Expression Profile and Genome Location of cDNA Clones from an Infant Human Trabecular Meshwork Cell Library

Abstract

purpose. To delineate the profile of genes expressed in infant human trabecular meshwork and identify candidate genes for glaucoma.

methods. Human trabecular meshwork cell cultures were established from six young donors. A cDNA library was made from the combined trabecular meshwork mRNA. The end-sequence of random clones was determined by direct sequencing. These sequences were then analyzed by a National Center for Biotechnology Information (NCBI, Bethesda, MD) database search. Nucleotide searches were performed using the BLASTN (ver. 2.1.3; against the nonredundant nucleic acid sequence) and dbEST databases (both provided by NCBI in the public domain at www.ncbi.nlm.nih.gov).

results. Sequences from 1118 clones from this nonamplified trabecular meshwork cDNA library were categorized. Of these, 877 expressed sequence tags (ESTs) (78.7%) were known genes. One hundred thirty-nine ESTs (12.5%) showed close identity to EST sequences reported in the public domain database (dbEST). Thirteen ESTs (1.2%) showed no significant similarity to known genes or ESTs in the public databases and were thus defined as novel ESTs. The most abundant genes expressed by the human trabecular meshwork included ferritin H, eukaryotic translation elongation factor 1-α, ferritin L, fibronectin, and TIMP-1. Ferritin H was the most abundant transcript, making up more than 4% of the genes expressed by the human trabecular meshwork. Extracellular matrix proteins were also highly expressed. The chromosome location of the trabecular meshwork ESTs is reported.

conclusions. A profile of genes expressed by human trabecular meshwork is presented. Thirteen novel ESTs were identified. The combined information obtained from expression analysis and chromosomal localization of trabecular meshwork cDNAs should be valuable in identifying candidate genes for glaucoma.

Regulation of intraocular pressure is critical for maintaining the correct shape of the eye for vision. ¹ Abnormal elevation of intraocular pressure often results in damage to the optic nerve and ultimately to glaucoma. ² The human trabecular meshwork is a unique ocular tissue that controls intraocular pressure by regulating the outflow of aqueous humor from the anterior chamber into the venous system. ³ Perturbation of the trabecular meshwork and its ability to regulate intraocular pressure may result in glaucoma. The trabecular meshwork is a tiny tissue weighing only 100 to 150 μg and containing approximately 200,000 to 300,000 cells per eye. ⁴ Consequently, molecular analysis of the trabecular meshwork has been limited. Identification of genes active in the trabecular meshwork is central to further understanding of the unique functions of this tissue in normal and glaucomatous eyes. 

Primary open-angle glaucoma (POAG) is one of the leading causes of blindness in the industrialized world. ⁵ A disease of the elderly, POAG will have a major impact on the quality of life of a population that is rapidly aging. The pathophysiology of POAG is not well understood. Six POAG genes have been mapped ^{6 7 8 9 10 11 12} and two of these have been identified, MYOC (GLC1A) and OPTN (GLC1E). ^{13 14} Developing better therapeutic strategies for this major blinding disease depends on breakthroughs in understanding the pathophysiology of POAG. 

To identify the molecules expressed specifically in the human trabecular meshwork, we used a method called expression profiling of active genes. This method entails sequencing randomly selected clones from a 3′-directed cDNA library that faithfully represents the original composition of mRNA species. ¹⁵ The active genes are then identified by their sequences, and the relative abundance of each transcript can be estimated by the frequency of the corresponding EST in the cDNA library. The resultant gene expression profile provides a quantitative estimate of the profile of the relative transcript abundance in human trabecular meshwork. This approach is very useful in the identification of cell tissue-type–specific genes by comparing the expression profiles obtained with those from other cell and tissue types. ^{16 17}  

This is the first analysis of a profile of gene expression in a human trabecular meshwork cell line to be reported. In this study, human trabecular meshwork cell lines from infant donors (age range, 0–2 years) were combined to prepare a cDNA library. An expression profile of the human trabecular meshwork was obtained. Identification of these human trabecular meshwork genes will help in developing a framework for characterizing gene expression in the normal trabecular meshwork. Candidate POAG genes (GLC1) were identified. 

Materials and Methods

Construction of the cDNA Library

All eyes used for cell culture were from patients without a history of ocular disease or surgery, diabetes, liver failure, sepsis, immune disease, or neurologic disease. Eyes were obtained from the Oregon Lions Sight and Hearing Foundation (Portland, OR), and protocols were approved by the Institutional Human Subjects Committee and conformed to the guidelines set forth in the Declaration of Helsinki and by the National Institutes of Health. 

The human cDNA library was made from mRNA isolated from trabecular meshwork cells established from eyes of six individuals, aged 2 weeks to 2 years. These cell cultures were initiated and maintained in medium with 10% fetal calf serum, as described earlier. ¹⁸ Confluent cells were harvested at passages 3 through 6. The cells were trypsinized, neutralized with media, flash frozen in liquid N₂ and stored at −70°C. The frozen cells (∼96 × 10⁶) were shipped on dry ice to Invitrogen (San Diego, CA) where a unidirectional cDNA library was constructed by using a pcDNA3 vector and TOP10F′ host cells. The isolated mRNA was reverse transcribed with an oligo-dT primer. To make the library unidirectional, the inserts were cut with the restriction enzymes BstXI for the 5′ end and NotI for the 3′ end. The size of the inserts ranged from 0.5 to 1.6 kb, with the average size being 0.85 kb. 

Template Preparation and Sequencing

Plasmid purifications were performed with one of three kits (Mini-prep 24 deom McConnell Research, San Diego, CA; the QIAwell 8 ultra plasmid kit from Qiagen, Valencia, CA; or the Perfectprep Plasmid Mini Kit from Eppendorf, Westbury, NY). The purified plasmid preps were then sequenced on an automated sequencer (model 377; Applied Biosystems, Foster City, CA) at the Molecular Microbiology and Immunology (MMI) Research Core Facility, Oregon Health and Sciences University, or the Veterans Administration Medical Center Molecular Biology Core Laboratory (Portland, OR) using the T7 primer. Because the trabecular meshwork cDNA library is unidirectional, all the ESTs were sequenced from the 5′ end of the cDNA clones. 

Sequence Analysis

Nucleotide searches were first performed using BLASTN (ver. 2.1.3; unless otherwise noted databases used as resources in this study are provided in the public domain by the National Center for Biotechnology Information [NCBI], Bethesda, MD, and are available at the Internet addresses shown, in this case: http://www.ncbi.nlm.nih.gov/BLAST/) against the nonredundant nucleic acid sequence database. Matching sequences with an Expect (E)-value equaling zero were identified as known genes. Sequences with E-values close to zero, were manually analyzed for sequencing errors, alternative splicing, chimeric clones or contamination with vector sequences. 

DNA sequences with no matches in the nonredundant GenBank database (http://www.ncbi.nlm.nih.gov/GenBank/) were subjected to a second search with BLASTN against the dbEST (http://www.ncbi.nlm.nih.gov/dbEST/) database. The dbESTs identified were searched for in the Unique Human Gene Sequence Collection database (UniGene; http://www.ncbi.nlm.nih.gov/UniGene/) to determine whether they had been placed in a gene cluster. BLAST searches were performed between September 2000 and September 2001. All the clones that had been identified as ESTs or unknown genes were BLASTed between August and September 2001. GeneRIF (http://www.ncbi.nlm.nih.gov/LocusLink/) was used to identify the functional role of each gene. Some genes had more than one function, and these were placed in one functional classification to avoid confusion. 

The sequences of the 1118 cDNA clones were each mapped by BLASTing the sequences with BLASTN against the human genome database. Chromosome locations were also identified in UniGene, and more than 99% agreement was found between the two methods. Sequence data from this article has been deposited with the European Molecular Biology Laboratory (EMBL) and GenBank Data Libraries (the European Molecular Biology Laboratory library is available in the public domain at http://www.embl-heidelberg.de/). 

Semiquantitative RT-PCR Analysis of IGFBP-5, -6, and -7

Human trabecular meshwork cDNA was harvested from infant human trabecular meshwork cell cultures containing 10% fetal calf serum. Isolation of mRNA and reverse transcription of the mRNA was performed, as described previously. ¹⁹ PCR amplification was performed in a final volume of 25 μL containing 10 mM Tris-HCl (pH 8.3); 50 mM KCl; 1.5 mM MgCl₂; 0.001% gelatin; 10 pmol of each primer; 200 μM of dATP, dCTP, dGTP, dTTP; 5 ng of trabecular meshwork cDNA; and 1.0 U Taq polymerase (REDTaq; Sigma, St. Louis, MO) using a commercial PCR system (model 9700; Applied Biosystems) with initial denaturation of 94°C for 10 seconds, 70°C to 60°C for 20 seconds, and 72°C for 40 seconds; 23 cycles of 94°C for 10 seconds, 55°C for 20 seconds, and 72°C for 40 seconds; and a final incubation at 72°C for 5 minutes. The PCR primers used to amplify each of the specific IGFBP mRNAs are shown in Table 1 . 

Touchdown amplification was used to eliminate nonspecific annealing. Different template concentrations and cycle numbers were used to make certain that reactions were in the linear phase of amplification. Different template concentrations, including 0.05, 0.5, 5, and 25 ng were used, whereas other parameters were kept constant. Five nanograms of template was the optimal concentration, with reactions in the linear phase. To further refine the reaction, different numbers of cycles from 20 to 25 were tested after 11 cycles of touchdown amplification. Optimal results within the linear range were established, with 23 cycles used for all three IGFBP PCR products. 

Identification of Glaucoma Candidate Genes

The genomic locations of the trabecular meshwork cDNA clones were identified as described above. If a clone mapped to chromosomes 2, 3, 8, or 7 (GLC1B, GLC1C, GLC1D, or GLC1F, respectively), the contig containing that sequence was noted in our database. The initial Human Genome Database sequencing the entire human genome was completed after the GLC1 genes were mapped. ^{20 21} With this new sequence data, the flanking markers of each of the GLC1 loci were identified in their respective contigs and the contigs encompassing each GLC1 locus were aligned. The contigs containing sequences from the trabecular meshwork clones were then compared with the GLC1 contigs. If a contig containing a gene identified by a trabecular meshwork clone was also in a GLC1 contig, then the gene was considered to be a glaucoma candidate gene. 

A second method used to determine whether a trabecular meshwork EST was a glaucoma candidate gene was to identify the GLC1 cytogenetic location using the Online Mendelian Inheritance in Man (OMIM) Morbid Map (http://www.ncbi.nlm.nih.gov/Omim/searchmorbid/). LocusLink was then queried with the gene’s accession number to determine whether the gene was within the GLC1 region as specified by the OMIM Morbid Map. 

Results

cDNA Library Analysis

A cDNA library was constructed from human trabecular meshwork cells, as has been described. A listing of the genes in the library is available online (see “cDNA Library of Trabecular Meshwork Genes”). No amplification of the mRNA was required, and the library was not screened or subtracted. Therefore, the recurrence of specific genes should represent the actual gene expression level in the human trabecular meshwork. A total of 1118 ESTs were categorized into five classes to determine the fidelity of the library (Table 2) . Class I (883 ESTs, 79.6%) showed identity with the sequences of the nonredundant GenBank database (using BLASTN and E values of zero). These ESTs were labeled as known genes. Class II (137 ESTs, 12.3%) showed close identity to EST sequences reported in the public-domain database (dbEST). These ESTs were named known ESTs-unknown genes. Class III (13 ESTs, 1.2%) showed no significant similarity to known genes or known ESTs in the public databases and were thus defined as novel ESTs. Class IV (43 ESTs, 3.8%) contained transcripts of mitochondrial origin. Class V (42 ESTs, 3.8%) included vector, ribosomal RNA (three sequences), or repetitive elements. The low number of class IV and V elements indicates that genomic and mitochondrial contamination of the cDNA library was low. 

The 883 known genes expressed by the human trabecular meshwork cell lines were categorized into eight subgroups based on their functions, according to the format of Adams et al. ²² The 883 clones representing known genes are listed online at the Internet address provided in the prior paragraph with their RefSeq accession numbers, how many times they were observed (frequency), and their chromosome location. With the near completion of the Human Genome Project, more than 95% of the genes have been mapped. 

Unknown Human Trabecular Meshwork Genes

Of the 1118 clones sequenced, 137 (12.3%) produced sequences matching ESTs from unknown genes previously reported in the dbEST databank. A listing of these ESTs is also available online (see “ESTs from Unknown Genes in the Trabecular Meshwork Library”). UniGene clusters have been established for 122 of these ESTs. The number of ESTs in each of these clusters is shown in the online listing of ESTs from unknown genes. The ESTs are sorted by their abundance in UniGene. The ESTs not matching UniGene clusters are shown at the bottom of the table. Sixteen of these clones matched an EST in dbEST but have no UniGene cluster. The remaining 13 clones were unique sequences not found in BLASTN, dbEST, or UniGene, which implies that these genes are expressed only in trabecular meshwork or are very rare transcripts that have not been found in previous EST projects. All the unknown genes were relatively rare trabecular meshwork transcripts, with only one or two clones being found. The majority (91.5%) of the unknown human trabecular meshwork sequences have been mapped. Thus, ESTs that were GLC candidate genes were established. 

Highly Expressed ESTs

The ESTs more highly represented (by six or more clones) in the human trabecular meshwork library are summarized in a listing of highly expressed genes (Table 3) . The three most abundant transcripts are ferritin H, eukaryotic translation elongation factor 1-α, and ferritin L chain. Ferritin H comprises 4.28% of the clones in the human trabecular meshwork library. ESTs shared with the organ culture human trabecular meshwork cDNA library ²³ are indicated by asterisks. Of the 17 highly expressed genes, 7 were also present in the organ culture trabecular meshwork cDNA library. ²³  

Interleukin-1β, which may mediate the response of trabecular meshwork cells to laser trabeculoplasty as shown by our group, ²⁴ was expressed in the trabecular meshwork library (See Trabecular Meshwork Gene listing online). 

Insulin-like Growth Factor–Binding Proteins

Most of the known insulin-like growth factor binding proteins (IGFBPs) ²⁵ were expressed in the human trabecular meshwork cDNA library. Two of the IGFBPs were highly expressed: IGFBP7 and IGFBP2 (Table 3) . Lower amounts of IGFBP4, IGFBP5, IGFBP6, and IGFBP8 were present. To determine whether the number of clones reflects the abundance of mRNA in the human trabecular meshwork, RT-PCR of IGFBP7, IGFBP6, and IGFBP5 was performed. All three IGFBP messages were present (Fig. 1) . The quantity of IGFBP7 mRNA appeared to be at least two to four times higher than IGFBP6 and IGBP5 mRNAs, reflecting the fourfold increase in IGFBP7 found in the trabecular meshwork cDNA library. 

Candidate Glaucoma Genes Expressed in the Human Trabecular Meshwork Library

Six loci for POAG have been mapped. ^{7 8 9 10 11 12} The genes for GLC1A and GLC1E have been identified, MYOC and OPTN, respectively. ^{13 14} The genomic location of all 1118 ESTs in the human trabecular meshwork library was established to determine whether any of these were glaucoma candidate genes for the four remaining GLC1 loci. As shown in Table 4 , 10 of the ESTs mapped to the GLC loci. Two different methods were used to determine whether the ESTs from the human trabecular meshwork library were GLC1 candidate genes. Agreement between the two methods was found for the GLC1C, GLC1D, and GLC1F candidate genes (see Table 4 , OMIM Morbid Map versus MapView; http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/map_search). However, discrepancy between the two methods was observed for three of the GLC1B candidate genes. This most likely is due to the refined mapping of the flanking marker, D2S2161, which, according to the Human Genome Database, is within NT_015805, which maps to 2p12-p11.1. Originally, D2S2161 was mapped to 2qcen. ¹¹ GLC1B had the most candidate genes including endobrevin, DKFzp586M0122, a homologue of mouse DNA-directed RNA polymerase I, ribosomal protein L31, and thymosin-β. ¹⁰ The gene for GLC1A, myocilin, was not present in the trabecular meshwork library, however, previous reports have shown that myocilin is expressed at low levels in trabecular meshwork cell lines unless the cells are stressed or exposed to glucocorticoids. ^{26 27 28 29 30} OPTN, the GLC1E gene, was also not present in the screened library. ¹⁴ Ten candidate genes for glaucoma have been identified in this human trabecular meshwork library. In addition, PITX2, which causes Reiger syndrome type I (RIEG1) was identified in the library. ^{31 32 33} However, CYP1B1, which is associated with congenital glaucoma was not found. ³⁴  

Discussion

Composition of Human Trabecular Meshwork cDNA Library

The majority (78.9%) of ESTs identified in the human trabecular meshwork library are known genes. With each succeeding cDNA library added to the public database, a greater number of genes are being identified. Thus, the high number of known genes is to be expected. Conversely, fewer (1.2%) unique genes were identified in our human trabecular meshwork library, again reflecting the growing database. However, these unique genes are probably of specific importance to trabecular meshwork function. The low number of unique genes also illustrates that this library reflects the total transcript level within the trabecular meshwork, because it is not subtracted. The low number (3.8%) of mitochondrial ESTs as well as of repetitive sequences, vector or ribosomal RNA (3.8%) shows that this library primarily consists of cDNA transcripts. 

Characterization of ESTs Present in Normal Human Trabecular Meshwork Cell Lines

The trabecular meshwork morphology is still developing in the period after birth up to 4 years of age. ³⁵ Thus, the cell lines, which were established from individuals aged 2 weeks to 2 years may represent a transitional stage with some fundamental differences in gene expression compared with adult cells. Of note, the two major transcripts in our cDNA library were also the two major transcripts in a mouse blastocyst cDNA library. ³⁶  

Glaucoma Genes

The transcript for myocilin, the GLC1A gene, was not observed in the sequenced clones. This is not unexpected, because myocilin is expressed at low levels in trabecular meshwork cell lines, unless they have been stressed or induced with glucocorticoids. ^{26 27 28 29 30} Candidate genes for GLC1B, GLC1C, GLC1D, and GLC1F were identified. Hepatocellular carcinoma-associated antigen 112 has been analyzed in affected members of GLC1F; however, no disease-causing mutations have been found. We are in the process of analyzing the remaining GLC1C and GLC1F candidate genes in affected family members. PITX2, the RIEG1 gene, was expressed in our library. ^{31 32 33}  

Abundant Human Trabecular Meshwork Transcripts

The most abundantly expressed transcript in the human trabecular meshwork library was ferritin H, representing 4.28% of the library clones sequenced. The ferritin L gene was the third highest expressed in the trabecular meshwork library. Ferritin is the major iron storage protein of eukaryotes and appears to be ubiquitously expressed. ³⁷ The ferritin protein is a spherical shell composed of 24 subunits of varying proportions of H and L chains, depending on the tissue type. ³⁸ H-rich ferritin predominates in erythropoietic tissues, where iron availability is required both for heme synthesis and host defense. Ferritin H accumulates and releases iron faster than L-rich ferritin. ³⁸ For long-term iron storage in the liver and spleen, L-rich ferritin is the preferred form. The human trabecular meshwork cells were cultured in 10% fetal calf serum, which is rich in iron. The levels of ferritin H transcripts may result from exposure of the cells to a high concentration of iron and/or serum, because both are known to induce synthesis of ferritin H. ³⁹ Potential functions for ferritin in the anterior chamber may include providing iron to apoenzymes, boosting host defense and neutralization of oxidative stress by the trabecular meshwork. Myocilin (GLC1A) may be a heat shock or oxidative stress protein. The similar role of ferritin H in macrophages during oxidative stress suggests that, under certain environmental stresses, ferritin H and myocilin may both be important in protecting the trabecular meshwork from reactive radicals. Numerous heat shock proteins, including αB crystallin, HSPB1, HSPA5, HSPCA, and chaperonin were found in the trabecular meshwork library in relatively abundant numbers. 

Ferritin was not as highly expressed in the human trabecular meshwork library synthesized from organ culture message, ²³ although ferritin L was an abundant transcript. This difference between the two human trabecular meshwork libraries could either reflect cell culture versus organ culture, the age difference between the samples, or serum versus serum-free conditions. The ratio of H to L chains varies during development. ^{38 40} Our cDNA library is from cells from infants in whom the trabecular meshwork is still maturing, which may be why this ratio is observed. 

Another gene, known for its role in protecting motoneurons from iron toxicity, macrophage inhibitory cytokine-1, was expressed in the trabecular meshwork library. ⁴¹  

IGFBP7 and IGFBP2 were highly expressed in the trabecular meshwork cDNA library, with eight and six clones, respectively. Lower expression of IGFBP5, IGFBP6, IGFBP8, and IGFBP4 was observed. In an earlier article, we described the expression of IGFBP5 in trabecular meshwork. ⁴² In the organ culture human trabecular meshwork, IGFBP3 and IGFBP5 were expressed. ²³ Thus, all the known IGFBPs with the exception of IGFBP1 have been shown to be expressed in human trabecular meshwork. 

Gene Expression and Mechanisms of Trabecular Meshwork Outflow

The trabecular meshwork has been implicated as the site of obstruction of aqueous outflow, which in turn leads to increased intraocular pressure and potentially to glaucoma. ^{4 43 44} Several of the genes expressed in our trabecular meshwork library have been explored by us and others as possible modifiers of aqueous outflow through the trabecular meshwork. Interleukin-1β, which appears to mediate the response of trabecular meshwork cells to laser trabeculoplasty as shown by our group, ²⁴ was expressed in the trabecular meshwork library (see Trabecular Meshwork Gene listing online). Endogenous production of interleukin-1β by glaucomatous trabecular meshwork cells also appears to protect the cells from responding to oxidative stress by induction of apoptosis. ⁴⁵  

The cytoskeletal proteins may also affect aqueous outflow. Several pharmacologic agents that cause changes in cytoskeletal structure in trabecular meshwork cells are used clinically to increase outflow facility. ^{46 47} Several cytoskeleton proteins, including α-smooth muscle actin, γ1 actin, three isoforms of myosin, and vinculin were expressed in our trabecular meshwork library. Expression of α-smooth muscle actin has previously been demonstrated in trabecular meshwork organ culture and cells. ^{48 49 50}  

Fibronectin, also highly expressed in our library, has been known to be a major component of trabecular meshwork extracellular matrix. ^{51 52} Increased fibronectin deposition in the aqueous outflow pathway has been shown to occur in elderly and glaucomatous eyes, ⁵¹ although a recent study found no differences between normal and glaucomatous eyes. ⁵³ The potential for fibronectin to influence the flow of aqueous humor through the juxtacanalicular trabecular meshwork and the inner walls of Schlemm’s canal has intrigued many glaucoma researchers over the years. ^{51 52 53 54 55 56 57}  

Another abundant gene expressed in the trabecular meshwork was GAPDH, which is known for its role in glycolysis and oxidative phosphorylation. Considering that glycolysis is the major energy pathway for the trabecular meshwork, ⁵⁸ expression of the glycolytic enzymes would be expected. However, although high levels of GAPDH transcript were found, other genes for glycolytic enzymes were expressed at lower levels: glucose-6-phosphate dehydrogenase and enolase 1, each of which had two clones compared with seven clones for GAPDH. Once considered a housekeeping protein, many new functions for GAPDH have been ascribed, including membrane, cytoplasmic and nuclear functions in endocytosis, mRNA regulation, tRNA export, DNA replication, and DNA repair. ⁵⁹ Thus, the presence of GAPDH in our cDNA library may reflect additional roles for GAPDH in the trabecular meshwork in addition to glycolysis. 

Comparison of Genes Highly Represented in Cell Line and Organ Culture Human Trabecular Meshwork cDNA Libraries

Four genes were highly expressed in both the organ culture library reported by Gonzalez et al. ²³ and our cell culture human trabecular meshwork cDNA library. These include ferritin L, translation elongation factor 1-α, GAPDH, and TIMP1. Approximately 25% of the transcripts were shared between the two libraries. Glycolysis provides the major source of energy to the trabecular meshwork; thus, high levels of GAPDH would be expected in both human trabecular meshwork cDNA libraries. ⁵⁸ The high levels of translation elongation factor 1-α in both cDNA libraries suggests that translational control may be an important method for regulating gene expression in the trabecular meshwork. 

Primary cultured cells and organ cultures each have their unique advantages and disadvantages. One advantage of using primary cultured cells is that more cells can be obtained, thus allowing RNA work without need for amplification. In addition, the cells can be frozen for storage and thus are readily available for experimentation, whereas experiments with organ cultures are dependent on the supply of eyes. Because the trabecular meshwork cells have been isolated, changes occurring within these cells are solely a result of the trabecular meshwork’s physiology and are not influenced by adjacent tissues. However, some limitations of the cell culture system do exist. One of these is that mechanical forces acting on the outflow pathway tissue are not present in traditional cell culture. In addition, changes in gene expression may occur in cultured cells that do not reflect in vivo conditions. Organ cultures are probably a closer approximation of what is actually happening in the eye, because this model preserves the architecture of the outflow tissue. However, a limited amount of tissue necessitates amplification of the isolated message. Thus, the resultant library may not accurately reflect actual levels of specific messages, because some may be amplified better than others. Both primary cultured cells and organ cultures have unique advantages and disadvantages that must be taken into consideration in analysis of resultant data. Analysis of the two systems will give a much fuller picture of what is actually occurring in vivo, then either one alone could do. 

Additional research is needed to generate a comprehensive profile of genes expressed in the trabecular meshwork during development and aging. The study reported herein and another recent publication ²³ provide a comprehensive reference point and an important resource of cloned “trabecular meshwork-expressed genes” that can be used for biological and functional investigations. Expression and chromosomal localization of trabecular meshwork ESTs should facilitate identification of mutations that result in or contribute to glaucoma. 

 Supported by National Eye Institute Grants R01 EY10555, R01 EY11650, R01 EY03279, R01 EY08247, and R01 EY10572, Research to Prevent Blindness, and Alcon Laboratories.

 Submitted for publication March 5, 2002; revised July 12, 2002; accepted July 23, 2002.

 Commercial relationships policy: F.

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

IGFBP Primers Used to RT-PCR the IGFBP cDNA

Primer Name	Primer Sequence	Expected PCR Product Size (bp)
IGFBP5U	5′TGA AGA AGG ACC GCA GAA 3′	285
IGFBP5L	5′GTC CAC GCA CCA GCA GAT 3′
IGFBP6U2	5′ACA GGA TGT GAA CCG CAG AG 3′	314
IGFBP6L2	5′AGG AGG AGC TTC CAT TGC CA 3′
IGFBP7U2	5′AGC AAG GTC CTT CCA TAG TG 3′	231
IGFBP7L2	5′GCA CCC AGC CAG TTA CTT CA 3′

Table 2.

 

Summary of ESTs Found in the HTM Library

	cDNA Category	Clones (n)	Clones (%)
I	Known genes	883	78.9
II	Known ESTs-unknown genes	137	12.3
III	No match in databases	13	1.2
IV	Mitochondrial transcripts	43	3.8
V	Uninformative sequences	42	3.8
Total			1118	100

Table 3.

 

Highly Expressed Genes in TM cDNA Library

Gene	Accession No.	Frequency	Total (%)
Ferritin H*	NM_002032	44	4.28
Eukaryotic translation elongation factor 1, α, †	NM_001402	15	1.46
Ferritin L, †	NM_000146	13	1.26
Fibronectin	M10905	11	1.07
Ribosomal protein L13a	NM_012423	11	1.07
TIMP-1, †	NM_003254	9	0.87
Prothymosin β4*	NM_021109	9	0.87
Ribosomal protein L18a	NM_000980	9	0.87
Ribosomal protein L17	NM_000985	8	0.78
Ribosomal protein S2*	NM_002952	8	0.78
Ribosomal protein S18	NM_022551	8	0.78
Insulin-like growth factor–binding protein 7	NM_001553	8	0.78
Tumor protein, translationally controlled 1	NM_003295	7	0.68
Glyceraldehyde-3-phosphate dehydrogenase, †	NM_002046	7	0.68
Insulin-like growth factor–binding protein 2	NM_000597	6	0.58
Amyloid β (A4) precursor-like protein 2	NM_001642	6	0.58
Ribosomal protein S23	NM_001025	6	0.58

*  Expressed or

†  highly expressed ²³ in HTM organ culture explants.

Figure 1.

 

RT-PCR of IGFBP5, IGFBP6, and IGFBP7 from cDNA isolated from confluent human infant trabecular meshwork cells grown in 10% fetal calf serum.

Table 4.

 

Candidate GLC1 Genes Expressed in the Trabecular Meshwork cDNA Library

Loci	OMIM Morbid Map*	Accession No.	Gene Name	LocusLink, †	Contig	MapView, ‡
GLC1B	2qcen-q13	NM_003761	Endobrevin	2p12-p11.2	NT_015805	2p12-p11.1
			DKFzp586M0122, homologous to mouse RNA
		NM_015425	Polymerase I, DNA-directed	2p24.3-p24.1	NT_015805	2p12-p11.1
		NM_000993	Ribosomal protein L31	2q11.2	NT_022171	2p11.1-q12.1
		NM_021103	Thymosin-β 10	2p11.2	NT_005120	2pcen
GLC1C	3q21-q24	M90657	Transmembrane 4 superfamily, member 1	3q21-q25	NT_033349	3q21-q25
		NM_006286	Transcription factor Dp-2	3q23	NT_0022502	3q23
GLC1D	8q23	NM_020189	DC6	8q23.2	NT_008048	8q23.2
GLC1F	7q35-q36	AI344746	Est	—	NT_007914	7q33-q36.1
		NM_018487	Hepatocellular carcinoma–associated antigen 112	7q35	NT_007914	7q33-q36.1
		NM_005614	Ras-related GTP-binding protein	7q36	NT_007914	7q33-q36.1

*  OMIM Morbid Map gives the cytogenetic location of the disease loci.

†  LocusLink displays the cytogenetic location of the genes. AI344746 has no reported map location in LocusLink.

‡  MapView was used to correlate the contig’s physical position with the cytogenetic map.

Supplementary Materials

cDNA Library of Trabecular Meshwork Genes [HTML] [PDF] 

ESTs from Unknown Genes in the Trabecular Meshwork Library [HTML] [PDF] 

 

Table 2. TM cDNA library expressed genes.

Gene Name	Accession #	F Frequency		Chromosome
Cell Division
cyclin-dependent kinase 4	NM_000075	3			12
enhancer of rudimentary, drosophila	NM_004450	2			14
thymosin, α	NM_002823	2			2
CDC28 protein kinase 2	NM_001827	1			9
cyclin dependent kinase inhibitor 2A	NM_000077	1			9
H1 histone family, member 2	NM_005319	1			6
histone H3, family 3A	NM_002107	1			1
chromobox homolog 6	NM_014292	1			22
nucleolar autoantigen	NM_006455	1			17
stromal antigen 3	NM_012447	1			7
growth arrest-specific 1	NM_002048	1			9
growth arrest-specific 6	NM_000820	1			?
transmembrane 4 superfamily protein	NM_005981	1			12
RAD23 (S. cerevisiae) homolog B	NM_002874	1			9
APEX nuclease	NM_001641	2			14
RAN, member RAS oncogene family	NM_006325	1			6
DNA helicase homolog	AF108138	1			15
Apoptosis
BCL2/adenovirus E1B 19kd-interacting protein 3	NM_004052	1			14
programmed cell death 5	NM_004708	1			19
acid-inducible phosphoprotein	NM_006107	1			17
NF-κB inhibitor, α	NM_020529	1			14
Cell signal/cell communication
insulin-like growth factor binding protein 7	NM_001553	8			4
insulin-like growth factor binding protein 2	NM_000597	6			2
guanine nucleotide binding protein, β polypeptide 2-1like	NM_006098	4			5
insulin-like growth factor binding protein 5	NM_000599	2			2
insulin-like growth factor binding protein 6	NM_002178	2			12
insulin-like growth factor binding protein 8	NM_001901	2			6
interleukin 8	NM_000584	2			4
prostate differentiation factor	NM_004864	2			19
calmodulin 2	NM_001743	1			2
catenin, δ1	NM_001331	1			11
integrin β1	NM_002211	1			10
insulin-like growth factor-binding protein 4	M62403	1			17
interleukin 1, β	NM_000576	1			2
midkine	NM_002391	1			11
small inducible cytokine A2	NM_002982	1			17
stanniocalcin 1	NM_003155	1			8
stanniocalcin 2	NM_003714	1			5
granulin	NM_002087	1			17
G protein-coupled receptor 1	NM_005279	1			2
progesterone membrane binding protein	NM_006320	1			4
melanocortin 1 receptor	NM_002386	1			16
laminin receptor 1	NM_002295	2			?
neuropilin 2	NM_003872	2			2
protein C receptor, endothelial	NM_006404	1			20
epidermal growth factor receptor pathway substrate 8	NM_004447	1			12
benzodiazapine receptor	NM_000714	1			22
COP9 subunit 6	NM_006833	1			7
prion protein	NM_000311	1			20
amyloid beta (A4) precursor protein binding, family B member 1	NM_001164	1			11
histidine triad nucleotide-binding protein; protein kinase C inhibitor	NM_005340	1			5
insulin receptor substrate 2	NM_003749	2			13
CD151	NM_004357	1			?
protein tyrosine phosphatase receptor π	NM_002847	1			7
Eph-like receptor tyrosine kinase	NM_004441	1			3
c-mer proto-oncogene tyrosine kinase	NM_006343	1			2
protein kinase inhibitor, γ	NM_007066	1			20
protein kinase c, ζ	NM_002744	1			1
ras-related GTP-binding protein	NM_005614	1			7
cerebral cell adhesion molecule	NM_016174	1			9
galectin-1	NM_002305	1			22
galectin-3	NM_002306	1			14
angio-associated, migratory cell protein	NM_001087	1			2
casein kinase 1, α1	NM_001892	1			13
Cell structure/motility
thymosin β 4	NM_021109	9			X
actin, γ1	NM_001614	5			17
myosin, light polypeptide 6, alkali,	NM_021019	4			12
profilin 1	NM_005022	3			17
transgelin	NM_003186	3			11
actin, α2, smooth muscle, aorta	NM_001613	1			10
actin, β	NM_001101	1			7
gelsolin	NM_000177	1			9
actinin, α1	NM_001102	1			14
cofilin 1	NM_005507	2			11
desmoplakin	NM_004415	1			?
GABA-A receptor-associated protein like 2	NM_007285	1			16
keratin 18	NM_000224	1			12
myristoylated alanine-rich protein kinase C substrate	NM_002356	2			6
protein phosphatase 2, regulatory subunit B (B56) α isoform	NM_006243	1			1
myosin, light polypeptide, regulatory,	NM_006471	2			18
myosin regulatory light chain	D50372	1			18
tropomyosin 2 (β)	NM_003289	1			9
tubulin, β2	NM_006088	1			?
tubulin, β5	NM_032525	1			?
vimentin	NM_003380	1			10
vinculin	NM_003373	1			10
calponin 2	NM_004368	1			?
lamin A/C	NM_005572	1			1
GABA (A) receptor-associated protein	NM_007278	1			17
thymosin, β10	NM_021103	2			2
brain acid-soluble protein 1	NM_006317	1			5
dynein, cytoplasmic, light intermediate polypeptide 2	NM_006141	1			16
claudin 1	NM_021101	2			3
ninjurin	NM_004148	1			9
regulator of G-protein signaling	NM_002925	1			1
Extracellular matrix maintenance
fibronectin	M10905	11	2
TIMP-1	NM_003254	9	X
matrix Gla protein	NM_000900	4	12
collagen,type I, α1	NM_000088	5	17
collagen, type III, αI	NM_000090	4	2
osteonectin	NM_003118	4	5
collagen, type V, α1	NM_000093	1	9
collagen, type V1, α1	NM_001848	1	21
collagen, type VI, α2	NM_001849	2	21
collagen, type VI, α3	NM_004369	1	2
collagen, XVI, α1	NM_001856	1	1
decorin	NM_001920	1	12
EGF-containing fibulin-like extracellular matrix protein 2	NM_016938	1	11
basigin	NM_001728	1	19
procollagen-proline, 2 oxoglutarate 4-dioxygenase β polypeptide	NM_000918	1	17
Gene/Protein Expression
eukaryotic translation elongation factor 1, α1	NM_001402	15	6
ribosomal protein, large PO	NM_001002	6	12
ribosomal protein large, P1	NM_001003	2	15
ribosomal protein, large, P2	NM_001004	2	?
ribosomal protein L3	NM_000967	1	22
ribosomal protein L4	NM_000968	1	?
ribosomal protein L5	NM_000969	1	1
ribosomal protein L6	BC004138	3	12
ribosomal protein L7a	NM_000972	4	9
ribosomal protein L8	NM_000973	3	8
ribosomal protein L9	NM_000661	2	15
ribosomal protein L10; Wilms' tumor-related protein	NM_006013	3	X
ribosomal protein L10a	NM_007104	2	6
ribosomal protein L11	NM_000975	2	1
ribosomal protein L12	NM_000976	5	9
ribosomal protein L13	NM_000977	1	16
ribosomal protein L13a	NM_012423	11	19
ribosomal protein L15	NM_002948	4	3
ribosomal protein L17	NM_000985	8	18
ribosomal protein L18a	NM_000980	9	19
ribosomal protein L19	NM_000981	1	?
ribosomal protein L21	U14967	3	?
ribosomal protein L22	NM_000983	2	3
ribosomal protein L23a	NM_000984	2	17
ribosomal protein L23	NM_000978	5	17
ribosomal protein L24	NM_000986	5	3
ribosomal protein L26	NM_000987	4	17
ribosomal protein L27a	NM_000990	1	11
ribosomal protein L27	NM_000988	3	17
ribosomal protein L28	NM_000991	4	19
ribosomal protein L29	NM_000992	4	3
60S ribosomal protein L30 isolog	NM_016304	3	15
ribosomal protein L31	NM_000993	5	2
ribosomal protein L32	NM_000994	3	3
ribosomal protein L34	NM_000995	2	4
ribosomal protein L35	NM_007209	2	9
ribosomal protein L35a	NM_000996	2	?
ribosomal protein L36	NM_015414	1	19
ribosomal protein L37	NM_000997	4	5
ribosomal protein L37a	NM_000998	3	2
ribosomal protein L39	NM_001000	1	X
ribosomal protein S2	NM_002952	8	16
ribosomal protein S3	NM_001005	5	11
ribosomal protein S3A	NM_001006	1	4
ribosomal protein S4, Y-linked	NM_001008	1	Y
ribosomal protein S4, X-linked	NM_001007	3	X
ribosomal protein S6	NM_001010	4	9
ribosomal protein S7	NM_001011	4	?
ribosomal protein S8	NM_001012	3	1
ribosomal protein S9	NM_001013	5	19
ribosomal protein S10	NM_001014	2	6
ribosomal protein S11	NM_001015	2	19
ribosomal protein S13	NM_001017	1	11
ribosomal protein S14	NM_005617	5	5
ribosomal protein S15	NM_001018	4	19
ribosomal protein S15a	NM_001019	4	16
ribosomal protein S16	NM_001020	1	19
ribosomal protein S17	NM_001021	1	15
ribosomal protein S18	NM_022551	8	6
ribosomal protein S19	NM_001022	1	19
ribosomal protein S20	NM_001023	2	8
ribosomal protein S21	NM_001024	1	20
ribosomal protein S23	NM_001025	6	5
ribosomal protein S24	NM_001026	4	10
ribosomal protein S25	NM_001028	1	11
40S ribosomal protein S27 isoform	NM_015920	1	15
ribosomal protein S27a	NM_002954	4	2
ribosomal protein S28	NM_001031	2	19
RNA binding motif protein 9	NM_014309	1	22
eukaryotic translation initiation factor 3, subunit 8	NM_003752	1	16
eukaryotic translation initiation factor 3, subunit 5	NM_003754	2	2
eukaryotic translation initiation factor 4A, isoform 1	NM_001416	2	17
mitochondrial translational initiation factor 2	NM_002453	1	2
nascent-polypeptide-associated complex α polypeptide	NM_005594	3	12
ATPase, H+ transporting, lysosome pump	NM_001694	2	16
RNA polymerse I, DNA-directed	NM_015425	1	2
Polymerase (RNA) II (DNA directed) polypeptide E	NM_002695	1	19
Polymerase (RNA) III (DNA directed) polypeptide K	NM_016310	1	16
glucocorticoid receptor DNA binding factor	NM_004491	2	19
cold shock domain protein A	NM_003651	1	12
basic transcription factor 3	NM_001207	1	5
DR1-associated protein 1	NM_006442	1	11
CCAAT/enhancer binding protein, δ	NM_005195	1	8
general transcription factor IIF, polypeptide 2	NM_004128	1	13
interleukin enhancer binding factor 3	NM_012218	1	19
interferon regulatory factor 3	NM_001571	1	19
Krupple-like zinc finger protein GLIS2	NM_032575	1	16
transcription elongation factor B (SIII)	NM_005648	1	8
transcription factor Dp-2	NM_006286	1	3
COP9	NM_006837	1	8
leucine rich repeat interacting protein 1	NM_004735	1	2
paired-like homeodomain transcription factor 2 (PITX2)	NM_000325	2	4
cofactor required for Sp1 transcriptional activation, subunit 2	NM_004229	1	X
cofactor required for Sp1 transcriptional activation, subunit 8	NM_004269	1	5
nuclear factor (erythroid-derived 2)-like 3	NM_004289	1	7
AE-binding protein 1	NM_001129	1	7
SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily c, member 2	NM_003075	1	12
nuclease sensitive element binding protein 1	NM_004559	1	1
regulatory factor X, 5	NM_000449	1	1
zinc finger protein 173	NM_003449	2	6
methyl-CpG binding domain protein 2	NM_003927	1	18
heterogeneous nuclear ribonucleoprotein R	NM_005826	1	1
small nuclear ribonucleoprotein polypeptide	NM_004596	1	19
splicing factor, arginine/serine-rich 3	NM_003017	1	11
poly(A)-binding protein, cytoplasmic 1	NM_002568	1	8
NS1-associated protein 1	NM_006372	1	6
Ewing Sarcoma mRNA, RNA-binding protein	NM_005243	1	22
non-POU-domain-containing octamer-binding	NM_007363	1	X
RNA binding motif protein 3	NM_006743	1	X
nucleophosmin (nucleolar phosphoprotein B23, numatrin)	NM_002520	2	5
M phase phosphoprotein M10	X98494	1	2
eukaryotic translation elongation factor 1 β2	NM_001959	4	2
eukaryotic translation elongation factor 1 δ	NM_001960	1	8
eukaryotic translation elongation factor 1 γ	NM_001404	5	7
seryl-tRNA synthetase	NM_006513	1	1
putative translation initiation factor (SUI1)	NM_005801	2	?
crystallin, αB	NM_001885	3	11
tubulin-specific chaperone a	NM_004607	1	X
peptidylprolyl isomerase B (cyclophilin B)	NM_000942	2	15
protein disulfide isomerase-related protein	NM_005742	2	2
peptidylprolyl isomerase E (cyclophilin E)	NM_006112	1	1
peptidylprolyl isomerase A (cyclophilin A)	NM_021130	3	?
protein (peptidyl-prolyl cis/trans isomerase) NIMA-interacting 1	NM_006221	1	19
chaperonin containing tcp1, subunit 5 (ε)	BC002971	1	5
C12orf8: chromosome 12 open reading frame 8	NM_006817	1	12
FK506-binding protein 1A	NM_000801	2	20
FK506-binding protein 2	NM_004470	1	11
calnexin	NM_001746	2	5
heat shock 27kD protein 1	NM_001540	1	7
heat shock 60kd protein 1	NM_002156	2	2
heat shock 70kD protein 5	NM_005347	3	9
heat shock 90kD protein 1, α	NM_005348	2	11
serine (or cysteine) proteinase inhibitor, clade H, member 2	NM_001235	2	11
chaperonin containing TCP1,subunit 4	NM_006430	1	2
glucose regulated protein	NM_005313	1	15
ubiquitin fusion degradation-1 like	NM_005659	1	22
ubiquitin-conjugating enzyme E2A (RAD6 homolog)	NM_003336	1	X
E3 ubiquitin ligase SMRF1	AF199364	1	7
ubiquitin protein ligase E3A	NM_000462	1	15
ubiquitin carboxyl-terminal esterase L1	NM_004181	4	4
ribophorin I	NM_002950	1	3
ribophorin II	NM_002951	1	20
v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog	NM_000222	1	4
SMT3	NM_006937	1	17
membrane bound transcription factor protease, site 1	NM_003791	1	16
dolichyl-dipphosphooligosaccharide-protein glycosyltransferase	NM_005216	1	1
protein kinase C-like	NM_002741	1	19
tyrosine kinase 2	NM_003331	1	19
upstream regulatory element binding protein 1	NM_031407	1	X
protein-L-isoaspartate (D-aspartate) O-methyltransferase	NM_005389	1	6
lysyl oxidase-like 2,	NM_002318	1	8
dolichyl phosphate glucosyltransferase	NM_013338	1	13
serum inducible kinase, serine threonine kinase	NM_006622	1	5
ubiquitin B	NM_018955	3	17
ubiquitin-conjugating enzyme E2L 6	NM_004223	1	11
peptidase D	NM_000285	1	19
ubiquitin A-52 residue ribosomal protein fusion product 1*	NM_003333	2	19
proteasome (prosome, macropain) subunit, α type, 7	NM_002792	1	20
proteasome (prosome, macropain) subunit, β type, 2	NM_002794	1	1
proteasome (prosome, macropain) subunit, α type, 6	BC002979	1	Y
proteasome (prosome, macropain) subunit β type, 7	NM_002799	2	9
cathepsin B	NM_001908	1	8
cathepsin D	NM_001909	1	11
LOC51035	NM_015853	1	11
sequestosome 1	NM_003900	3	5
caveolin 1	NM_001753	1	7
clathrin-associated protein	NM_004069	1	19
clathrin, light polypeptide	NM_001833	1	9
KDEL endoplasmic reticulum retention receptor 1	NM_006801	1	19
KDEL endoplasmic reticulum protein retention receptor 2	NM_006854	1	7
endobrevin	NM_003761	1	2
coatomor protein complex, subunit β 2	NM_004766	1	?
mannose-6-phosphate receptor	NM_002355	1	12
intersectin	NM_003024	1	21
ADP-ribosylation factor-like 1	NM_001177	1	12
ADP-ribosylation factor 1	NM_001658	1	1
ADP-ribosylation factor GTPase activating protein 1	NM_014570	1	22
RAB1, member RAS oncogene family	NM_004161	2	2
syntaxin 7	NM_003569	1	6
signal recognition particle	NM_003135	1	5
signal sequence receptor, β (translocon-associated protein β)	NM_003145	2	1
signal sequence receptor, delta (translocon-associated protein δ)	NM_006280	2	X
eIF4E-transporter	NM_019843	1	22
nucleoporin-like protein 1	NM_007342	2	7
major vault protein	NM_017458	1	16
proteolipid protein 2	NM_002668	1	X
ArsA (bacterial) arsenite translocating ATP-binding, homolog 1	NM_004317	1	19
ATP-binding cassette, sub-family B (MDR/TAP), member 7	NM_004299	1	X
chloride intracellular channel 1	NM_001288	1	6
solute carrier family 7, member 11	NM_014331	1	4
solute carrier family 3, member 2	NM_002394	1	11
solute carrier family 16, member 3	NM_004207	1	17
solute carrier family 25, member 4	NM_001151	1	4
solute carrier family 25, member 3	NM_002635	1	12
ATPase, H+ transporting, lysosome pump	NM_001696	1	22
ATP synthase, H+ transporting, miochondrial F0 complex, subunit c (subunit 9), isoform 2	NM_005176	1	12
ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c (subunit 9) isoform 3	NM_001689	2	2
ATP synthase, H+ transporting, mitochondrial F1 complex, δ subunit	NM_001687	1	19
angiopoietin-like 2	NM_012098	1	9
endothelial differentiation-related factor 1	NM_003792	1	3
transferrin receptor	NM_003234	1	3
cystatin B	NM_000100	1	21
serine (or cysteine) proteinase inhibitor, clade F, member 1	NM_002615	1	17
pescadillo homolog 1	NM_014303	1	22
small EDRK-rich factor 1B	NM_021967	1	5
p75NTR-assoc. cell death executor	NM_014380	1	X
cysteine and glycine-rich protein 1	NM_004078	1	1
exostosis (multiple)-like 1	NM_004455	1	1
Cell/organism defense
ferritin, heavy polypeptide	NM_002032	44	11
ferritin, light polypeptide	NM_000146	13	19
tumor protein, translationally controlled 1	NM_003295	7	13
amyloid β (A4) precursor-like protein 2	NM_001642	6	11
metallothionein 2A	NM_005953	2	16
glutathione S-transferase π	NM_000852	2	11
catalase	NM_001752	1	11
superoxide dismutase 1, soluble	NM_000454	1	21
adenotin	NM_003299	1	12
CD59	NM_000611	1	11
CD68	NM_001251	1	17
interferon induced transmembrane protein 2	NM_006435	2	11
cornichon-like protein	NM_005776	1	14
lectin, galactoside-binding, soluble, 3 binding protein	NM_005567	1	17
macrophage migration inhibitory factor (glycosylation inhibiting factor)	NM_002415	3	22
microsomal glutathione S-transferase 3	NM_004528	2	1
D component of complement (adipsin)	NM_001928	1	19
major histocompatibility complex, class I, A	M24095	1	6
major histocompatibility complex, class I, C	X58536	1	6
major histocompatibility complex, class I, E	NM_005516	1	6
major histocompatibility complex, class 1, β	NM_005514	1	6
b-2-microglobulin	NM_004048	1	15
interferon, α-inducible protein	NM_002038	1	1
interferon induced transmembrane protein (18-U)	NM_021034	1	?
CD63	NM_001780	1	12
CD81	NM_004356	1	11
Metabolism
glyceraldehyde-3-phosphate dehydrogenase	NM_002046	7	12
annexin A2	NM_004039	4	15
annexin A1	NM_000700	3	9
nucleoside-diphosphate kinase (NM23B)	NM_002512	4	17
aldo-keto reductase family 1, member B1	NM_001628	3	7
5-methyltetrahydrofolate-homocysteine methyltransferase reductase	NM_002454	1	5
phosphoserine aminotransferase	NM_021154	1	1
succinate dehydrogenase complex, subunit B, iron sulfur	NM_003000	1	1
cytochrome c oxidase subunit VIIa polypeptide 2	NM_001865	1	4
cytochrome c oxidase subunit IV	NM_001861	1	16
oxidase (cytochrome c) assembly 1-like	NM_005015	1	14
NADH dehydrogenase (ubiquinone) flavoprotein 2	NM_021074	1	18
NADH dehydrogenase (ubiquinone) 1 α subcomplex, 6	NM_002490	1	22
NADH dehydrogenase (ubiquinone) 1 β subcomplex, 8	NM_005004	1	10
NADH dehydrogenase (ubiquinone) 1 β subcomplex, 9	NM_005005	1	8
ATPase inhibitor precursur	NM_016311	1	1
aldolase A, fructose-biphosphate	NM_000034	1	16
aldo-keto reductase family 1, member B10	NM_020299	1	7
aldo-keto reductase family 1, member A1 (aldehyde reductase)	NM_006066	1	1
alcohol dehydrogenase 5 (class III), chi polypeptide	NM_000671	1	4
glucose-6-phosphate dehydrogenase	NM_000402	2	X
phosphoglycerate kinase 1	NM_000291	1	X
galactosidase, α	NM_000169	1	X
UDP-Gal: βGlcNAc β 1,4- galactosyltransferase, polypeptide 1	NM_001497	1	9
transaldolase 1	NM_006755	1	11
enolase 1	NM_001428	2	1
glucosidase, α; acid	NM_000152	1	17
glucosidase, β; acid	NM_000157	1	1
iduronate 2 sulfatase	NM_000202	1	X
transketolase	NM_001064	1	3
lactate dehydrogenase B	NM_002300	1	12
malate dehydrogenase 2, NAD	NM_005918	1	7
NADPH quinone oxidoreductase homolog	NM_004881	1	2
1-acylglycerol-3-phosphate O-acyltransferase 2	NM_006412	1	9
prostaglandin D2 synthase	NM_000954	1	9
high density lipoprotein binding protein (vigilin)	NM_005336	1	2
prosaposin; sphingolipid activator proteins 1 and 2 process mutant	NM_002778	1	10
prostatic binding protein	NM_002567	2	12
annexin A5	NM_001154	2	4
uncoupling protein 3	NM_003356	1	11
glutathione peroxidase 4	NM_002085	1	19
cytochrome P450, 51	NM_000786	1	7
2-hydroxyphytanoyl-CoA lyase	NM_012260	1	3
mevalonate (dephospho) decarboxylase	NM_002461	1	16
prostaglandin E synthase	NM_004878	1	9
sterol-C4-methyl oxidase-like	NM_006745	1	4
adipose differentiation-related protein	NM_001122	1	9
dihydropyrimindase-like 3	NM_001387	1	5
thioredoxin	NM_003329	1	9
thioredoxin reductase 1	NM_003330	1	12
glutaredoxin 2	NM_016066	1	1
nicotinamide N-methyltransferase	NM_006169	1	11
carbonic anhydrase XII	NM_001218	2	15
sialidase 1 (lysosomal sialidase)	NM_000434	1	6
a-L-iduronidase	NM_000203	1	4
spermidine synthase	NM_003132	1	1
methylene tetrahydrofolate dehydrogenase (NAD+ dependent), methenyltetrahydrofolate cyclohydrolase	NM_006636	1	2
orithine decarboxylase antizyme	NM_004152	2	19
pyrophosphatase (inorganic)	NM_021129	1	10
PTD004	NM_013341	1	?
v-Ki-ras2 Kirsten rat sarcoma 2 viral oncogene homolog	NM_004985	1	?
MLL Septin-like fusion gene	NM_006640	1	17
Nit protein 2	NM_020202	1	3
lysophospholipase II	NM_007260	1	1
HU-K4	NM_012268	1	19
MGC15224	NM_017547	1	11
farnesyl pyrophosphate synthetase	NM_002004	1	1
Unclassified genes
p8 protein (candidate of metastasis 1)	NM_012385	4	16
reticulon 4	NM_007008	3	2
putative heme-binding protein (SOUL)	NM_014320	3	6
tumor differentially expressed 1	NM_006811	3	20
GCN5	NM_001487	1	12
Tax interaction protein 1 ;glutaminase-interacting protein 3	NM_014604	1	17
cig41	AF026943	1	10
endoplasmic reticulum glycoprotein	NM_006816	2	?
signal peptidase complex (18kD)	NM_014300	1	15
transmembrane 9 superfamily	NM_006405	2	14
lysosomal-associated protein transmembrane 4	NM_014713	1	2
transmembrane 4 superfamily member 7	NM_003271	1	?
transmembrane 4 superfamily, member 1; tumor antigen (L6)	M90657	1	3
porimin	AL110202	1	11
methyl-CpG binding domain protein 3	NM_003926	1	19
leishmanylysin-like (metallopeptidase M8 family)	AJ312398	1	9
homocysteine-inducible, endoplasmic reticulum stress-inducible ubiquitin-like domain member 1	NM_014685	1	16
small membrane protein 1	NM_014313	1	1
calcineurin B	NM_000945	1	?
synaptobrevin-like 1	NM_005638	1	X
88-kDa Golgi protein	NM_015003	1	15
lysosomal-associated membrane protein	NM_005561	1	13
nucleolar protein family A, member 3	NM_018648	1	15
adrenal gland protein AD--003	NM_014064	1	9
necdin-like protein 1	NM_019066	1	15
amino acid transporter 2	NM_018976	1	12
follicular lymphoma variant translocation 1	NM_002035	1	18
suppression of tumorigenicity 5	NM_005418	1	11
enigma	NM_005451	1	5
Sec 15 (S. cerevisiae)-like	AL137438	1	10
Sec61 gamma	XM_044650	1	7
maternally expressed 3	BE551364	1	14
WD repeat domain 1 protein	NM_017491	1	4
SH3BGRL3-like protein, P1725	NM_031286	2	1
growth arrest-specific 5	BE734306	2	1
EGL nine (C.elegans) homolog 1	NM_022051	1	1
homolog of yeast Golgi membrane protein Yif1p	NM_020470	1	11
CGI-99 protein: LOC51637	NM_016039	1	14
epsilon/beta	AF001893	1	11
similar to rat nuclear ubiquitous casein	NM_022731	1	1
ubiquitin-like 5 (UBL5)	NM_024292	1	19
MUM2 protein	NM_021210	1	?
neuronal calcium binding protein	AF193758	1	16
Wilms tumor 1-associating protein	NM_004906	1	6
myeloid leukemia factor 2	NM_005439	1	12
jumping translocation breakpoint	NM_006694	1	1
ubiquitin-like 3 (UBL3)	NM_007106	1	13
tryptophan rich basic protein (WRB)	NM_004627	2	21
phospholipase C, epsilon 2	AK023546	1	3
37kDa leudine-rich repeat protein	NM_005824	1	7
GL004 protein	NM_020194	1	?
F-box only protein 9	NM_012347	1	6
large tumor suppressor, Drosophila homology	AB028019	1	13
HSPC035 protein	NM_016127	2	8
T-cell lymphoma-associated tumor	NM_022117	1	?
divalent cation tolerant protein CUTGA	NM_015921	1	6
MORF-related gene	NM_012286	1	X
mannose-P-dolichol utilization defect 1	NM_004870	1	17
CGI-18 protein	NM_015947	1	10
arsenate resistance protein ARS2	NM_015908	1	7
HNOEL-iso protein	NM_020190	2	1
hypothetical protein, estradiol-induced (E2IG5)	NM_014367	1	3
putative nucleotide binding protein	NM_014366	1	3
HS1-binding protein HAX-1	NM_006118	1	1
acinus	NM_014977	1	14
protein kinase D2	NM_016457	1	19
PAI-1 mRNA-binding protein	NM_015640	1	1
DC6 protein	NM_020189	2	8
zinc finger protein 277	NM_021994	1	7
CMP-N-acetylneuraminic acid synthase	NM_018686	1	12
nadrin	NM_018054	1	16
epididymal secretory protein	NM_006432	1	14

 

Table 3. EST�s from Unknown Genes Represented in HTM library

Gene	Accession #	Freq	Chromosome		ESTs in UniGene (n)
glioma tumor candidate region	NM_015710	1	19	1078
FLJ10241	NM_018035	1	19	951
MGC2747	NM_024104	1	19	683
DC 2	AF161425	1	4	671
DKFZp564M182	AJ007398	1	16	601
DKFZp5641563	AA765979	2	8	579
FLJ20500	NM_019058	1	10	577
PRO1843	NM_018507	1	12	548
MGC10753	NM_016628	1	10	526
KIAA1265	BI492941	1	20	438
HSPc186	NM_016456	1	1	395
FLJ13052	NM_023018	1	1	384
FLJ20113	NM_017670	1	11	376
DKFZp566C243	NM_015388	1	6	368
MGC3133	NM_031287	1	6	348
PRO2975	AI382020	1	5	340
FLJ22597	NM_016589	1	3	314
est	AA314294	1	X	308
KIAA1162	NM_021156	1	20	301
MGC11352	AK026621	1	10	293
MGC2683	AF007157	1	1	293
MGC14156	NM_032906	1	4	290
MGC5306	NM_024116	1	11	287
KIAA1067	AB028990	1	?	286
putative nuclear protein ORF1-FL49	NM_032412	1	5	272
MGC2835	NM_024072	1	12	262
MGC2749	NM_024069	1	19	261
LOC55831	NM_018447	1	3	260
FLJ13732; similar to tensin	NM_022748	1	7	256
HSPC071	NM_014161	1	6	243
HSPC023 protein	NM_014047	1	19	240
MGC3181	NM_032627	1	19	217
KIAA0063	NM_014876	1	22	212
FLJ20727	NM_017944	1	11	211
FLJ14225	NM_024874	1	1	206
FLJ20752	NM_019048	1	2	202
MGC3402	NM_016494	1	2	202
FLJ11856	NM_024531	1	8	196
FLJ14153	NM_022736	2	3	193
FLJ10803	NM_018224	1	7	190
FLJ22175	NM_025161	1	?	190
KIAA1077	AB029000	2	8	188
MGC26282	NM_032520	1	16	185
FLJ11729	NM_024960	1	20	183
E21G4	NM_015516	1	11	177
FLJ14041	AK024103	1	6	175
MGC2941	NM_024297	1	17	170
FLJ00008 protein	NM_031213	1	19	168
MGC1223	NM_030969	2	6	168
est	BC009927	1	1	166
est	BF245622	1	9	161
est	BG654408	1	4	160
MGC5564	AI376862	1	6	158
DKFZp586I1518	AL049378	1	5	156
est	BG913357	1	12	154
est, Hs.3017111	BE784012	2	12	154
FLJ12892	BE893521	1	3	154
HSPC002	NM_015362	1	17	152
FLJ10415	NM_018089	1	2	151
DKFZp566D213	NM_015513	1	3	150
KIAA1532 protein	AB040965	2	19	140
est	BG291346	1	4	139
est	BF513991	1	17	135
MGC1346	NM_032758	1	22	133
FLJ12438	NM_021933	1	1	132
hepatocellular carcinoma-associated antigen 112	NM_018487	1	7	129
DKFZp761I141	NM_031488	1	22	127
est	AI344746	1	7	127
est	AF288394	1	1	126
AF140225	BC007829	1	5	125
hypothetical nuclear factor SBBI22	BI255383	1	4	119
est	BE677948	1	4	118
PRO1914 protein	NM_014106	1	15	114
KIAA0909	AB020716	1	17	114
est	BF204739	1	7	112
MGC2404	NM_032360	1	1	111
KIAA1143 protein	AB032969	1	14	108
MGC15749	NM_032881	1	1	104
FLJ20539	NM_017870	1	11	99
FLJ14825	NM_032847	1	8	94
KIAA1737	AB05152	1	14	94
HSPC034 protein	NM_016126	1	1	94
MGC2654	NM_024109	1	16	89
est	AI810399	2	22	82
MGC2821	NM_024054	1	7	75
FLJ22649	NM_021928	1	4	73
DKFZp434J194	AL110227	1	19	73
FLJ21820	NM_021925	1	2	73
KIAA0860 protein	NM_014948	1	20	71
FLJ22621	NM_025103	1	9	70
est	AA416894	1	15	69
est	BE501080	1	2	66
DKFZp586F1322	AL050172	1	11	62
FLJ10846	NM_018241	1	4	57
MGC15737	NM_032926	1	X	57
FLJ21130	NM_020466	1	6	46
est	BF439215	1	7	45
FLJ20512	NM_017854	1	19	45
FLJ20245	NM_017723	1	?	44
LOC88523	AV726785	1	13	42
est	AW183161	1	?	42
FLJ14218	AK024280	1	11	38
HSPC230	NM_016487	1	6	37
FLJ22463	NM_014632	1	11	37
est	AA150501	1	12	27
DKFZp564N1116	AW960473	1	5	24
est	AI809953	1	2	24
FLJ10302	AK001164	1	?	13
est	BE542166	1	1	10
est	BF060661	1	10	9
est	BE877420	1	3	7
est	AW272279	1	2	6
est FLJ22805	AK026458	2	19	0
est	BG993458	1	?	0
est	AF086471	1	11	0
est	AF088058	2	17	0
FLJ20002	AK000009	2	13	0
est	BF677892	1	?	0
est	AL049356	1	?	0
est	AW969629	1	10	0
FLJ21087	AK024740	1	6	0
est	BG541576	1	3	0
est	BE394862	1	17	0
FLJ21020	AK024673	1	?	0
FLJ10936	XM_049050	1	12	0
est	AW051913	1	3	0
est FLJ21770	AK025423	1	22	0
TM132		1	12	0
TMT95		1	16	0
TM246		1	14	0
TM184		1	13	0
TM155		1	1	0
TM7		1	?	0
TM553		1	22	0
TMT527		1	?	0
TM366		1	12	0
TMM228		1	9	0
TM516		1	15	0
TMM103		1	12	0
TM37		1	?	0

The authors thank the Oregon Lions Sight and Hearing Foundation for providing eyes and the MMI Research Core Facility at Oregon Health and Sciences University and the Portland VAMC Molecular Biology Core Laboratory for sequencing.