WO2000068423A2

WO2000068423A2 - Improved predictive power of rna analysis for protein expression

Info

Publication number: WO2000068423A2
Application number: PCT/EP2000/004095
Authority: WO
Inventors: Matthias W. Hentze
Original assignee: The European Molecular Biology Laboratory
Priority date: 1999-05-05
Filing date: 2000-05-05
Publication date: 2000-11-16
Also published as: WO2000068423A3; AU4562700A

Abstract

The invention relates to the field of RNA expression analysis. In particular, this invention relates to the isolation of polysomal messenger RNA and of non-polysomal ribonucleoprotein particles on which subsequent expression analyses are performed. The polysomal messenger RNA fraction is analyzed for more accurate prediction of protein expression levels in an organism. In particular, changes in the translational state of an organism in response to a perturbation can be determined by examining the abundance of an RNA of interest in the polysomal RNA fraction of the organism before and after it is exposed to the perturbation. The effect of a perturbation on an organism can be determined by examining the ratio of an RNA of interest in the polysomal RNA fraction to an RNA of interest in the total RNA fraction. A change in the abundance of the RNA of interest in the polysomal fraction, or in the ratio of the RNA of interest in the polysomal fraction to the RNA of interest in the non-polysomal fraction indicates a translational effect of the perturbation.

Description

IMPROVED PREDICTIVE POWER OF R\ A ANALYSIS FOR PROTEIN EXPRESSION

1. FIELD OF THE INVENTION This invention relates to the use of polysomal messenger RNA and of messenger πbonucleoprotein particles to perform expression analyses In one embodiment, the polysomal messenger RNA fraction is analyzed for more accurate prediction of protein expression levels in an organism In a preferred embodiment polysomal messenger RNA is analyzed bv hybridization to a microarray of nucleic acid probes in order to better predict protein expression levels

2. BACKGROUND

Genetic information becomes cellular function through the synthesis of proteins whose blueprints are contained in the DNA and RNA The amounts and types of proteins synthesized at any given time in a cell can be controlled transcπptionally, post- transcπptionally, translationallv, or post-translationally Modulation of transcription initiation through, e g , promoter availability, affinity of polymerase for a promoter, frequency of binding to a promoter, of mR A splicing and post-transcπptional modification, of mRNA stability, of mRNA translation, and of the post-translational fate of proteins, e g , folding, transport, and stability, can all contribute to variability in the amounts and types of proteins present in a cell

Translation is the process by which the genetic information encoded in messenger RNA (hereinafter referred to as "mRNA") is converted into proteins mRNAs can be polycistronic, / e , coding for several proteins as in the case of pro aryotes, or they can be monocistronic, having only one open reading frame ("ORF") per molecule, as in the case of eukaryotes. The process of translation from a linear arrangement of nucleotides to an assembly of covalently linked amino acids occurs in the cell cytoplasm on πbosomes, compact πbonucleoprotein particles containing both proteins and πbosomal RNA ("rRNA") A πbosome attaches to mRNA 5' of the coding region and moves along toward the 3' end translating each triplet codon into an ammo acid along the way Each codon is matched to a particular amino acid v ia transfer RNA ("tR A ) which can be covalentlv linked to one amino acid, and which contains an anticodon loop complementary to the mRNA codon representing that amino acid A πbosome accommodates two aminoacvl- tRNAs at the same time, allowing the formation of a peptide bond between the nascent polypeptide chain on one tRNA having 'n" amino acids and the n-H " amino acid on the second tRNA \ single mRNA molecule may be translated manv times w ith the result that mRN A which is activ ek being translated is associated simultaneouslv w ith several πbosomes (Levv in denes II 1990 Oxford university Press)

Although irtuallv all mRN A is associated w ith proteins not all mRN A is actively ^"" being translated at the same time In prokaryotes transcription and translation are often coupled, and mRN As are typically unstable In eukaryotes, nevv lv synthesized pre- messenger RNA is bound rapidly by proteins to form pre-messenger πbonucleoprotein particles ( 'pre-mRNP Y ) in which the pre-mRNA portion is capped at the 5' nucleotide, polyadenylated at the 3 '-end, and spliced to form messenger πbonucleoprotein particles 10 ( mRNP's") Mature mRNA is accompanied by proteins through the nuclear pores and into the cytoplasm (McCarthy & Kollmus, 1995, I t enth Btochem Set 20(5) 191 -7)

Through the association of mRNA with proteins, genetic control can be exerted at the translational lev el In systems such as developing embryonic cells that depend on the precise temporal expression of genes mRNA may be stored in mRNPs for translation at ' 5 another time or under different circumstances (Lewin 1990, Standart & Jackson, 1994, Biochimte 76 867-879) For example, the glp-1 C elegans membrane receptor gene is temporally and spatially regulated in early embryogenesis at the translational level (Evans et al , 1994, Cell 11 183- 194) Negative translational regulation of some mRN As is achieved by protein binding to specific structural or sequence elements in either the 5' or 3' 20 untranslated regions ("UTR") The translation of proteins involved in iron storage and metabolism, such as ferπtin, erythroid δ-aminolevuhnate synthase, and pig heart (mitochondπal) cis-acomtase is regulated by the binding of iron regulatory protein to iron responsive elements in the 5'-UTR of these mRN As (Hentze & Kuhn, 1996 Pt oc Natl Λcad Set USA 93( 16) 8175-82) Other proteins, such as thvmidylate synthase, 25 dihydrofolate reductase, and the yeast L32 πbosomal protein autoregulate translation by binding to their own mRN As (McCarthy & Kollmus 1995) Translational control can also be exerted through initiation factors, elongation factors, and termination factors by e g , varying concentrations of the factors, phosphorvlation, or dephosphorvlation in response to hormones, growth factors, heat shock, or viral infection (Dav & Tuite, 1998, / ³⁰ Endocnn 157 361 -371 ).

Protein expression in cells and tissues is often predicted from an analvsis of cellular RNA. e g , by Northern blot analvsis, in situ hybridization, or reverse transcription followed by polymerase chain reaction Many techniques used bv those skilled in the art for isolating RNA produce either total cellular RNA or total cytoplasmic RNA (Current - Protocols in Molecular Bιolog\ , Volume I Ausubel el al , eds , 1994 John Wiley & Sons,

. i lnc ) Howev er, only mRNA w hich is a small fraction ot the total RNA in both prokaryotes and eukarvotes ( Lewin, denes Il ^~). is translated into protein Furthermore, as is evidenced by the above examples, the presence of mRNA in a cell does not necessarily indicate that the protein for which it codes is being synthesized Rather the mRNA can be stored or suppressed for translation at a different time or under different circumstances or it can be degraded without ever being translated Other techniques for isolating RN A produce the polysomal RNA fraction (Aziz & Munro, 1 86, Nucl Acids Re.s 14(2) 91 5-927, Rogers & Munro. 1987 Proc Natl Acad Set USA 84 2277-81 , Melefors et al . 1993 J Btol Chern 268 5974-78) A need exists for more accurate methods of performing expression analysis in order to determine the effects, side effects, and targets of drugs, as well as to discover lead candidates for drugs Current methods of expression analvsis using nucleotide arrays do not specifically querv the translational state of a cell Nucleotide arrav expression analysis studies are done using cellular RNA that is not fractionated into polysomal or non- polysomal portions (see, e g , International Publication No WO 92/10588 dated June 25, 1992), the amount of which may poorly correlate with protein expression Therefore, there is a need in the art for methods of analysis that specifically measure quantities that better correlate with levels of protein expression in a cell

Citation of a reference herein shall not be construed as indicating that such reference is prior art to the present invention

3. SUMMARY OF THE INVENTION

The present invention provides methods of expression analysis that measure quantities that correlate well with the level of protein expression in a cell The present invention relates to methods for predicting the amount of a protein of interest expressed by a cell, comprising measuring the amount in the cell of RNA encoding said protein that is within the polysomal fraction of RNA of the cell In particular, the invention relates to methods for isolating the polysomal fraction of RNA from a cell and for measuring the amount of RNA encoding a protein of interest in the polysomal fraction In a preferred embodiment, the invention relates to methods for determining the effects of various perturbations on cells or organisms, including but not limited to the effects of drugs, of environmental changes, and of the presence of a disease state, by measuring the level of an RNA of interest in the polysomal RNA fraction from a cell or organism exposed to the perturbation, and comparing such level in the polysomal RNA fraction from a cell or organism not exposed to the perturbation Furthermore, the translational state of a cell or organism exposed to different levels of a perturbation or to different perturbations, e.g.. two different drugs, can be measured in the same manner. In particular, the invention provides a method for determining the translational state of each of a plurality of RNA species within the cell. The present invention also relates to methods of determining whether a perturbation to which a cell is exposed affects translation, RNA abundance, or fractional translational efficiency. In particular, the methods relate to measuring the abundance of RNAs of interest in the polysomal RNA fraction of a cell and in the total RNA fraction of a cell, and to measuring the ratio of polysomal to non-polysomal RNA before and after it is exposed to a perturbation For example, an increase in the abundance of RNAs of interest in the polysomal RNA fraction of a cell but not proportionately in the total RNA fraction (or in the non-polysomal fraction) of the cell indicates that the perturbation has an effect of activating translation. An increase in the abundance of an RNA of interest in the total RNA fraction (or in the non-polysomal fraction) of a cell but not proportionately in the polysomal RNA fraction of the cell after it is exposed to a perturbation indicates that the perturbation has a translational deactivating effect. In particular, a method of determining whether a perturbation upon a cell or an organism affects translation, RNA abundance, or fractional translational efficiency comprises: (a) measuring abundances of each of a plurality of species of RNA within a polysomal RNA fraction in a first cell or organism exposed to a perturbation; (b) comparing the abundances of each of said plurality of species of RNA to the abundances of each of said plurality of species of RNA within a polysomal RNA fraction in a second cell or organism not exposed to said perturbation or exposed to a different amount of said perturbation, to detect any change in said abundances; (c) measuring abundances of each of a plurality of species of RNA within total cellular or cytoplasmic RNA or non-polysomal fraction in said first cell or organism exposed to said perturbation to obtain abundances of each of said plurality of species of RNA within the total cellular or cytoplasmic RNA; (d) comparing the abundances of each of said plurality of species of RNA to the abundances of each of said plurality of species of RNA within total cellular or cytoplasmic RNA in said second cell or organism not exposed to said perturbation or exposed to a different amount of said perturbation to detect any change in said abundances; and (e) comparing the change in abundances detected in step (b) with the change in abundances detected in step (d) for at least one species of RNA within said plurality, so as to determine whether said perturbation affects translation, abundance, or relative translational efficiency of said at least one species, wherein a change in abundance of a species of RNA within the polysomal fraction and not proportionately of said species of RNA within the total cytoplasmic or cellular RN A indicates a translational effect of the perturbation, a change in the abundance of said species of RN A within the total cellular or cytoplasmic RNA indicates an effect on total RNA abundance and a change in the ratio ot polysomal R A ot said species to non-polysomal RNA ot said species indicates an effect on the fractional translational efficiency of the perturbation on said cell or organism when exposed to the perturbation The abundance of total RNA of a particular species can be determined by adding polysomal plus non-polysomal RNA levels of that species or bv direct measurement of total cellular or cytoplasmic RN A of that species

The invention also relates to methods of measuring the abundance of RNAs of interest in a cell In a preferred embodiment the abundance of RNAs of interest in a particular RNA fraction of a cell is measured using hybridization to a microarrav of nucleic acid probes

In particular the invention relates to a method for comparing the effects of a first perturbation and a second, different perturbation upon a cell or organism comprising (a) measuring abundances of each of a plurality of species of RNA within a polysomal fraction in a first cell or organism exposed to a first perturbation, (b) comparing the abundances of each of said plurality of species of RNA to the abundances of each of said plurality of species of RNA within a polysomal fraction in a second cell or organism not exposed to said first perturbation, to detect any change in said abundances (c) measuring abundances of each of said plurality of species of RNA within a polysomal fraction in a third cell or organism exposed to a second perturbation different from said first perturbation, (d) comparing the abundances of each of said plurality of species of RNA measured in step c to the abundances of each of said plurality of species of RNA within a polysomal fraction in a second cell or organism not exposed to said second perturbation to detect any change in said abundances, and (e) comparing the change in abundances detected in step (b) with the change in abundances detected in step (d), wherein a difference in the amount of change in abundance of a species of polysomal RNA detected in step b relative to step d indicates differences in effects of the first perturbation and the second perturbation upon expression of the protein encoded by said species The invention further comprises a method for comparing the effects of a first perturbation and a second different perturbation upon a cell or organism, wherein the abundances of each of said plurality of RNA species are measured bv a method comprising contacting a microarray with a polysomal fraction of RNA from said cell or organism or with cDNA derived therefrom wherein said microarray comprises a solid phase surface ith attached nucleic acid probes said nucleic acid probes being capable of hybridizing with said plurality of RNA species or with cDNA derived therefrom The inv ention further provides microarravs to which is hybridized RNA or cDNA derived therefrom from a polysomal fraction

4. DETAILED DESCRIPTION OF THE INVENTION 4.1 EXPRESSION ANALYSIS METHODS

The present invention provides methods of RNA expression analysis that measure quantities that better correlate with the level of protein expression in a cell In one embodiment the present invention is directed to methods for predicting the amount of a protein of interest expressed by a cell comprising measuring the amount of an RNA transcript in the polysomal RNA fraction of the cell In particular, the polysomal RNA fraction is isolated from a cell using density gradient centπfugation or other separation techniques, and subsequently, the amount of an RNA transcript coding for a particular protein in the polysomal RNA fraction is measured

The present invention is directed to methods for determining the effect of a perturbation upon a cell or organism by analyzing the abundance of one or more RNA species in the polysomal RNA fraction in a cell and comparing it to the abundance of the same species of RNA in the polysomal RNA fraction in a cell not exposed to the perturbation, or alternatively, by comparing it to the abundance of the same species of RNA in the polysomal RNA fraction in the cell before it is exposed to the perturbation This comparison elucidates a change in the abundance of an RNA species ithin the polysomal fraction, which indicates a change in the amount of protein encoded by the RNA species, expressed bv the cell or organism when exposed to the particular perturbation Such perturbations include, but are not limited to, changes in pH, temperature, radiation exposure, salt concentration, food source, cell density, and exposure to drugs (including but not limited to hormones or growth factors), as well as alterations in developmental state, differentiation state, and disease state of a cell or organism The present invention is further directed to methods for comparing the effects of different perturbations upon a cell or organism In particular, the abundances of RNA species in the polysomal fraction in a first cell exposed to a first perturbation are measured and are compared to the abundances of RNA species in the polysomal fraction in a second cell not exposed to the perturbation in order to detect any changes in the abundances In addition the abundances of RNA species in the polysomal fraction in a third cell exposed to a second perturbation are measured and are compared to the abundances of RN A species in the polysomal fraction in the second cell not exposed to the perturbation in order to detect anv changes in the abundances The changes in abundances of RNA species in the polvsomal fractions of the two cells exposed to the two different perturbations are then compared to determine the effects of these perturbations on the expression of proteins encoded by the RNA species The two perturbations whose effects on protein expression levels in a cell can be determined include but are not limited to, exposure to different drugs (including but not limited to hormones or growth factors) to different pHs, to different salt concentrations, to different food sources, to different temperatures, as well as being in different developmental states differentiation states, or disease states

The present invention relates to methods for determining the translational state of a cell by measuring the abundances of RNA species in the polysomal fraction of RNA within the cell In a preferred embodiment the abundances of a plurality of RNA species are measured by contacting a gene transcript array with a polvsomal fraction of RNA from a cell or organism, wherein the gene transcript array comprises a solid surface to which nucleic acids are attached In another preferred embodiment, the nucleic acids of the transcript array are capable of hybridizing to the majority of gene transcripts expressed by a cell or organism

The invention further relates to microarrays to which are hybridized RNA from a polysomal fraction or cDNA derived therefrom The invention provides a microarray that is a solid phase comprising on its surface a plurality of nucleic acid probes attached to said surface, wherein the density of the probes of the microarray is greater than 60 probes per cm², each of which probes hybridizes to a gene transcript of one or more organisms or cDNA derived therefrom, wherein at least a portion of said probes are hvbπdized to a plurality of RNA species, or cDNA derived therefrom derived from a polysomal fraction from said one or more organisms, and wherein substantially none of said probes is hybridized to RNA, or to cDNA derived therefrom, derived from a non-polysomal fraction The invention further relates to an embodiment of the above-described microarray, to which is simultaneously hybridized a plurality of a first RNA species or cDNA derived therefrom of a first organism exposed to a first perturbation and a plurality of a second RNA species or cDNA derived therefrom of a second organism exposed to a second perturbation, and wherein the first and second RNA species or cDNA derived therefrom are distinguishably labeled In a different specific embodiment, the invention relates to a microarray, to which is simultaneously hybridized a plurality of a first RNA species or cDNA derived therefrom of a first organism exposed to a first perturbation and a plurality of a second R A species or

- 7 -

SUBSTTTUTE SHEET (RULE 26) cDNA derived therefrom of a second organism not exposed to the perturbation, and wherein the first and second RN A species or cDNA derived therefrom are distinguishably labeled

The inv ention also relates to methods of determining whether a perturbation upon a cell or an organism affects translation RN A abundance and/or fractional translational efficiency The relativ e contributions of effects ot a perturbation on the translational state of a cell, on RN A abundance and on fractional translational efficiency can be conveniently determined bv, e g measuring total mRNA (or RNA) abundances and mRNA (or RNA) abundances in the polvsomal RNA and non-polvsomal RNA fractions and comparing the ratio of the two before and after a cell or an organism is exposed to a perturbation in order to characterize the effect of the perturbation as affecting translation, RNA abundance, or fractional translational efficiency Total cytoplasmic RNA consists of a polvsomal fraction plus a non-polysomal fraction mRNP (messenger πbonucleoprotein ) is translationally inactive RNA in the non-polysomal fraction If the amount of total cellular or cytoplasmic mRNA in a cell remains constant before and after a perturbation, but the amount of mRNA in the polysomal fraction increases in response to the perturbation, then a redistribution of mRNA into the polysomal fraction has occurred and the perturbation has resulted in translational activation Similarly, if there is an increase in the amount of total mRNA in a cell after the cell has experienced a perturbation, but there is not a proportionate increase of mRNA in the polysomal fraction, then the perturbation has a translational deactivating effect Examination of the ratio of a species of RNA or mRNA of interest in the polysomal fraction to that in the non-polysomal fraction of a cell exposed to a perturbation in comparison to a cell not exposed to the peπurbation indicates whether there has been an increase in translation in the cell of that species of RNA or mRNA due to the perturbation (increase in the ratio), or whether there has been a decrease in translation in the cell of that species of RNA or mRNA due to the perturbation (decrease in the ratio)

Although, for simplicity this disclosure often makes reference to a cell" (e g , "RNA is isolated from a cell"), it will be understood by those of skill in the art that any particular step of the invention will also be construed as covering use of a plurality of cells, e g , from a tissue sample from an organism, or from a cultured cell line Such cells can be genetically similar cells, called herein a "cell type" Such cells can be from naturally single celled organisms or derived from multi-cellular higher organisms The cell can be a cell of a plant or an animal (including but not limited to mammals, primates, humans, and non- human animals such as dogs, cats, horses, cows, sheep, mice, rats, etc ) Moreover, in the methods of the invention the abundances of either RNA or mRNA (only polyadenylated

- 8 -

SUBSTΓΓUTE SHEET (RULE 26) RNA) can be measured in the polvsomal, non-polysomal or total cellular or cvtoplasmic fractions

This section presents a detailed description of the inv ention and its application to monitoring the biological state and more specifically to the translational state, of a cell and changes in the biological state, and more specifically to the translational state, of a cell exposed to one or more conditions

The following subsections present the methods of the invention in greater detail In particular. Section 4.2 first describes methods for isolating polvsomal and non-polvsomal

RNA from prokarvotic and eukaryotic cells Section 4 3 describes, in detail, how perturbations experienced by a cell or an organism lead to alterations in protein expression

Section 4 4 describes, in detail, a preferred embodiment of the invention for measuring the amount of particular mRNA transcripts in a cell or an organism using microarrays Finally,

Section 4.5 describes other methods for measuring the amount of paπicular mRNA transcripts in a cell or an organism This description is by way of several exemplary illustrations, in increasing detail and specificity, of the general methods of this invention These examples are non-limiting, and related variants that will be apparent to one of skill in the art are intended to be encompassed by the appended claims

4.2 ISOLATION OF POLYSOMAL AND NON-POLYSOMAL

FRACTIONS OF mRNA

The methods of the invention involve analyses using the polvsomal fraction of RNA and in some instances, non-polysomal (mRNP) and/or total RN A, as described above Exemplary protocols that can be used are described below and in the Examples (infra) Protocols for isolating RNA should be performed in solutions that are substantially free of πbonuclease (RNase) Solutions that come in contact with RNA are preferably treated with an Rnase inhibitor, such as diethylpyrocarbonate (DEPC), to inhibit RNase activity, gloves are preferably worn at all times RNase inhibitors that can be used are detailed in references cited hereinbelow or can be any known in the art Isolation of prokarvotic RNA

Prokarvotic total cellular RNA can be isolated by any method known to those skilled in the art Exemplary' protocols for isolation of RNA from bacteria can be found in ( ^'urrenr Protocols in Molecular Biology Volume /, 1994, Ausubel et al eds John Wiley & Sons, Inc , pp 4 4 1 -4 4 7 One example of such methods is used to isolate RNA from gram negative bacteria It involves Ivsis of the bacteria in a sucrose/detergent solution, followed by phenol/chloroform extraction of contaminating proteins, ethanol precipitation of nucleic acids and purification of RNA on a CsCl gradient \ second example of such methods is also used to isolate RNA from gram negative bacteria It is a rapid isolation technique involv ing lysozvme digestion of the bacterial cell walls, ivsis of the remaining protoplasts in a detergent-containing buffer, precipitation of contaminating detergent, protein, and DNA, and subsequent ethanol precipitation of RNA A third example of such methods is used to isolate RNA from gram positive bacteria Because the cell walls of gram positive bacteria are thicker, sonication is used to break the cell wall, followed bv deteigent Ivsis of the cell membrane, protease digestion and phenol/chloroform extraction to remove contaminating proteins, DNase digestion to remove contaminating DNA, and ethanol precipitation of RNA

The recovery of RNA from bacteria using these protocols yields total RNA Methods that can be used to yield polysomal and non-polysomal RNA fractions are described below Isolation of eukarvotic RNA

Isolation of total cellular RNA or various RNA fractions can be accomplished by any method known to those skilled in the art Exemplary methods of isolation of total RNA can be found in Current Protocols in

Molecular Biology , Volume /, 1994, Ausubel et al , eds , John Wilev & Sons, Inc , pp 4 1 2-4 3 4, Chirgwin et al , 1979, Bioc e istn 18(24) 5294-5299 These protocols involve lysing cells in a gua dine solution, which quickly denatures all proteins, and separating out the water soluble RNA either bv collecting the aqueous phase in the presence of phenol/chloroform (single-step method), or bv ultracentπfugation through a CsCl gradient Variations on the guamdine solution cell lysis may involve the presence of detergent in the guamdine lysis solution, or the physical homogenization of tissues in the gua dine solution

The isolation of cytoplasmic RNA can be accomplished by lysing eukaryotic cells using a non-ionic detergent, leaving the nuclei intact (Current Protocols in Molecular Biology , Volume 1, 1994, Ausubel et al , eds John Wiley & Sons, Inc ) Although this protocol results in the co-purification of tRNA, rRNN and mRNA, DNA does not contaminate the preparation since it is removed bv sedimentation of the intact nuclei

The isolation of polysomal RNA can be accomplished by cell Ivsis, followed by ultracentπfugation of the cell Ivsate by ultracentπfugation through a 10-50% linear sucrose gradient ( Aziz &. Munro 1986 Xntl itith Res 14(2) 91 5-927 Rogers & Munro 1987 Proc Natl Acad Set (XSA 84 2277-81 Meiefors et al 1993 I Btol Chem 268 5974-78) As detailed in these references mRN A molecules associated with different numbers of πbosomes hav e different densities and therefore are separable bv the sucrose gradient method

The sucrose gradient is fractionated to separate monosomal RNA, polvsomal RNA and mRNP using absorbance of 254 nm radiation to monitor the profile RNA in each fraction is then extracted from protein using, e g , a phenol-chloroform-isoamv l alcohol (50 50 1 ) extraction procedure (Aviv & Leder 1972, Proc Natl Acad Sci US.4.

10 69(6) 1408- 12)

Isolation of polyfAV RNA

Messenger RNA can also be isolated by exploiting the fact that eukarvotic mRNAs

15 and some prokaryotic mRNAs undergo post-transcnptional polyadenylation at the 3' end This post-translational modification can be exploited to further isolate mRNA from other cellular RNAs, particularly when using protocols that isolate total cellular or cytoplasmic RNA These isolation procedures are known to those skilled in the art and inv olve the binding of isolated RNA (see above) to oligo-dT cellulose and then destabilization of the 0 dA dT duplex by removing salt from the solution (Current Pr otocols in Molecular Biology , Volume /, 1994, Ausubel et al , eds John Wiley & Sons, Inc , pp 4 5 1 -4 3 Aviv & Leder 1972 Pr oc Natl Acad Sci USA 69 1408-12)

4.3 THE EFFECTS OF PERTURBATIONS ON PROTEIN EXPRESSION 5 The present invention is particularly useful for determining the effects of perturbations on the translational state of a cell or an organism These perturbations include, but are not limited to, changes in environment, such as pH, temperature food source, radiation exposure, salt concentration, and cell density changes in disease state, changes in developmental state changes in differentiation state, and exposure to drugs 0 (including but not limited to hormones or growth factors) Such analyses are of great value in determining e g , the effects side effects and genetic targets of drugs and other perturbations assessing the effect of a disease state upon protein expression and thus allowing more effective therapeutic strategies to be designed as well as to discover lead candidates for drugs by detecting their effect on expression of proteιn(s) of interest Similarities and differences in perturbation effects can also be deduced Drug Action and Biolomcal State

According to the current invention drugs are any compounds of any degree of complexity that perturb a biological system, whether by known or unknown mechanisms and whether or not thev are used therapeuticallv Drugs thus include typical small molecules of research or therapeutic interest, naturally-occurring factors such as endocrine paracπne or autocπne factors or factors interacting with cell receptors of all types, mtracellular factors, such as elements of intracellular signaling pathways, factors isolated from other natural sources, and so forth The biological effect of a drug may be a consequence of, inter alia drug-mediated changes in the rate of transcription or degradation of one or more species of RNA, the rate or extent of translation or post-translational processing of one or more polypeptides, the rate or extent of the degradation of one or more proteins, the inhibition or stimulation of the action or activity of one or more proteins, and so forth In fact, most drugs exert their affects bv interacting with a protein

In addition to drugs, this invention is equally applicable to those changes in or aspects of the physical environment that perturb a biological system in targeted manners Such environmental changes can include moderate changes of temperature (e g , a temperature elevation of 2-3 ^CC), moderate changes of pH, exposure to moderate doses of radiation, or changes in cell density Other environmental aspects include the nutritional environment, such as the presence of only particular sugars, amino acids, and so forth This invention is also applicable to disease states that perturb a biological system in targeted manners As used herein, disease state refers to the condition of a cell that results in a detrimental alteration in normal cellular function Such disease states include but are not limited to cancer, autoimmune dysfunction viral or bacterial infection, senescence, hereditary disorders, and metabolic disorders The biological effects of a drug (or a physical environmental change or a disease state) are measured in the instant invention by observations of changes in the translational state of a cell The translational state of a cell includes the identities and abundances of the constituent protein species in the cell under a given set of conditions It can be conveniently determined by, e g , measuring RNA or mRNA abundances in the polysomal RNA fraction by any of several existing RNA detection technologies Most preferably, a substantial fraction of all mRNAs for all constituent protein species in the cell are measured but preferably at least a sufficient fraction is measured to characterize the action of a perturbation of interest

4.4 EXPRESSION ANALYSIS USING MICROARRAYS Preferably measurement of the translational state of a cell is made bv hybridization to transcript arrays which are described in this subsection Other methods of translational state measurement are described in Section 4 5 Transcript Arrays Generally In a preferred embodiment the present invention makes use of "transcript arrays"

Transcript arrays can be employed for analyzing the translational state of a cell, and especially for measuring the translational states of a cell exposed to a perturbation, e g , a drug of interest, environmental change, or disease state

In one embodiment, transcript arrays are utilized by hybridizing detectably labeled polynucleotides representing the RNA or mRNA transcripts present in the polysomal mRNA fraction of a cell (e g , fluorescently labeled cDNA synthesized from polysomal mRNA or RNA) to a microarray of nucleic acid probes In another embodiment, total mRNA transcripts can be analyzed by, e g , isolating all polv(A) mRNA in a cell and hybridizing it to a microarray In yet another embodiment, cDNA synthesized only from the polysomal, non-polysomal, or total cytoplasmic RNA or mRNA of a cell can be hybridized to microarrays A microarray is a surface with an ordered array of binding (e g , hybridization) sites for products of many of the genes in the genome of a cell or organism, preferably most or almost all of the genes Microarrays can be made in a number of ways, of which several are described below However produced, microarrays share certain characteristics the arrays are reproducible, allowing multiple copies of a given array to be produced and easily compared with each other Preferably the microarrays are small, usually smaller than 5 cm², and they are made from materials that are stable under binding (e.g nucleic acid hybridization) conditions In one embodiment, the microarray is a high density array, preferably having a density greater than about 60, 100, 500, or 1000 different probes per 1 cm² A given binding site or unique set of binding sites in the microarray will preferably specifically bind the product of a single gene in the cell Although there may be more than one physical binding site (hereinafter "site") per specific mRNA, for the sake of clarity the discussion below will assume that there is a single site

It will be appreciated that when cDNA complementary to the RNA of a cell is made and hybridized to a microarray under suitable hybridization conditions, the level of hybridization to the site in the array corresponding to any particular gene will reflect the prevalence in the cell, or in the polysomal fraction of mRN¹ A transcπbed from that gene For example, when detectably labeled (e.g , with a fluorophore) cDNA complementary to the polysomal mRNA fraction is hybridized to a microarray, the site on the array corresponding to a gene (/ e . capable of specifically binding the product of the gene) that is not actively associated with πbosomes and therefore is not actively being translated in the cell will have little or no signal (e < fluorescent signal), and a gene for which the encoded mRNA is prevalent in the polysomal fraction will have a relatively strong signal

In preferred embodiments cDN As from two different cells are hybridized to the binding sites of the microarray In the case of drug responses one cell is exposed to a drug and another cell of the same type is not exposed to the drug The cD A derived from each of the two cell types are differently labeled so that they can be distinguished In one embodiment for example, cDNA from a cell treated with a drug (or exposed to a pathway perturbation) is synthesized using a fluorescein-labeled dNTP, and cDNA from a second cell, not drug-exposed, is synthesized using a rhodamine-labeled dNTP When the two cDNAs are mixed and hybridized to the microarray, the relative intensity of signal from each cDNA set is determined for each site on the array, and any relative difference in abundance of a particular mRNA detected

In the example described above, the cDNA from the drug-treated cell will fluoresce green when the fluorophore is stimulated and the cDNA from the untreated cell will fluoresce red As a result, when the drug treatment has no effect, either directly or indirectly, on the relative abundance of a particular mRNA in a cell, the mRNA will be equally prevalent in both cells and, upon reverse transcription, red-labeled and green- labeled cDN A will be equally prevalent When hybridized to the microarray, the binding sιte(s) for that species of RNA will emit wavelengths characteristic of both fluorophores (and appear brown in combination) In contrast, when the drug-exposed cell is treated with a drug that, directly or indirectly, increases the prevalence of the mRNA in the polysomal fraction of the cell, the ratio of green to red fluorescence ill increase When the drug decreases the polysomal mRNA prevalence the ratio w ill decrease The use of a two-color fluorescence labeling and detection scheme to define alterations in gene expression has been described, e g , in Shena et al 1995, Quantitative monitoring of gene expression patterns with a complementary DNA microarray, Science 270 467-470 An advantage of using cDNA labeled with two different fluorophores is that a direct and internally controlled comparison of the mRNA levels corresponding to each arrayed gene in two cell states can be made and variations due to minor differences in experimental conditions ( g , hybridization conditions) will not affect subsequent analyses However, it will be recognized that it is also possible to use cDNA from a single cell, and compare for example, the absolute amount of a particular polysomal mRNA in, e g , a drug-treated cell and an untreated cell Preparation of Microarrays Microarrays are known in the art and consist of a surface to which probes that correspond in sequence to gene products (e g , cDN As mRNAs, cRN As, and fragments thereof), can be specifically hybridized or bound at a known position In one embodiment, the microarray is an array (/ e a matrix) in which each position represents a discrete binding site for a product encoded bv a gene (e g , an RNA or cDNA) and in which binding sites are present for products of most or almost all of the genes in the organism's genome In a preferred embodiment, the "binding site" (hereinafter, "site") is a nucleic acid or nucleic acid analogue to which a particular cognate cDNA can specifically hybridize The nucleic acid or analogue of the binding site can be, e g , a synthetic o gomer, a full-length cDNA, a less-than full length cDNA, or a gene fragment Another example of a suitable nucleic acid analogue is peptide nucleic acid (see, e g , Egholm el al , 1993, PNA hybridizes to complementary ohgonucleotides obeying the Watson-Crick hydrogen-bonding rules, Nature 365 566-568, see also U S Patent No 5,539,083)

Although in a preferred embodiment the microarray contains binding sites for products of all or almost all genes in the target organism's genome, such comprehensiveness is not necessarily required Usually the microarray will have binding sites corresponding to at least 25% of the genes m the genome, often at least 50%, more often at least 75%, and most often at least 90% or 95% In another embodiment, the microarray has less than 5000 and/or more than 100 binding sites Preferably, the microarray has binding sites for genes relevant to the action of a drug or disease of interest A "gene" is identified as an open reading frame (ORF) of preferably at least 50, 75, or 99 amino acids from which a messenger RNA is transcribed in the organism (e g , if a single cell) or in some cell in a multicellular organism The numbei of genes in a genome can be estimated from the number of mRNAs expressed by the organism, or by extrapolation from a well- characterized portion of the genome When the genome of the organism of interest has been sequenced, the number of ORFs can be determined and mRNA coding regions identified by analvsis of the DNA sequence For example, the Saccharomyces cer evisiae genome has been completely sequenced and is reported to have approximately 6275 open reading frames (ORFs) longer than 99 amino acids Analvsis of these ORFs indicates that there are 5885 ORFs that are likely to specify protein products (Goffeau et al , 1996, Science 274 546- 567) In contrast, the human genome is estimated to contain approximately 10^' genes

- 15 - SUBSmUTE SHEET (RULE 26) Preparing Nucleic Acids for Microarrays

As noted above, the "binding site" to which a particular cognate cDNA specifically hybridizes is usually a nucleic acid or nucleic acid analogue attached at that binding site In one embodiment, the binding sites of the microarray are DNA polynucleotides corresponding to at least a portion of each gene in an organism's genome These DNAs can be obtained by, v-g . polymerase chain reaction (PCR) amplification of gene segments from genomic DNA, cDNA (e.g. , by RT-PCR), or cloned sequences PCR primers are chosen, based on the known sequence of the genes or cDNA, that result in amplification of unique fragments (/ e. fragments that do not share more than 10 bases of contiguous identical sequence with any other fragment on the microarray) Computer programs are useful in the design of primers with the required specificity and optimal amplification properties See, e.g. , O/igo version 5 0 (National Biosciences) Typically each gene fragment on the microarray will be between 50 bases and 2000 bases, more typically between 100 bases and 1000 bases, and usually between 300 bases and 800 bases in length PCR methods are well known and are described, for example, in Innis et al eds., 1990, PCR Protocols A Guide to Methods and Applications, Academic Press Inc San Diego, CA It will be apparent that computer controlled robotic systems are useful for isolating and amplifying nucleic acids

An alternative means for generating the nucleic acid for the microarray is by synthesis of synthetic polynucleotides or o gonucleotides, e.g. , using N-phosphonate or phosphoramidite chemistries (Froehler et al. , 1986, Nucleic Acid Res 14 5399-5407, McBride et al. , 1983, Tetrahedron Lett. 24 245-248) Synthetic sequences can be, e.g. , single stranded nucleic acids of greater than 50 bases in length or greater than 100 bases in length Synthetic sequences are typicallv be between 10 and 100 or between 10 and 50 bases in length In some embodiments, synthetic nucleic acids include non-natural bases, e.g. , inosine As noted above, nucleic acid analogues may be used as binding sites for hybridization An example of a suitable nucleic acid analogue is peptide nucleic acid (see, e.g. , Egholm et al , 1993, Nature 365 566-568, see also U S Patent No 5,539,083) In an alternative embodiment, the binding (hybridization) sites are made from plasmid or phage clones of genes, cD As (e.g , expressed sequence tags), or inserts therefrom (Nguyen et al. , 1995, denomics 29 207-209) In yet another embodiment, the polynucleotide of the binding sites is RNA

- 16 -

SUBSTΓΓUTE SHEET (RULE 26) Attaching Nucleic Acids to the Solid Surface

The nucleic acid or analogue are attached to a solid support w hich mav be made from glass plastic (e g polypropylene ny lon) polyacry lamide nitrocellulose or other materials A preferred method for attaching the nucleic acids to a surface is bv printing on 5 glass plates as is described generally by Schena et l I °95 Science 270 467-470 This method is especially useful for preparing microarrays ot cDN A Sec also DeRisi et al 1996, Nature Genetics 14 457-460, Shalon et al 1996 Genome Res 6 639-645 and Schena et al 1995 Proc Natl Acad Set USA 93 10539- 1 1286

A second preferred method for making microarravs is by making high-densitv 10 oligonucleotide arravs Techniques are known for producing arrays containing thousands of oligonucleotides complementary to defined sequences at defined locations on a surface using photolithographic techniques for synthesis in situ (see Fodor et al , 1991 Science 251 767-773, Pease et al 1994 Proc Natl Acad Sci USA 91 5022-5026, Lockhart et al 1996 Natm e Biotech 14 1675 U S Patent Nos 5 578 832 5 556 752 and 5 5 10 270) or 15 other methods for rapid synthesis and deposition of defined oligonucleotides (Blanchard et al , 1996, Biosensor s & Biυe/ectronic s 1 1 687-90) When these methods are used, oligonucleotides (e g , 20-mers) of known sequence are synthesized directly on a surface such as a deπvatized glass slide Usually, the array produced is redundant, with several oligonucleotide molecules per RNA Oligonucleotide probes can be chosen to detect alternatively spliced mRNAs Other methods of making microarrays are known in the art and can be used

Other methods for making microarrays e g , bv masking (Maskos and Southern 1992, N c Acids Res 20 1679- 1684) may also be used In principal any type of arrav for example dot blots on a nylon hybridization membrane ( see Sambrook et al , Molecular ^ Cloning - A Laboratory Manual (2nd Ed ), Vol 1 -3, Cold Spring Harbor Laboratory Cold Spring Harbor, New York, 1989) could be used, although as will be recognized by those of skill in the art, very small arravs will be preferred because hybridization volumes will be smaller

Generating Labeled RNA or DNA for Hybridization to Microarravs ^J0 Methods for isolating the RNA or mRNA fractions for analysis using microarravs are described in Section 4 1 and are w ell known to those skilled in the art Generation of labeled nucleic acids from these preparations for use in hvbπdization to labeled probes are described below

In one embodiment isolated RNA fractions can be directly labeled by, e g , using ^{J :)} DNA-independent RNA polvmerases For example polv(A) polvmerase catalyzes the incorporation of AMP residues onto the free 3'-hydroxyl terminus of RNA using ATP In order to radiolabel the RNA, [α^,2P] ATP can be utilized in this reaction (( ^"urrent Protocols in Molecular Biolog , I ^'o/ume 1 Ausubel et al eds , 1994, John Wilev & Sons, Inc , pp 3 9 1 -3 9 2) In another embodiment labeled cDN A is prepared from mRNA bv oligo dT-pπmed or random-primed reverse transcription, both of which are well know n in the art (see e.g , Klug and Berger, 1987 ^', Methods Enzymol 152 3 16-325) Reverse transcription may be carried out in the presence of a dNTP conjugated to a detectable label, most preferably a fluorescently labeled dNTP Alternatively, isolated mRNA can be converted to labeled antisense RNA synthesized by //; vitro transcription of double-stranded cDNA in the presence of labeled dNTPs (Lockhart et al , 1996, Nature Biotech 14 1 675) In alternative embodiments, the cDNA or RNA probe can be synthesized in the absence of detectable label and mav be labeled subsequently, c.g , by incorporating biotinylated dNTPs or rNTP, or some similar means (e g , photo-cross-linking a psoralen derivative of biotin to RNAs), followed by addition of labeled streptavidin (e.g , phycoerythrin-conjugated streptavidin) or the equivalent

When fluorescently-labeled probes are used, many suitable fluorophores are known, including fluorescein, lissamine, phycoerythπn, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3 5, Cy5, Cy5 5, Cy7, FluorX (Amersham) and others (see, e.g , Kπcka, 1992, Nonisotopic DNA Probe Techniques, Academic Press San Diego, CA) It will be appreciated that pairs of fluorophores are chosen that have distinct emission spectra so that they can be easily distinguished

In another embodiment, a label other than a fluorescent label is used For example, a radioactive label, or a pair of radioactive labels w ith distinct emission spectra, can be used (see Zhao et al. , 1995, Gene 156 207, Pietu et al , 1996, Genome Re 6 492) However, because of scattering of radioactive particles, and the consequent requirement for widely spaced binding sites, use of radioisotopes is a less-preferred embodiment

In one embodiment, labeled cDNA is synthesized by incubating a mixture containing 0 5 mM dGTP, dATP and dCTP plus 0 1 mM dTTP plus fluorescent deoxvπbonucleotides (e g , 0 1 mM Rhodamine 1 10 UTP (Perken Elmer Cetus) or 0 1 mM Cv3 dUTP ( Amersham)) w ith rev erse transcπptase ( e g , Superscript™ II, LT1 Inc ) at

Hybridization to Microarravs

Nucleic acid hybridization and wash conditions are chosen so that the probe "specifically binds" or "specifically hybridizes" to a specific arrav site, / e the probe hvbπdizes, duplexes or binds to a sequence arrav site with a complementary nucleic acid sequence but does not hybridize to a site with a non-complementarv nucleic acid sequence .As used herein, one polynucleotide sequence is considered complementary to another when, if the shorter of the polynucleotides is less than or equal to 25 bases, there are no mismatches using standard base-pairing rules or. if the shorter of the polynucleotides is longer than 25 bases, there is no more than a 5% mismatch Preferably, the polynucleotides are perfectly complementary (no mismatches) It can easily be demonstrated that specific hybridization conditions result in specific hybridization by carrying out a hybridization assay including negative controls (see. e g , Shalon et al , supra, and Chee et al , supra) Optimal hybridization conditions will depend on the length (e g , ohgomer versus polynucleotide greater than 200 bases) and type (e g , RNA, DNA, PNA) of labeled probe and immobilized polynucleotide or oligonucleotide General parameters for specific (/ e., stringent) hybridization conditions for nucleic acids are described in Sambrook et al , supra, and in Ausubel et al , 1987, Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York When the cDNA microarrays of Schena et al are used, typical hybridization conditions are hybridization in 5 X SSC plus 0 2% SDS at 65° C for 4 hours followed by washes at 25° C in low stringency wash buffer ( 1 X SSC plus 0 2% SDS) followed by 10 minutes at 25° C in high stringency wash buffer (0 1 X SSC plus 0 2% SDS) (Shena et al , 1996, Proc Nat! Acad Set USA, 93 10614) Useful hybridization conditions are also provided in, e g , T essen, 1993, Hybridization With Nucleic Acid Probes, Elsevier Science Publishers B V and Kπcka, 1992, Nonisotopic DNA Probe Techniques, Academic Press San Diego, CA Signal Detection and Data Analysis

When fluorescently labeled probes are used, the fluorescence emissions at each site of a transcript array can be, preferably, detected by scanning confocal laser microscopy In one embodiment, a separate scan, using the appropriate excitation line, is carried out for each of two fluorophores used (one for each cell exposed to a perturbation) Alternatively, a laser can be used that allows simultaneous specimen illumination at wavelengths specific to the two fluorophores and emissions from the two fluorophores can be analyzed simultaneously (see Shalon et al , 1996, Genome Research 6 639-645) In a preferred embodiment, the arrays are scanned with a laser fluorescent scanner with a computer controlled X-Y stage and a microscope obiective Sequential excitation of the two fluorophores is achieved with a multi-line, mixed gas laser and the emitted light is split by wavelength and detected with two photomultipher tubes Fluorescence laser scanning devices are described in Schena et al , 1996 Genome Res 6 639-645 and in other references cited herein Alternatively the fiber-optic bundle described bv Ferguson et al 1996 Nature Biotech 14 1681 - 1684 may be used to monitor mRNA abundance levels at a large number of sites simultaneously

Signals are recorded and, in a preferred embodiment, analyzed by computer For any particular hybridization site on the transcript array a ratio of the emission of the two fluorophores can be calculated The ratio is independent of the absolute expression level of the cognate gene, but is useful for genes whose expression is significantly modulated by drug administration, or any other tested event Measurement of Drug Response Data

To measure the translational state of a cell in response to exposure to drugs, the cells are exposed to the drug or drug candidate of interest Translational data for cells not exposed to the drug can be compared to translational data for cells exposed to the drug Furthermore, the cells may be exposed to different levels of the drug, and translational data for cells exposed to each of these levels may be compared When the cells are grown in vitro, the compound is usually added to their nutrient medium In some cases a drug will be solubilized in a suitable solvent The RNA of cells exposed to the drug and the RNA of cells not exposed to the drug are used to hybridize to transcript arrays, which are measured to find species of polysomal RNA, non-polysomal RNA, or total RNA having altered concentrations due to exposure to the drug Thereby the drug response is obtained

It is preferable for drug responses, in the case of two-color differential hybridization, to measure also with reversed labeling

4.5 OTHER METHODS OF EXPRESSION ANALYSIS

The translational state of a cell may be measured bv other gene expression technologies known in the art For example, analysis of specific RNA sequences in preparations can be done by

Northern and slot blot hybridization (Cur rent Protocols in Molecular Biology, Volume /, Ausubel et al , eds , 1994, John Wiley &. Sons, Inc , p 4 9 1 -4 9 16) In Northern blotting, RNA is first fractionated by size on a gel, and is then transferred onto nitrocellulose The membrane is subsequentlv studied using labeled RNA or DNA probes Slot or dot blot techniques are similar to Northern blotting techniques, except that RNA is not fractionated

- 20 -

SUBSTΠTΠΈ SHEET (RULE 26) by size, but is deposited directlv onto a membrane, and therefore, information about the molecular weight of the RNA of interest is lost

A second exemplary method for examining RNA transcripts is differential display. which attempts to fingerprint a mixture of expressed genes This fingerprint seeks to establish whether two samples are the same or different No attempt is made to determine the quantitative, or even qualitative, expression of particular, determined genes (Liang et al 1995, Current Opinions in Immunology 7 274-280, Liang et al , 1992. Science 257 967-71 , Welsh et al , Nucleic Acid Res , 1992, 20 4965-70, McClelland et al , 1993, Exs. 67 103- 15, Lisitsyn, 1993, Science, 259 946-50) Differential display uses the polymerase chain reaction ("PCR") to amplify cDNA subsequences of various lengths (derived from total RNA or mRNA by reverse transcription), which are defined by being between the hybridization sites of arbitrarily selected primers Ideally, the pattern of lengths observed is characteristic of the tissue from which the library was prepared Typically, one primer used in differential display is olιgo(dT) and the other is one or more arbitrary oligonucleotides designed to hybridize within a few hundred base pairs of the poly-dA tail of a cDNA in the library Thereby, on electrophoretic separation, the amplified fragments of lengths up to a few hundred base pairs should generate bands characteristic and distinctive of the sample Changes in tissue gene expression may be observed as changes in one or more bands Other methods of differential expression analysis known in the art may also be used (see, e.g., U.S. Patent No 5,871,697 dated February 16, 1999, Velculescu et al , 1995, Science 270 484-487)

5. EXAMPLE: ISOLATION OF TOTAL. POLYSOMAL. AND NON- POLYSOMAL RNA FRACTIONS FROM HUMAN CELLS Tissue Culture. HeLa cells are maintained m minimum Eagle's medium (MEM) supplemented with glutamine, penicillin, streptomycin, and 10% fetal calf serum at 37° C in 5% CO₂

Isolation of Total Cellular RNA. Cells are lysed in 4M guanidium thiocyanate, and the lysate is precipitated with 0 75 volume of ethanol and centrifuged at 10,000 rpm (Sorvall SA 600) for 10 min. The pellet is resuspended in 1-2 ml of 7 5M guanidinium hydrochloride and precipitated with 0 5 volume of ethanol The RNA pellet is phenol- extracted and ethanol precipitated

Isolation of Polysomal RNA Fractions Cells are lysed by incubation for 10 mm at 4°C in 0 15 M KCI/10 mM Tris-HCl, pH 7 2/0 5% Nonidet P-40/150 μg of cycloheximide per ml/20 mM dithiothreitol/10 mM MgCl₂/100 units per ml of ribonuclease inhibitor (RNasin) Mitochondria and nuclei are pelleted bv centriftigation for 10 min in a Eppendorf centrifuge and the supernatant diluted with KC1 to 0 25M, is lavered onto 32 ml of a 10%- 50% sucrose gradient buffered with 20 mM HEPES, pH 7 2/0 25 M Cl/10 mM MgCl₂/20 mM dithiothreitol containing 150 μg of cvcloheximide per ml 100 units of RNasin per ml, and 0 5 μg of hepaπn per ml in a Beckman model SW-27 ultracentπfuge tube (25 x 89 mm) and spun for 4 hr at 26,000 rpm Gradients are separated into fractions using an ISCO (Lincoln NB) fractionator with an absorbance monitor to trace the poly some profile (absorbance at 254 nm) of the gradient Polvsomal RNA sediments to the more dense regions of the sucrose gradient Isolation of Non-Poly somal RN Ti actions Fractions corresponding to the less dense regions of the sucrose gradient described above yield cytoplasmic RNA that is either associated ith monosomes or that is in mRNP particles

LXctract ion of RNA RNA in each fraction is extracted from associated proteins as follows RNA/protein complexes are suspended in 0 1 M Tπs-HCl (pH 9 0), 0 1 M NaCl, ImM EDTA at a concentration of 20 optical density units (260 nm absorbance) per ml and sodium dodecyl sulfate (SDS) is added to 1% An equal volume of phenol-chloroform- isoamyl alcohol (50 50 1) is added, the mixture is shaken vigorously for 10 min at room temperature chilled to 5°C, and the phases are separated by centπfugation at 12,000 x g for 10 min The aqueous phase is removed, extracted again as above, and potassium acetate (pH 5 5) is added to 2% Crude RNA is precipitated by the addition of two volumes of ethanol and is allowed to stand at -20°C overnight The RNA is then collected by centπfugation at 12,000 x g at -20°C for 20 min The RNA pellet is washed twice with ethanol/0 2M NaCl (2 1 ) and dissolved in H_:O or in 0 01 M Tπs-HCl (pH 7 5)/0 5 M KC1

6. REFERENCES CITED

All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application as specifically and individually indicated to be incorporated bv reference in its entirety for all purposes Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art The specific embodiments described herein are offered by way of example only and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled

Claims

WHAT IS CLAIMED IS

1 A method for predicting the amount of a protein of interest expressed bv a cell comprising measuring the amount in said cell of R A encoding said protein that is within a polysomal fraction of RNA of said cell

2 A method for predicting the amount of a protein of interest expressed by a cell comprising

(a) recovering a polysomal fraction of RNA from said cell, and (b) measuring the amount of RNA encoding said protein in said polysomal fraction

3 A method for determining the effect of a perturbation upon a cell or organism comprising (a) measuring abundances of each of a plurality of species of RNA within a polysomal RNA fraction in a first cell or organism exposed to a perturbation, and

(b) comparing the abundances of each of said plurality of species of RNA to the abundances of each of said plurality of species of RNA within a polysomal RNA fraction in a second cell or organism not exposed to said perturbation or exposed to a different amount of said perturbation, to detect any change in said abundances, wherein a change in abundance of a species of RNA within the polysomal fraction indicates a change in the amount of protein encoded by said species that is expressed bv said cell or organism when exposed to said perturbation

4 The method of claim 3, wherein the perturbation is exposure to a drug

5 The method of claim 4, wherein the drug is a growth factor or hormone

6 The method of claim 3, wherein the perturbation is an environmental change

7 The method of claim 6, wherein the environmental change is selected from the group consisting of a change in pH, food source temperature, radiation exposure, and cell density

8 The method of claim 3 w herein the perturbation is a disease state

9 The method of claim 3 wherein the perturbation is a change in developmental state or differentiation state

10 A method for comparing the effects of a first perturbation and a second different perturbation upon a cell or organism comprising

(a) measuring abundances of each of a plurality of species of RNA within a polysomal fraction in a first cell or organism exposed to a first perturbation, (b) comparing the abundances of each of said plurality of species of RNA to the abundances of each of said plurality of species of RNA within a polysomal fraction in a second cell or organism not exposed to said first perturbation, to detect any change in said abundances,

(c) measuring abundances of each of said plurality of species of RNA within a polysomal fraction in a third cell or organism exposed to a second perturbation different from said first perturbation,

(d) comparing the abundances of each of said plurality of species of RNA measured in step c to the abundances of each of said plurality of species of RNA within a polvsomal fraction in a second cell or organism not exposed to said second perturbation to detect any change in said abundances, and

(e) comparing the change in abundances detected in step b with the change in abundances detected in step d, wherein a difference in the amount of change in abundance of a species of polysomal RNA detected in step b relative to step d indicates differences in effects of the first perturbation and the second perturbation upon expression of the protein encoded bv said species

1 1 The method of claim 10 wherein the perturbation is exposure to a drug

12 The method of claim 1 1 wherein the drug is a growth factor or hormone

13 T he method of claim 10 wherein the perturbation is an environmental change

14 The method of claim 10 wherein the perturbation is a disease state

- 24 -

15 The method of claim 10, wherein the perturbation is a change in developmental state or differentiation state

16 A method for determining the translational state of a cell comprising measuring abundances of each of a plurality of RNA species within a polvsomal fraction of RNA within the cell

17 The method according to claim 3, 4, 8 , 10, or 15 wherein the abundances of each of said plurality of RNA species are measured by a method comprising contacting a microarray with a polysomal fraction of RNA from said cell or organism or with cDNA derived therefrom, wherein said microarray comprises a solid phase surface with attached nucleic acid probes said nucleic acid probes being capable of hybridizing with said plurality of RNA species or with cDNA derived therefrom

18 The method of claim 17, wherein the attached nucleic acid probes are capable of hybridizing to at least 25% of the genes in the genome of an organism

19 The method of claim 17, wherein the attached nucleic acid probes are capable of hybridizing to at least 50% of the genes in the genome of an organism

20 The method of claim 1 7, wherein the attached nucleic acid probes are capable of hybridizing to at least 75% of the genes in the genome of an organism

21 The method of claim 1 7, wherein the density of the nucleic acid probes of the microarray is greater than 60 probes per cm²

22 The method of claim 1 7, wherein the density of the nucleic acid probes of the microarray is greater than 500 probes per cm²

23 The method of claim 17, wherein each probe of the microarray comprises a polynucleotide sequence of between 50 and 2000 nucleotide bases

24 The method of claim 1 7, wherein each probe of the microarray comprises a single stranded oligonucleotide of 10 to 100 bases

- 25 - SUBSππJTE SHEET (RULE 26)

25 The method of claim 17 wherein each probe of the microarray comprises a single stranded polynucleotide of greater than 50 bases

26 A method of determining whether a perturbation upon a cell or an organism affects translation RNA abundance, or fractional translational efficiency comprising

(a) measuring abundances of each of a plurality of species of RNA within a polysomal RNA fraction in a first cell or organism exposed to a perturbation,

(b) comparing the abundances of each of said plura tv of species of RNA to the abundances of each of said plurality of species of RNA within a polysomal RNA fraction in a second cell or organism not exposed to said perturbation or exposed to a different amount of said perturbation, to detect any change in said abundances,

(c) measuring abundances of each of a plurality of species of RNA within a total cellular or cytoplasmic RNA or non-polysomal fraction in said first cell or organism exposed to said perturbation to obtain abundances of each of said plurality of species of RNA within the total cellular or cytoplasmic RNA,

(d) comparing the abundances of each of said plurality of species of RNA to the abundances of each of said plurality of species of RNA within total cellular or cytoplasmic RNA in said second cell or organism not exposed to said perturbation or exposed to a different amount of said perturbation to detect any change in said abundances, and

(e) comparing the change in abundances detected in step b with the change in abundances detected in step d for at least one species of RNA within said plurality, so as to determine whether said perturbation affects translation RNA abundance or fractional translational efficiency of said at least one species, wherein a change in abundance of a species of RNA within the polysomal fraction and not proportionately of said species of RNA within the total cellular or cytoplasmic RNA indicates a translational effect of the perturbation, a change in the abundance of said species of RNA within the total cellular or cytoplasmic RNA indicates an effect on total RNA abundance, and a change in the ratio of polysomal RNA of said species to non-polysomal RNA of said species indicates an effect on the fractional translational efficiency of the perturbation on said cell or organism when exposed to the perturbation

27 The method of claim 26, wherein step (e) comprises comparing the change in abundances detected in step (b) with the change in abundances detected in step (d) for species of RN corresponding to at least ot the genes in the genome of an organism

28 The method of claim 26 wherein the perturbation is selected from the roup consisting of drug exposure env ironmental change and disease state

29 The method according to claim 26, 27 or 28 wherein the abundances of said plurality of RNA species are measured by a method comprising contacting a microarray with a polvsomal fraction of RNA from said cell or organism or with cDNA derived therefrom and contacting said microarray with a total fraction of RNA from said cell or organism or with cDNA derived therefrom, wherein said microarray comprises a solid phase surface with attached nucleic acids, said nucleic acids being capable of hybridizing with said plurality of RNA species, or with cDNA derived therefrom

30 The method of claim 29, wherein the attached nucleic acid probes are capable of hybridizing to at least 25% of the genes in the genome of an organism

3 1 The method of claim 29, wherein the attached nucleic acid probes are capable of hybridizing to at least 50% of the genes in the genome of an organism

32 The method of claim 29, wherein the attached nucleic acid probes are capable of hybridizing to at least 75% of the genes in the genome of an organism

33 The method of claim 29, wherein the density of the nucleic acid probes of the microarray is greater than 60 probes per cm²

34 The method of claim 29 wherein the density of the nucleic acid probes of the microarray is greater than 500 probes per cm²

35 The method of claim 29, wherein each probe of the microarray comprises a polynucleotide sequence of between 50 and 2000 nucleotide bases

36 The method of claim 29 w herein each probe of the microarray comprises a single stranded oligonucleotide of 10 to 100 bases

- 27 - SUBSTITUTE SHEET (PULE 26)

37 The method of claim 29 w herein each probe of the microarray comprises a single stranded polynucleotide of greater than 50 bases

38 A microarray that is a solid phase comprising on its surface a plurality ot nucleic acid probes attached to said surface wherein the density of the probes of the microarray is greater than 60 probes per cm² each of which probes hybridizes to a gene transcript of one or more organisms or cDNA derived therefrom, wherein at least a portion of said probes are hybridized to a plurality of RNA species, or cDNA derived therefrom, from a polysomal fraction of RNA from said one or more organisms, and wherein substantially none of said probes is hybridized to RNA or cDNA derived therefrom derived from a non-polysomal fraction of RNA

39 The solid phase of claim 38, to which is simultaneously hybridized a plurality of a first RNA species or cDNA derived therefrom of a first organism exposed to a first perturbation and a plurality of a second RNA species or cDNA derived therefrom of a second organism exposed to a second perturbation, and wherein the first and second RNA species or cDNA derived therefrom are distinguishably labeled

40 The solid phase of claim 38, to which is simultaneously hybridized a plurality of a first RNA species or cDNA derived therefrom of a first organism exposed to a first perturbation and a plurality of a second RNA species or cDNA derived therefrom of a second organism not exposed to the perturbation, and wherein the first and second RNA species or cDNA derived therefrom are distinguishably labeled

41 The solid phase of claim 38, 39 or 40, wherein the perturbation is exposure to a drug

42 The solid phase of claim 41 , wherein the drug is a growth factor or hormone

43 The solid phase of claim 38, 39 or 40, wherein the perturbation is an environmental change

- 28 - SUBSTTTUTE SHEET (RULE 26)

44 The solid phase of claim 43. wherein the environmental change is selected from the group consisting of a change in pH, food source temperature, radiation exposure, and cell density

45 The solid phase of claim 38, 39 or 40 w herein the perturbation is a disease state

46 The solid phase of claim 38, 39 or 40, wherein the perturbation is a change in developmental state or differentiation state

47 The solid phase of claim 38, 39 or 40, wherein each probe of the microarray comprises a single stranded oligonucleotide of 10 to 100 bases

48. Use of a method according to any one of claims 1 , 2, 3, 10, 16 or 26 or a solid phase according to claim 38 for determining that a specific cellular constituent present in a cell type is a target of a drug.

49. Use of a method according to any one of claims 1 , 2, 3, 10, 16 or 26 or a solid phase according to claim 38 for screening for a molecule that directly or indirectly modulates a specific cellular constituent.

50. Use of a method according to any one of claims 1 , 2, 3, 10, 16 or 26 or a solid phase according to claim 38 for the screening of a drug molecule.