US 2004O229367A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0229367 A1 Berka et al. (43) Pub. Date: Nov. 18, 2004 (54) METHODS FOR MONITORING MULTIPLE Related U.S. Application Data GENE EXPRESSION (60) Division of application No. 09/533,559, filed on Mar. (75) Inventors: Randy M. Berka, Davis, CA (US); 22, 2000, which is a continuation-in-part of applica Michael W. Rey, Davis, CA (US); tion No. 09/273,623, filed on Mar. 22, 1999, now Jeffrey R. Shuster, Davis, CA (US); abandoned. Sakari Kauppinen, Smoerum (DK); Ib Groth Clausen, Hillerod (DK); Peter Publication Classification Bjarke Olsen, Copenhagen (DK) 51)1) Int. Cl.C.7 ............................. C12N 15/745/74; C12N 1/16 Correspondence Address: (52) U.S. Cl. ......................................... 435/.484; 435/254.3 NOVOZYMES BIOTECH, INC. (57) ABSTRACT 1445. DREWAVE The present invention relates to methods for monitoring DAVIS, CA 95616 (US) differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more Second filamentous fungal cells using (73) Assignees: Novozymes Biotech, Inc., Davis, CA; microarrays containing filamentous fungal expressed Novozymes A/S, Inc., Bagsvaerd (DK) Sequenced tags. The present invention also relates to fila mentous fungal expressed Sequenced tags and to computer (21) Appl. No.: 10/653,047 readable media and Substrates containing Such expressed Sequenced tags for monitoring expression of a plurality of (22) Filed: Aug. 29, 2003 genes in filamentous fungal cells. US 2004/0229367 A1 Nov. 18, 2004 METHODS FOR MONITORING MULTIPLE GENE organisms whose genomes have not been Sequenced. First, EXPRESSION one Spot on an array equals one gene or open reading frame, So redundancy is eliminated. Second, Since Sequence infor CROSS-REFERENCE TO RELATED mation is available So that redundancy and follow-up char APPLICATION acterization is minimized. Third, EST microarrays can be organized based on function of the gene products to facilitate 0001. This application is a divisional of pending U.S. analysis of the results (e.g., ESTs encoding enzymes from application Ser. No. 09/533,559 filed Mar. 22, 2000, which the Same metabolic pathway can be arranged or grouped is a continuation-in-part of U.S. application Ser. No. 09/273, accordingly). 623 filed Mar. 22, 1999, now abandoned, which applications are fully incorporated herein by reference. 0011 Ruan et al., 1998, The Plant Journal 15: 821-833, disclose the use of microarrays containing Arabidopsis thaliana. EST Sequences for determining the temporal BACKGROUND OF THE INVENTION expression of Arabidopsis thaliana genes in root, leaf, and 0002) 1. Field of the Invention two stages of floral development. 0003. The present invention relates to methods for moni 0012 Iyer et al., 1999, Science 283; 83-87, disclose the toring expression of a plurality of genes in filamentous use of microarrays containing human EST Sequences for fungal cells. The present invention also relates to expressed determining the temporal expression of human fibroblast Sequenced tags and to Substrates and computer readable cells in response to Serum. media containing Such expressed Sequenced tags for moni 0013 Hayward et al., 2000, Molecular Microbiology 35: toring expression of a plurality of genes in filamentous 6-14, disclose shotgun DNA microarrays and Stage-specific fungal cells. gene expression in Plasmodium falciparum malaria. 0004 2. Description of the Related Art 0014 Filamentous fungi are increasingly being used as 0005 Microarray technology is increasingly becoming host microorganisms for the industrial production of the method of choice for the quantitative and Simultaneous enzymes and other proteins whether endogenous or heter analysis of the expression levels of many thousands of ogenous to the microorganisms. There is a need in the art to genes. Microarray analyses typically follow the Steps of provide methods for monitoring the global expression of gene Selection, microarray Synthesis, Sample preparation, genes from filamentous fungal cells to improve the produc array hybridization, detection, and data analysis (Watson et tion potential of these microorganisms. al., 1998, Current Opinion in Biotechnology 9: 609-614). 0015. It is an object of the present invention to provide 0006 PCR-amplified coding sequences of genomic DNA alternative methods for monitoring expression of a plurality are particularly useful in microarrays for obtaining global of genes in filamentous fungal cells. expression profiles where the genome of the organism has been fully Sequenced. SUMMARY OF THE INVENTION 0007 Chu et al., 1998, Science 282: 699-705 disclose the 0016. The present invention relates to methods for moni use of microarrayS containing PCR-amplified genomic cod toring differential expression of a plurality of genes in a first ing Sequences for determining the temporal expression of filamentous fungal cell relative to expression of the same Saccharomyces cerevisiae genes during Sporulation. genes in one or more Second filamentous fungal cells, 0008 For other organisms whose genomes have not been comprising: Sequenced, global expression profiles may be obtained with 0017 (a) adding a mixture of fluorescence-labeled arraying (1) random genomic DNA segments or clones (e.g., nucleic acids isolated from the filamentous fungal from a genomic DNA library); (2) random cDNA clones cells to a Substrate containing an array of filamentous (e.g., from one or more cDNA libraries) that are uncharac fungal ESTs under conditions where the nucleic terized at the DNA sequence level; or (3) EST clones that acids hybridize to complementary Sequences of the have been Sequenced and partially characterized with ESTs in the array, wherein the nucleic acids from the respect to putative identification and function. first filamentous fungal cell and the one or more 0009. However, there are disadvantages with using ran Second filamentous fungal cells are labeled with a dom genomic or cDNA clones from organisms whose first fluorescent reporter and one or more different genomes have not been fully Sequenced. These disadvan Second fluorescent reporters, respectively; and tages include (1) more than one gene may be represented on a single clone; (2) no gene(s) may be encoded on a single 0018 (b) examining the array by fluorescence under clone; (3) extensive characterization and DNA sequencing is fluorescence excitation conditions wherein the rela required to follow-up array Spots that appear interesting, and tive expression of the genes in the filamentous fungal cells is determined by the observed fluorescence (4) duplicity, multiplicity, and reduncancy add to the follow emission color of each spot in the array in which (i) up work. the ESTs in the array that hybridize to the nucleic 0010 Expressed sequenced tags (ESTs) are partial cDNA acids obtained from either the first or the one or more Sequences of expressed genes. Simply Stated, an EST is a Second filamentous fungal cells produce a distinct Segment of a Sequence from a cDNA clone that corresponds first fluorescence emission color or one or more to the mRNA of a specific gene. The use of sequenced ESTs Second fluorescence emission colors, respectively, in microarrays compared to genomic clones or random and (ii) the ESTs in the array that hybridize to the cDNA clones provides Several advantages especially for nucleic acids obtained from both the first and one or US 2004/0229367 A1 Nov. 18, 2004 more Second filamentous fungal cells produce a assembled into a contig. In the methods of the present distinct combined fluorescence emission color. In a invention, the filamentous fungal ESTs described herein preferred embodiment, the filamentous fungal EST's preferably represent a plurality of genes present in the two are selected from the group consisting of SEQ ID or more filamentous fungal cells to be evaluated. NOs. 1-7860, nucleic acid fragments of SEQ ID 0026. ESTs are generally generated as follows: Total NOS. 1-7860, and nucleic acid Sequences having at polyadenylated mRNA is isolated from a filamentous fungal least 90%, preferably at least 95%, more preferably cell and reverse transcribed into total cDNA. The total at least 99%, and most preferably at least 99.9% cDNA is digested with a restriction endonuclease, size homology to the sequences of SEQID NOS. 1-7860. Selected by agarose gel electrophoresis, isolated, and ligated 0019. The present invention further relates to isolated into a vector, e.g., p7ErO-2.1. The ligation mixture is ESTs obtained from Fusarium venenatum (SEQ ID NOS. transformed into competent E. coli cells and transformants 1-3770), Aspergillus niger (SEQ ID NOS. 3771-4376), are Selected under Selective preSSure, e.g., kanamycin Selec Aspergillus Oryzae (SEQ ID NOS. 4377-7401), and Tricho tion. The cDNA libraries isolated from the selected trans derma reesei (SEQ ID NOS. 7402-7860). formants are amplified, isolated, and partially Sequenced. The partial Sequences are then compared to Sequences in 0020. The present invention also relates to computer various publicly available databases for identification. readable media and Substrates containing an array of Such filamentous fungal ESTs for monitoring differential expres 0027. Any method known in the art may be used for Sion of a plurality of genes in a first filamentous fungal cell generating ESTs (see, for example, Adams et al., 1991, relative to expression of the same genes in one or more Science 252: 1651-1656; Fields, 1996, Tibtech 14:286-289; Second filamentous fungal cells. Weinstock et al., 1994, Current Opinion in Biotechnology 5: 599-603; Matsubara and Okubo, 1993, Current Opinions in DETAILED DESCRIPTION OF THE Biotechnology 4: 672-677; Nelson et al., 1997, Fungal INVENTION Genet. Biol. 21: 348-363; Roe at al., http://www.genome.o- u.edu/fungal.html). 0021. The present invention relates to methods for moni toring differential expression of a plurality of genes in a first 0028. In the methods of the present invention, the fila filamentous fungal cell relative to expression of the same mentous fungal ESTs are preferably at least about 50 bp in genes in one or more Second filamentous fungal cells.