 2.times.SSC, 0.2% SDS preferably at least at45.degree. C. (very low stringency), more preferably at least at50.degree. C. (low stringency), more preferably at least at55.degree. C. (medium stringency), more preferably at least at60.degree. C. (medium-high stringency), even more preferably atleast at 65.degree. C. (high stringency), and most preferably atleast at 70.degree. C. (very high stringency).

For shorter nucleic acid probes which are about 50 nucleotides toabout 100 nucleotides in length, stringency conditions are definedas prehybridization, hybridization, and washing post-hybridizationat 5.degree. C. to 10.degree. C. below the calculated T.sub.m usingthe calculation according to Bolton and McCarthy (1962, Proceedingsof the National Academy of Sciences USA 48:1390) in 0.9 M NaCl,0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1.times. Denhardt'ssolution, 1 mM sodium pyrophosphate, 1 mM sodium monobasicphosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml followingstandard Southern blotting procedures for 12 to 24 hours optimally.

For shorter nucleic acid probes which are about 50 nucleotides toabout 100 nucleotides in length, the carrier material is washedonce in 6.times.SCC plus 0.1% SDS for 15 minutes and twice each for15 minutes using 6.times.SSC at 5.degree. C. to 10.degree. C. belowthe calculated T.sub.m.

The choice of hybridization conditions will depend on the degree ofhomology between the Trichoderma reesei array features and thenucleic acids obtained from the two or more filamentous fungalcells. For example, where the cells are the same cell from whichthe array features were obtained, high stringency conditions may bemost suitable. Where the cells are from a genus or speciesdifferent from which the Trichoderma reesei array features wereobtained, low or medium stringency conditions may be more suitable.

In a preferred embodiment, the hybridization is conducted under lowstringency conditions. In a more preferred embodiment, thehybridization is conducted under medium stringency conditions. In amost preferred embodiment, the hybridization is conducted underhigh stringency conditions.

The entire solid support is then reacted with detection reagents,if needed, and analyzed using standard calorimetric, radioactive,or fluorescent detection means. All processing and detection stepsare performed simultaneously to all of the microarrays on the solidsupport ensuring uniform assay conditions for all of themicroarrays on the solid support.

Detection

The most common detection method is laser-induced fluorescencedetection using confocal optics (Cheung et al., 1998, Nat. Genet.18: 225-230). The array is examined under fluorescence excitationconditions such that (i) the Trichoderma reesei array features inthe array that hybridize to the nucleic acid probes obtained fromone of the first cell and one or more second cells produces adistinct first fluorescence emission color or one or secondfluorescence emission colors, respectively, and (ii) theTrichoderma reesei ESTs or SSH clones, or a combination thereof, inthe array that hybridize to substantially equal numbers of nucleicacid probes obtained from the first cell and one of the one or moresecond cells produce a distinct combined fluorescence emissioncolor, respectively; wherein the relative expression of the genesin the two or more cells can be determined by the observedfluorescence emission color of each spot in the array.

The fluorescence excitation conditions are based on the selectionof the fluorescence reporters. For example, Cy3 and Cy5 reportersare detected with solid state lasers operating at 532 nm and 650nm, respectively.

However, other methods of detection well known in the art may beused such as standard photometric, calorimetric, or radioactivedetection means, as described earlier.

In a preferred embodiment, the methods comprise (a) adding amixture of fluorescence-labeled nucleic acids isolated from thefilamentous fungal cells to a substrate containing an array ofTrichoderma reesei ESTs or SSH clones, or a combination thereof,selected from the group consisting of SEQ ID NOs. 1-1188, nucleicacid fragments of SEQ ID NOs. 1-1188, and nucleic acid sequenceshaving at least 90% homology to SEQ ID NOs. 1-1188, underconditions where the nucleic acids hybridize to complementarysequences of the ESTs or SSH clones; or a combination thereof; inthe array, wherein the nucleic acids from the first filamentousfungal cell and the one or more second filamentous fungal cells arelabeled with a first fluorescent reporter and one or more differentsecond fluorescent reporters, respectively; and (b) examining thearray by fluorescence under fluorescence excitation conditionswherein the relative expression of the genes in the filamentousfungal cells is determined by the observed fluorescence emissioncolor of each spot in the array in which (i) the Trichoderma reeseiESTs or SSH clones, or a combination thereof, in the array thathybridize to the nucleic acids obtained from either the first orthe one or more second filamentous fungal cells produce a distinctfirst fluorescence emission color or one or more secondfluorescence emission colors, respectively, and (ii) theTrichoderma reesei ESTs or SSH clones; or a combination thereof; inthe array that hybridize to the nucleic acids obtained from boththe first and one or more second filamentous fungal cells produce adistinct combined fluorescence emission color.

Data Analysis

The data obtained from the scanned image may then be analyzed usingany of the commercially available image analysis software. Thesoftware preferably identifies array elements, subtractsbackgrounds, deconvolutes multi-color images, flags or removesartifacts, verifies that controls have performed properly, andnormalizes the signals (Chen et al., 1997, Journal of BiomedicalOptics 2: 364-374).

Several computational methods have been described for the analysisand interpretation of microarray-based expression profilesincluding cluster analysis (Eisen et al., 1998, Proc. Nat. Acad.Sci. USA 95: 14863-14868), parametric ordering of genes (Spellmanet al., 1998, Mol. Biol. Cell 9: 3273-3297), and supervisedclustering methods based on representative hand-picked orcomputer-generated expression profiles (Chu et al., 1998. Science282: 699-705). Preferred methods for evaluating the results of themicroarrays employ statistical analysis to determine thesignificance of the differences in expression levels. In themethods of the present invention, the difference in the detectedexpression level is at least about 10% or greater, preferably atleast about 20% or greater, more preferably at least about 50% orgreater, even more preferably at least about 75% or greater; andmost preferably at least about 100% or greater.

One such preferred system is the Significance Analysis ofMicroarrays (SAM) (Tusher et al., 2001, Proc. Natl. Acad. Sd. USA98: 5116-5121). Statistical analysis allows the determination ofsignificantly altered expression of levels of about 50% or evenless. The PAM (or predictive analysis for microarrays) representsanother approach for analyzing the results of the microarrays(Tibshirani et al., 2002, Proc. Natl. Acad. Sci. USA 99:6567-6572).

Cluster algorithms may also be used to analyze microarrayexpression data. From the analysis of the expression profiles it ispossible to identify co-regulated genes that perform commonmetabolic or biosynthetic functions. Hierarchical clustering hasbeen employed in the analysis of microarray expression data inorder to place genes into clusters based on sharing similarpatterns of expression (Eisen et al., 1998, supra). This methodyields a graphical display that resembles a kind of phylogenetictree where the relatedness of the expression behavior of each geneto every other gene is depicted by branch lengths. The programsCluster and TreeView, both written by Michael Eisen (Eisen et al.,1998 Proc. Nat Acad. Sci. USA 95: 14863-14868) are freelyavailable. Genespring is a commercial program available for suchanalysis (Silicon Genetics, Redwood City, Calif.).

Self-organizing maps (SOMs), a non-hierarchical method, have alsobeen used to analyze microarray expression data (Tamayo et al.,1999, Proc. Natl. Acad. Sci. USA 96: 2907-2912). This methodinvolves selecting a geometry of nodes, where the number of nodesdefines the number of clusters. Then, the number of genes analyzedand the number of experimental conditions that were used to providethe expression values of these genes are subjected to an iterativeprocess (20,000-50,000 iterations) that maps the nodes and datapoints into multidimensional gene expression space. After theidentification of significantly regulated genes, the expressionlevel of each gene is normalized across experiments. As a result,the expression profile of the genome is highlighted in a mannerthat is relatively independent of each gene's expression magnitude.Software for the "GENECLUSTER" SOM program for microarrayexpression analysis can be obtained from the Whitehead/MIT Centerfor Genome Research. SOMs can also be constructed using theGeneSpring software package.

Computer Readable Media

The Trichoderma reesei array features described herein may be"provided" in a variety of mediums to facilitate theiruse. The term "provided" refers to a manufacturecomprising an array of Trichoderma array features. Suchmanufactures provide a large portion of the genomes of Trichodermareesei and parts thereof (e.g., an open reading frame (ORF)) in aform which allows one skilled in the art to examine the manufactureusing means not directly applicable to examining the genome or asubset thereof as it exists in nature or in purified form.

Thus, the present invention also relates to such a manufacture inthe form of a computer readable medium comprising an array ofTrichoderma reesei ESTs or SSH clones, or a combination thereof. Ina preferred embodiment, the computer readable medium comprises anarray of Trichoderma reesei ESTs or SSH clones, or a combinationthereof, selected from the group consisting of SEQ ID NOs. 1-1188,nucleic acid fragments of SEQ ID NOs. 1-1188, or nucleic acidsequences having at least 95%, preferably at least 99% and mostpreferably at least 99.9% homology to a sequence of SEQ ID NOs.1-1188. In another preferred embodiment, the computer readablemedium comprises an array of Trichoderma reesei ESTs or SSH clones,or a combination thereof, selected from the group consisting of SEQID NOs. 1-1188.

In one application of this embodiment, the Trichoderma reesei arrayfeatures can be recorded on computer readable media. The term"computer readable media" is defined herein as any mediumwhich can be read and accessed directly by a computer. Suchcomputer readable media include, but are not limited to, magneticstorage media, e.g., floppy discs, hard disc storage medium, andmagnetic tape; optical storage media, e.g., CD-ROM, DVD; electricalstorage media, e.g., RAM and ROM; and hybrids of these categories,e.g., magnetic/optical storage media. One skilled in the art canreadily appreciate how any of the presently known computer readablemedia can be used to create a manufacture comprising computerreadable medium having recorded thereon nucleotide sequences of theTrichoderma reesei array features of the present invention.Likewise, it will be clear to those of skill how additionalcomputer readable media that may be developed also can be used tocreate analogous manufactures having recorded thereon nucleotidesequences of the Trichoderma reesei array features.

As used herein, "recorded" refers to a process forstoring information on computer readable medium. One skilled in theart can readily adopt any of the presently known methods forrecording information on computer readable medium to generatemanufactures comprising the nucleotide sequence information of thepresent invention.

A variety of data storage structures are available for creating acomputer readable medium having recorded thereon the Trichodermareesei array features of the present invention. The choice of thedata storage structure will generally be based on the means chosento access the stored information. In addition, a variety of dataprocessor programs and formats can be used to store the nucleotidesequence information of the present invention on computer readablemedium. The sequence information can be represented in a wordprocessing text file, formatted in commercially-available softwaresuch as WordPerfect and Microsoft Word, or represented in the formof an ASCII file, stored in a database application, such as DB2,Sybase, Oracle, or the like. A skilled artisan can readily adaptany number of data-processor structuring formats (e.g., text fileor database) in order to obtain computer readable medium havingrecorded thereon the nucleotide sequence information of the presentinvention.

Various computer software are publicly available that allow askilled artisan to access sequence information provided in acomputer readable medium. Thus, by providing in computer readableform an array of Trichoderma reesei ESTs or SSH clones, or acombination thereof, selected from the group consisting of SEQ IDNOs. 1-1188, nucleic acid fragments of SEQ ID NOs. 1-1188, andnucleic acid sequences having at least 90%, preferably at least95%, more preferably at least 99%, and most preferably at least99.9% homology to SEQ ID NOs. 1-1188 enables one skilled in the artto routinely access the provided sequence information for a widevariety of purposes.

Software utilizing the BLAST (Altschul et al., 1990, Journal ofMolecular Biology 215: 403-410) and BLAZE (Brutlag et al., 1993,Comp. Chem. 17: 203-207) search algorithms may be used to identifyopen reading frames (ORFs) within a genome of interest, whichcontain homology to ORFs or proteins from Trichoderma reesei andfrom other organisms. Among the ORFs discussed herein are proteinencoding fragments of the Trichoderma reesei genome useful inproducing commercially important proteins, such as enzymes, and inthe production of commercially useful metabolites.

The present invention further provides systems, particularlycomputer-based systems, which contain the sequence informationdescribed herein. Such systems are designed to identify, amongother things, genes and gene products--many of which could beproducts themselves or used to genetically modify an industrialexpression host through increased or decreased expression of aspecific gene sequence(s).

The term "a computer-based system" is defined herein as ahardware means, software means, and data storage means used toanalyze the nucleotide sequence information of the presentinvention. The minimum hardware means of the computer-based systemsof the present invention comprises a central processing unit (CPU),input means, output means, and data storage means. One skilled inthe art can readily appreciate that any currently availablecomputer-based system is suitable for use in the present invention.

As stated above, the computer-based systems of the presentinvention comprise a data storage means having stored thereinnucleic acid sequences of the Trichoderma reesei array features andthe necessary hardware means and software means for supporting andimplementing a search means.

The term "data storage means" is defined herein as memorywhich can store nucleotide sequence information of the presentinvention, or a memory access means which can access manufactureshaving recorded thereon the nucleotide sequence information of thepresent invention.

The term "search means" is defined herein as one or moreprograms which are implemented on the computer-based system tocompare a target sequence or target structural motif with thesequence information stored within the data storage means. Searchmeans are used to identify fragments or regions of the presentsequences which match a particular target sequence or target motif.A variety of known algorithms are disclosed publicly and a varietyof commercially available software for conducting search means areand can be used in the computer-based systems of the presentinvention. Examples of such software includes, but is not limitedto, MacPattern (Fuchs, 1991, Comput Appl. Biosci. 7: 105-106),BLASTN and BLASTX (NCBI). One skilled in the art can readilyrecognize that any one of the available algorithms or implementingsoftware packages for conducting homology searches can be adaptedfor use in the present computer-based systems.

The term "target sequence" is defined herein as any DNAor amino acid sequence of six or more nucleotides or two or moreamino acids. One skilled in the art can readily recognize that thelonger a target sequence is, the less likely a target sequence willbe present as a random occurrence in the database. The mostpreferred sequence length of a target sequence is from about 10 to100 amino acids or from about 30 to 300 nucleotide residues.However, it is well recognized that searches for commerciallyimportant fragments, such as sequence fragments involved in geneexpression and protein processing, may be of shorter length.

The term "a target structural motif" or "targetmotif" is defined herein as any rationally selected sequenceor combination of sequences chosen based on a three-dimensionalconfiguration which is formed upon the folding of the target motif.There are a variety of target motifs known in the art. Proteintarget motifs include, but are not limited to, enzyme active sitesand signal sequences, substrate and cofactor binding domains,transmembrane domains, and sites for post-translationalmodifications. Nucleic acid target motifs include, but are notlimited to, promoter sequences, hairpin structures and inducibleexpression elements (protein binding sequences), repeats,palindromes, dyad symmetries, intron-exon boundaries, transcriptionand translation start and stop sites, and polyadenylation signals.

A variety of structural formats for the input and output means canbe used to input and output the information in the computer-basedsystems of the present invention. A preferred format for an outputmeans ranks fragments of the nucleic acid sequences possessingvarying degrees of homology to the target sequence or target motif.Such presentation provides one skilled in the art with a ranking ofsequences which contain various amounts of the target sequence ortarget motif and identifies the degree of homology contained in theidentified fragment.

A variety of comparing means can be used to compare a targetsequence or target motif with the data storage means to identifysequence fragments of a genome. For example, implementing softwarewhich utilize the BLAST and BLAZE algorithms, described in Altschulet al., 1990, Journal of Molecular Biology 215: 403-410, may beused to identify open reading frames within the Trichoderma reeseigenome or the genomes of other organisms. A skilled artisan canreadily recognize that any one of the publicly available homologysearch programs can be used as the search means for thecomputer-based systems of the present invention. Of course,suitable proprietary systems that may be known to those of skillalso may be employed in this regard.

Substrates

The present invention also relates to substrates as describedherein comprising an array of Trichoderma reesei ESTs or SSHclones, or a combination thereof. In a preferred embodiment, thesubstrate comprises an array of Trichoderma reesei ESTs or SSHclones, or a combination thereof, selected from the groupconsisting of SEQ ID NOs. 1-1188, nucleic acid fragments of SEQ IDNOs. 1-1188, or nucleic acid sequences having at least 95%,preferably at least 99% and most preferably at least 99.9% homologyto a sequence of SEQ ID NOs. 1-1188. In a more preferredembodiment, the substrate comprises an array of Trichoderma reeseiESTs or SSH clones, or a combination thereof, selected from thegroup consisting of SEQ ID NOs. 1-1188.

Claim 1 of 8 Claims
1. A method for monitoring differential expression of a pluralityof genes in a first filamentous fungal cell relative to expressionof the same genes in one or more second filamentous fungal cells,comprising: (a) adding a mixture of detection reporter-labelednucleic acids isolated from the first and one or more secondfilamentous fungal cells to a substrate containing an array ofTrichoderma reesei ESTs or SSH clones, or a combination thereof,selected from the group consisting of SEQ ID NOs. 1-1188, underconditions where the nucleic acids hybridize to complementarysequences of the ESTs or SSH clones, or a combination thereof, inthe array, wherein the nucleic acids from the first filamentousfungal cell and the one or more second filamentous fungal cells arelabeled with a first detection reporter and one or more differentsecond detection reporters, respectively; and (b) examining thearray under conditions wherein the relative expression of the genesin the first and one or more second filamentous fungal cells isdetermined by the observed detection signal of each spot in thearray in which (i) the Trichoderma reesei ESTs or SSH clones, or acombination thereof, in the array that hybridize to the nucleicacids obtained from either the first or the one or more secondfilamentous fungal cells produce a distinct first detection signalor one or more second detection signals, respectively, and (ii) theTrichoderma reesei ESTs or SSH clones, or a combination thereof, inthe array that hybridize to the nucleic acids obtained from boththe first and one or more second filamentous fungal cells produce adistinct combined detection signal; wherein the hybridizationconditions are selected from the group consisting of very low, low,low-medium, medium, medium-high, high, and very high stringencyconditions.

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