Gene expression profile in rat pancreatic islet and RINm5F cells

in Journal of Molecular Endocrinology

To clarify tissue-specificity of pancreatic β cells, comparison of mRNA expression in various conditions of the tissue of multiple organisms is important. Although the developed methodologies for mRNA monitoring such as microarray, rely on the growth of dbEST (database of expressed sequence tag), a large number of unknown genes in the genome, especially in the rat, have not been shown to be expressed. In this study, we have established the first database of ESTs from rat pancreatic islet and RINm5F cells. Two cDNA libraries were constructed using mRNAs from rat pancreatic islet and RINm5F cells to cover a wider spectrum of expressed genes. Over 40 000 clones were randomly selected from the two libraries and partially sequenced. The sequences obtained were subjected to BLAST database analyses. This large-scale sequencing generated 40 710 3′-ESTs. Clustering analysis and homology search of nucleotide and peptide databases using both 3′- and 5′-ESTs revealed 10 406 non-redundant transcripts representing 4078 known genes or homologs and 6328 unknown genes. To confirm actual expression, the unknown sequences were further subjected to dbEST search, resulting in the identification of 5432 significant matches to those from other sources. Interestingly, of the remaining sequences showing no match, 779 were found to be encoded by exon–intron organization in the corresponding genomic sequences, suggesting that these are newly found as actually expressed in this study. Since many genes are up- or down-regulated in differing conditions, applications of the expression profile should facilitate identification of the genes involved in cell-specific functions in normal and disease states.

Abstract

To clarify tissue-specificity of pancreatic β cells, comparison of mRNA expression in various conditions of the tissue of multiple organisms is important. Although the developed methodologies for mRNA monitoring such as microarray, rely on the growth of dbEST (database of expressed sequence tag), a large number of unknown genes in the genome, especially in the rat, have not been shown to be expressed. In this study, we have established the first database of ESTs from rat pancreatic islet and RINm5F cells. Two cDNA libraries were constructed using mRNAs from rat pancreatic islet and RINm5F cells to cover a wider spectrum of expressed genes. Over 40 000 clones were randomly selected from the two libraries and partially sequenced. The sequences obtained were subjected to BLAST database analyses. This large-scale sequencing generated 40 710 3′-ESTs. Clustering analysis and homology search of nucleotide and peptide databases using both 3′- and 5′-ESTs revealed 10 406 non-redundant transcripts representing 4078 known genes or homologs and 6328 unknown genes. To confirm actual expression, the unknown sequences were further subjected to dbEST search, resulting in the identification of 5432 significant matches to those from other sources. Interestingly, of the remaining sequences showing no match, 779 were found to be encoded by exon–intron organization in the corresponding genomic sequences, suggesting that these are newly found as actually expressed in this study. Since many genes are up- or down-regulated in differing conditions, applications of the expression profile should facilitate identification of the genes involved in cell-specific functions in normal and disease states.

Keywords:

Introduction

Pancreatic islets play the critical role in the regulation of blood glucose by secreting hormones from endocrine cells that differentiate from common progenitor cells during fetal development (postnatal origin of β-cell replenishment remains controversial) (Edlund 2002, Bonner-Weir & Sharma 2002, Dor et al. 2004). Functional defects of β-cells lead to the development of diabetes mellitus. Since a number of genes are involved in the pathogenesis of impaired insulin secretion, it is important to characterize the expression profile of a set of genes that endow β-cells with the tissue-specific functions of insulin synthesis and secretion.

Recent genome projects demonstrated a similar number of 30 000–40 000 genes in human and mouse chromosomes, including ~27 000 protein-encoding transcripts for which there was strong corroborating evidence and ~10 000 computationally derived genes with weak supporting evidence (International Human Genome Sequencing Consortium 2001, Venter et al. 2001, Mouse Genome Sequencing Consortium 2002). Comparison with the transcriptome revealed almost all of the human genes known to be expressed to have orthologues in the mouse genome. The other putatively novel genes in the genome were detected using computer algorithms for transcript prediction. To estimate the accuracy of the power of new gene detection, the results of the gene annotation done by the two human genome efforts were previously compared (Hogenesch et al. 2001). Surprisingly, although a similar number of the total genes was demonstrated, there is little agreement regarding the new genes predicted by the two projects, suggesting that a significant fraction of tissue-restricted transcripts for novel genes remain undiscovered, possibly due to limitations in the computer prediction methods.

As expression analysis of the genes in multiple organisms becomes a major focus in the new era of biology, functional genomics will rely largely on the vast sources of subsets of partial cDNA sequences from various tissues that have proven enormously valuable and are deposited as expressed sequence tags (ESTs) in the public databases. The Endocrine Pancreas Consortium has recently constructed human and mouse cDNA libraries from various conditions of endocrine pancreas and generated over 100 000 ESTs (Bernal-Mizrachi et al. 2003). We also have collected ~20 000 ESTs from human normal pancreatic islets and islet tumors, resulting in the identification of ~3000 new genes expressed in the islets (Takeda et al. 1993, Jin et al. 2003). Such systematic sequencing efforts complement each other and should improve the various methodologies including DNA microarray technology (Scearce et al. 2002) for monitoring differential gene expression in normal and disease states. In addition, the laboratory rat is an indispensable model organism of human diseases, providing a useful tool in experimental medicine and drug discovery. As various spontaneous diabetic rats such as the GK and OLETF rats and the experimental streptozotocin-induced diabetic rat are widely used in pancreatic islet studies, it is important to establish an additional source of rat expressed sequences. However, although ~26 millions of ESTs have so far been deposited in the database (dbEST release 031105), approximately 40% of which are derived from human and mouse, only 2.6% of the sequences are from rat, and none are from pancreatic islets except for the present deposition. In this study, toward elucidation of the entire transcriptome in rat pancreatic islets, we have made two cDNA libraries, one from rat normal pancreatic islets and the other from RINm5F tumor cells having undergone less differentiation (Gazdar et al. 1980, Philippe et al. 1987), and performed a large-scale collection of ESTs. Since a number of the genes are up- or down-regulated in different conditions, a collection of ESTs from these distinct cDNA sources should more effectively cover a wider spectrum of expressed genes, generating a larger pool of non-redundant sequences. In addition, since the insulin content of the less-differentiated RINm5F cells used in this study was previously found to be much lower than that of normal islets (Kayo et al. 1997), direct comparison of the expression profiles of the two cDNA libraries should facilitate the identification of the genes involved in insulin synthesis and secretion as well as in β-cell differentiation and tumorigenesis.

Materials and methods

Preparation of rat pancreatic islets

Pancreatic islets were prepared from male Wister rats by a collagenase digestion method as described previously (Ma et al. 1996). Briefly, under pentobarbital anesthesia, the pancreas was distended by an injection of 10 ml Hank’s solution containing 0.3 mg/ml collagenase (type XI, Sigma-Aldrich, StLouis, MO, USA). Islets were separated by the Ficoll (Amersham, Piscataway, NJ, USA) density gradient method with four layers (27%, 23%, 20.5%, and 11% of Ficoll dissolved in Hank’s solution). After centrifugation at 450 g for 15 min, pancreatic islets were concentrated at the interface between the 11% and 20.5% Ficoll layers. Islets were then harvested by a pick-up method under a stereomicroscope. Purity of the islets was estimated to be ~99% by counting the cells immunoreactive to insulin and glucagon antibodies after trypsin treatment of a fraction of the islets collected. The high purity also could be estimated by the frequency of cDNA for major exocrine molecules, such as α-amylase, in the entire islet ESTs identified, as described below.

Large-scale cDNA sequencing

Two unidirectional cDNA libraries were constructed in the Uni-ZAP XR vector (Stratagene, La Jolla, CA, USA) using mRNAs from rat normal pancreatic islets and RINm5F cells. A large set of plasmid DNAs for sequencing was prepared as described (Takeda et al. 1993, Jin et al. 2003). Briefly, the cDNA libraries were excised en masse from the λ phage into phagemid particles using the ExAssist phage system (Stratagene), and subsequently transfected into E. coli SOLR (Stratagene) for conversion to plasmid forms. Plasmid DNAs were extracted from E. coli colonies randomly selected from LB-Amp plates using the Biomek 2000 mini-prep system (Beckman, Fullerton, CA, USA). Single-pass DNA sequencing from the 3′-end of the inserts was performed using a BigDye Terminator Cycle Sequencing FS Ready Reaction Kit and DNA Sequencer model 3700 (Applied Biosystems, Foster City, CA, USA). Vector sequences were removed from the results using Assembly LIGN software (Oxford Molecular Group PLC, Oxford, UK). Quality assessment of the sequences obtained was performed using PE Sequencing Analysis 3.3 software (Applied Biosystems).

Database analysis of rat pancreatic islet and RINm5F ESTs

We compared a total of ~40 000 sequences from rat pancreatic islet and RINm5F cells with non-redundant nucleotide and peptide sequences extracted in silico from databases at the National Center for Biotechnology Information (NCBI). Before comparisons, interspersed repetitive sequences such as LINEs were unmasked and removed from the pool using software RepeatMasker (http://ftp.genome.washington.edu/RM/RepeatMasker.html). To assemble sequences sharing a stretch of nucleotide identity, the LaboServer system (World fusion, Tokyo, Japan) was applied to make contigs. Representative sequences from each contig then were subjected to BLASTN analysis for sequence homology at nucleotide level against a merged database by the Kiroku program (World fusion). If a query sequence shared over 95% nucleotide identity and showed a score of more than 400 with any sequences in the database, they were grouped together. The clones without significant match to known sequences in the nucleotide database were re-sequenced from the other end to compare the sequences with those in the peptide database at NCBI using BLASTX program (Altschul et al. 1997), which conceptually translates the query sequence in all six reading frames for comparison. The ESTs identical or highly similar to functionally annotated genes were first classified into seven major categories according to the general functions of the proteins encoded, and then further classified into subcategories according to their specific functions.

Semi-quantitative RNA expression analysis

To ascertain the level of mRNA expression of the ESTs from rat normal islets and RINm-5F cells, real-time quantitative reverse transcriptase (RT)-PCR was carried out using an ABI PRISM 7900HT Sequence Detection System (Applied Biosystem). Total RNA was extracted from pooled islets isolated from normal rats and RINm5F cells, using an RNeasy Mini Preparation Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. TaqMan primers and probes were designed using Primer Express software purchased from Applied Biosystems. TaqMan reactions were performed in a reaction volume of 20 μl using components supplied in a TaqMan PCR reagent kit. Each reaction consisted of 10 μl TaqMan Universal Master Mix, 900 nM of each amplification primer, and 250 nM corresponding TaqMan probe. Each sample was run for an initial 2 min at 50 °C and 10 min at 95°C, followed by 40 cycles at 95 °C for 15 s and at 60 °C for 1 min. Amplification data were collected by the 7900HT Sequence Detector and analyzed using Sequence Detection System software. The RNA concentration was determined from the threshold cycle at which fluorescence is first detected, cycle number being inversely related to RNA concentration.

In situ hybridization

ESTs showing marked differences in frequency between islet and RINm5F cells were subjected to analysis of mRNA distribution in the pancreas by in situ hybridization. Paraffin embedded blocks and sections of normal rat pancreas for in situ hybridization (ISH) were obtained from GENOSTAFF, Inc. (Tokyo, Japan). The pancreases of male Wistar rats 8 weeks old (CREA Tokyo, Japan, Inc.) were dissected after perfusion, fixed by Tissue Fixative (GENOSTAFF, Cat No.STF-01), and embedded in paraffin by the proprietary procedures. ISH was performed with the Ventana HX system (Ventana Medical Systems, Inc., Tucson, AZ, USA). Entire EST inserts were amplified by PCR using ExTaq (TaKaRa, Kyoto, Japan) in a 50 μl reaction mixture using M13 forward and reverse primers. Amplification was performed as follows: 3 min at 94 °C for initial denaturation, 35 cycles of 94 °C denaturing for 30 s 60 °C annealing for 30 s, and 72 °C extension for 1 min, followed by a final extension at 72 °C for 10 min. Quality and quantity of the purified PCR product was confirmed by 1.2% agarose gel electrophoresis. Anti-sense and sense RNA probes were labeled using the T7/T3 digoxigenin RNA labeling kit (Roche Diagnostics, Indianapolis, IN, USA), according to the manufacturer’s instructions. Sections were pre-treated and hybridized with a Ventana RiboMap kit (Ventana Medical Systems) on the automated Ventana HX system Discovery. Detection of hybrids was performed with a digoxigenin nucleic acid detection kit (Boehringer Mannheim, Germany) following the manufacturer’s instructions. Sections were then dehydrated through an ethanol series (80, 90 and 100% ethanol, for 1 min each) and washed for 1 min in xylene before mounting in malinol mounting medium (Muto Pure Chemicals Ltd, Tokyo, Japan).

Results and discussion

The expression profile of genes in pancreatic islets of experimental animals will greatly complement human studies of functional genomics of the tissue and the genetics of its disease states. This study both establishes a first molecular inventory of rat pancreatic islets and reveals a number of novel genes, the expression of which has not previously been described. These should provide important insights into the entire transcriptome of endocrine pancreas and be an immensely valuable aid to the improvement of genomic annotation.

Large-scale collection of ESTs from rat pancreatic islet and RINm5F cells

A total of 40 710 clones randomly selected from the two cDNA libraries were partially sequenced from the 3′-end. Sequences containing less than 1% ambiguous bases longer than 200 bp were subjected to BLASTN database search. Contaminated genomic sequences, e.g. repetitive sequences, (1967 clones) and mitochondrial DNAs (4633 clones), were removed from the pool of sequences, resulting in a collection of 34 110 ESTs comprising 22 310 known and 11 800 unknown sequences. Our previous study of 1000 ESTs from human pancreatic islets, the purity of which was estimated to be ~90% by microscopic examination and protein analysis, identified 13, 12, 6, and 9 ESTs for major exocrine genes for α-amylase, elastase, pancreatic lipase, and trypsinogen respectively (Takeda et al. 1993). Since only 2, 8, 2, and 5 clones for these exocrine genes respectively, were found in the ~20 000 EST sequences of this study, the possibility of contamination from exocrine cells appears to be quite negligible (~0.1%). This estimation of purity is consistent with that of the protein analysis described above. Because large-scale sequencing based on random isolation of clones generates high redundancy, clustering analysis was performed to assemble the sequences into non-redundant sequence groups. A total of 6030 and 6260 independent groups were obtained from pancreatic islet and RINm5F cells respectively, and the pattern of redundancy was similar between the two sources (Table 1 & 2). Together, 10 406 non-redundant sequences comprising 4859 clusters of sequences and 5547 singletons were obtained representing 4078 known genes and 6328 unknown genes. Since only 1896 distinct genes (18%) were found to overlap in the normal islets and RINm5F cells, this large-scale sequencing using two distinct cDNA sources was quite effective in identifying a larger number of non-redundant sequences. Studies of the number of different mRNA sequences in a cell suggest that a typical higher eukaryotic cell synthesizes 10 000 to 20 000 different proteins (Alberts et al. 1994), so this approach covered at least 50% of the possible protein-coding genes. As in similar large-scale cDNA sequencing studies carried out in other tissues, about 50% of the clones obtained are derived from genes not functionally annotated. These unknown clones were re-sequenced from the 5′-end, and the 5512 clones sequenced successfully were also subjected to database search. As a result, 1404 sequences representing 502 distinct transcripts showed perfect identity to known genes, so the 3′-end sequences of these clones clearly are not contained in the cDNA sequences deposited in the nucleotide databases. These ESTs were then assigned to the known group. All representative EST sequences obtained from each cluster were deposited in the public database to be freely available to all researchers (DDBJ accession No. BP464981–BP504629).

Characterization of known genes in pancreatic islet and RINm5F cells

The ESTs showing identity or high similarity to known genes were classified into seven major categories on the basis of putative general functions of the protein encoded, as described previously (categories: cell division, cell signaling/communication, cell structure/ motility, cell/organism defense, gene/protein expression, metabolism, and unclassified). In total, 3951 out of 4078 known genes were represented in the classified data set (online supplement). The largest category of genes was gene/protein expression (26.4%). Successively smaller categories were cell signaling and communication (19.0%), metabolism (16.8%), cell structure/ motility (7.7%), cell/organism defense (7.5%), and cell division (5.6%). ESTs lacking sufficient information to be classified constituted the remainder, unclassified (16.9%). To further analyze the molecular complexity, each major category was subdivided according to the putative specific functions of the proteins (Table 3, also see online supplement). For example, the largest category, gene/protein expression, was subdivided into eight subgroups. Of these, transcription factor constituted the largest number of non-redundant genes (416 genes by 1209 ESTs). The transcription factors include PDX-1, BETA2/NeuroD, HNF-4α, Nkx-2.2, Nkx-6.1, and Isl-1 etc, all of which are important for pancreatic development and islet-specific functions, and the first three of which are the causal genes for monogenic forms of diabetes, MODY4, MODY6, and MODY1 (Fajans et al. 2001). The other genes for transcription factors also are plausible candidates for diabetogenes or genes responsible for β-cell specific functions.

In this study, 60.8% of the non-redundant ESTs did not match any of the known genes in the nucleotide database. To identify novel rat genes encoding proteins structurally related to the known proteins, we performed BLASTX similarity search in the peptide databases using 5512 distinct ESTs. Of these, 127 represent rat homologs of genes identified in other species or new members of structurally related families in rat, the cut off for significant similarity being P value of 107 and similarity of 50% (Table 4). Functional analyses of the proteins encoded by these ESTs should clarify their novel roles in pancreatic islets.

To identify genes that had not been determined to be expressed, the sequences showing no significant match to any of the annotated genes were further compared with dbEST entries from other tissues, revealing 896 expressed genes that have not appeared in the database. These 896 putatively novel genes were analyzed in the context of recently determined rat genomic sequences (Rat Genome Sequencing Project Consortium 2004). Of these genes, 779 were encoded by exonintron organization in the corresponding genomic sequences. However, since the transcripts of many of these genes were barely detectable by in situ hybridization, they may be expressed at low levels, at least in adult islets. Because chemiluminescence-based in situ detection of mRNAs is not sufficiently sensitive, a large-scale RT-PCR analysis is presently in progress in our laboratory to elucidate the tissue distribution. The expression of 71 of the other 117 sequences was uncertain due to their ambiguous genomic structure. The remainder could be derived from possible pseudogenes or retroposons, as the corresponding sequences in the genome are flanked by AT-rich sequences that were recognizable by oligo-dT priming in the process of cDNA synthesis.

Characterization of differentially expressed genes

The immunoreactive insulin (IRI) content of rat normal β-cells has been reported to be ~8000 pmol/106 cells, while the RINm5F cell line used in this study has been estimated to contain a very low level of IRI (0.43 pmol/106 cells) and a much lower number of secretory granules (Kayo et al. 1997). Accordingly, the expression levels of the genes involved in insulin synthesis and secretion in RINm5F cells should markedly differ from those of the normal β-cells. In addition, since the RINm5F cells were derived from radiation-induced tumor cells and exhibited a decrement of well-differentiation (Gazdar et al. 1980, Philippe et al. 1987, Kayo et al. 1997), the expression levels of the genes involved in cell differentiation and tumorigenesis may also be altered. The relative frequencies of ESTs have been shown to reflect the average level of expression of the corresponding mRNAs in the tissues examined (Lee et al. 1995). As pancreatic islet cells are mostly β-cells, the expression profile of insulin-related genes in the two cDNA sources (of same size) can be compared to identify differentially expressed genes. The EST frequencies for most of the house-keeping genes were similar in the two cDNA libraries, suggesting that such comparison of EST frequencies is reasonable. Over 2-fold differences in frequency between the two libraries were found in 204 genes (higher EST> 10 times). The direction of change in mRNA levels in these ESTs observed by comparison of the EST frequency and the TaqMan semi-quantitative analysis was quite parallel, while the magnitude of the change was not correlated. The representative results for some of the ESTs (> 15 times) are shown in Fig. 1. Previously, similar comparative analysis using ~6000 ESTs from two different conditions of PC-12 cells was performed (Lee et al. 1995). The study found the ratio of EST frequencies between the two cDNA sources to be correlated with the Northern blot analysis, except for the low-frequency ESTs. Thus, the genes of interest that are expressed at least at moderate levels also should be examined by semi-quantitative analysis before further analysis.

We focus on the seven genes exhibiting high expression (> 15 times) in islet but no expression in RINm5F cells (Table 5), since they may well play a specific role in insulin synthesis and secretion. Expression of all of the genes examined was confirmed in pancreatic β cells by in situ hybridization, although the expression was not restricted to β cells (Fig. 2A). The expression of the high EST clones (> 15 times) in RINm5F cells and none in islets was found only outside the islets by in situ hybridization (Fig. 2B). These patterns of expression are consistent with a previous assumption that over 90% of the genes are house-keeping and are expressed at various levels in many tissues. Receptors of the insulin/insulin-like growth factor (IGF) family have been implicated both in the regulation of pancreatic β-cell growth and insulin secretion. IRR, an orphan receptor of the insulin receptor subfamily, is expressed at a considerably higher level in pancreatic β cells (Hirayama et al. 1999), and a decrease in the mRNA level was found in diabetic GK rats (N Shihara, Y Horikawa, J Takeda, unpublished observations). However, since glucose-stimulated insulin secretion and embryonic β cell development have been shown to occur normally in mice lacking IRR (Kitamura et al. 2001), decreased expression of IRR alone may be insufficient for the development of diabetes. To understand the functional properties of IRR in pancreatic β cells, it is important to identify its possible ligand and functional partners. Mesothelin, produced by mesothelial cells, has been suggested to play a role in cellular adhesion in ovarian cancer cells (Scholler et al. 1999). Since this EST was not found in cultured RINm5F cells, methothelin may well be unimportant in single-cell growth without cell adhesion and not directly involved in endocrine tumorigenesis. CD74, which is transitorily associated with class II histocompatibility antigens during intra-cellular transport (Claesson et al. 1983), also was found to be highly expressed in human islet tumor in our previous study (Jin et al. 2003). Since this islet tumor exhibited features of moderately differentiated islet cells, despite markedly reduced insulin secretion, in contrast to RINm5F cells, CD74 might be related to the degree of cell differentiation. Osteonectin is reported to be abundantly expressed in adipose tissue. Although the molecule has not been reported to be expressed in islets, in situ hybridization showed that its expression is moderate in islet cells and diffuse throughout the pancreas of normal Wistar rat. It has been reported that absence of osteonectin leads to an increase in the size of individual adipocytes as well as in the number of adipocytes per fat pad (Bradshaw et al. 2003). Thus, osteonectin might be related to β cell mass and growth rather than insulin synthesis and secretion. As these genes might contribute to the development of islet cell specificity, their functional significance should be examined in various conditions of pancreatic islets.

Applications of rat ESTs for islet studies

In this study, we describe a collection of 40 710 rat pancreatic islet-related ESTs representing 10 406 different transcripts. This is the first report describing a systematic collection of rat expressed genes from pancreatic islets and a β-cell line. Since DNA microarray technology relies largely on the rapid growth of the EST databases, these newly identified expressed genes should facilitate analysis of differential gene expression in pancreatic islets under various conditions. At present, only the PanChip microarray, which was prepared using 3400 cDNA sequences from mouse whole pancreas, is available as a tissue-specific microarray for islet studies (Scearce et al. 2002). Accordingly, the establishment of islet-specific DNA microarrays for the rat should be especially important in the analysis of the transcriptome of diabetic rats such as GK and OLETF, and is presently underway in our laboratory. Another advantage of the large-scale collection of EST clones is that the cDNA fragments obtained can be used as hybridizing probes for Northern blotting or in situ hybridization to analyze the size and number of alternatively spliced transcripts and their local tissue distribution. However, it is possible that a small fraction of the ESTs obtained might be contaminated from other cell types such as endothelial cells or blood. Indeed, our preliminary trial of non-isotopic in situ hybridization using rat ESTs was found to be quite effective for analysis of mRNA expression in pancreatic islets. A large-scale in situ hybridization of rat islet mRNAs is also presently in progress in our laboratory. Functional analysis of a wide spectrum of islet-specific genes and genes highly abundant or less abundant in islets identified by this approach might clarify the molecular mechanisms underlying the differentiation of islet cells, tumorigenesis, and the pathogenesis of diabetes, as well as lead to new therapies for the improvement and regeneration of β-cell function through manipulation of gene expression and gene products.

In addition, as the genome sequence analysis of the Brown Norway rat recently has been completed (Rat Genome Sequencing Project Consortium 2004), the results of this study should be helpful in annotating the genes actually expressed in the rat genome and thus provide further insight into mammalian evolution of genes involved in tissue-specificity of endocrine pancreas.

Table 1

Redundancy of 3′-ESTs from rat pancreatic islet and RINm5F cells

IsletRINm5FIslet & RINm5F
The 40 710 ESTs were generated by sequencing cDNA clones from the
3′-ends. After clustering analysis, a total of 6030 and 6260 independent groups were obtained from pancreatic islet and RINm5F cells, respectively.
Redundancy
> 1000101
101–1000137
51–1003615
31–5061228
21–30213169
11–2087138253
6–10228299533
3–5103911131878
2155615932075
1308830655547
Table 2

Summary of non-redundant 3′-ESTs and/or 5′-ESTs

KnownUnknownTotal
The 6030 and 6260 non-redundant sequences from pancreatic islet and RINm5F cells, respectively, were subjected to BLASTN database search at the National Center for Biotechnology Information.
Islet only116529814146
RINm5F only156928074376
Islet & RINm5F13445401884
Total4078632810 406
Table 3

Functional categories of proteins encoded by non-redundant ESTs

SubcategoryIslet onlyRINm5F onlyIslet & RINm5FTotal
The ESTs showing identity or high similarity to known genes were classified into seven major categories on the basis of putative general functions of the protein encoded and each major category was further subdivided according to the putative specific functions of the proteins.
Functional category
Cell divisionGeneral18132152
DNA synthesis118524
Apoptosis13151846
Cell cycle13361968
Chromosomal structure815932
Subtotal539772222
Cell signalingCell adhesion20181250
Channel15211955
Effectors23242471
Hormone31282584
Intracellular transducers505362165
Metabolism2136
Protein modification564355154
Receptor605751168
Subtotal257245251753
Cell structureGeneral9171642
Contractile protein98724
Cytoskeletal23263988
Extracellular matrix2410842
Microtubule-associated17231757
Vesicular transport15191953
Subtotal97103106306
Cell defenseHomeostasis (general)21162057
DNA repair11171139
Carrier protein22242268
Stress response9121435
Immunology47232797
Subtotal1109294296
Gene expressionRNA polymerase661224
RNA processing237062155
Transcription factor136173107416
Targeting374965151
Protein turnover342847109
Ribosomal proteins172087124
tRNA synthesis19818
Translation factor9122950
Subtotal2633674171047
MetabolismGeneral44513
Amino acid12151845
Cofactors1315
Energy313756124
Lipid395149139
Nucleotide18301361
Protein modification7252052
Sugar198130130
Transport25363192
Subtotal156282223661
Unclassified197300169666
Total No. of unique genes1133148613323951
Table 4

Rat homologs of known genes and new members of gene families

GeneSpecies%SIMPvalueIsletRINm5F
The unknown clones after BLASTN search in the nucleotide databases were re-sequenced from the 5′-end and then subjected to BLASTX search in the peptide databases. The cut off used for significant similarity was P value of 10−7 and similarity of 50%. The clone IDs RBC and RIN show ESTs from pancreatic islet and RINm5F cells, respectively.
Clone ID
RBC00545breast carcinoma amplified sequence 3 homologHomo sapiens0.722E-2111
RBC00858retrovirus-related POL polyproteinMus musculus0.744E-4210
RBC01084eukaryotic translation initiation factor 4 gamma 3Homo sapiens0.665E-4410
RBC01744protein C20orf149 homologRattus norvegicus0.741E-5110
RBC03066DNA transformation protein comFPseudomonas stutzeri0.743E-6310
RBC03516hypothetical protein in acoE 3′ regionRhodobacter sphaeroides0.857E-4810
RBC04593speckle-type POZ protein-like 1; POZ 56 proteinRattus norvegicus0.723E-3210
RBC05170hypothetical protein 3Rattus norvegicus0.787E-3511
RBC0561014 kDa phosphohistidine phosphataseRattus norvegicus0.649E-2830
RBC06005transposase for insertion sequence element IS904Pseudomonas putida0.832E-4910
RBC06162aurora-A kinase interacting proteinRattus norvegicus0.792E-4520
RBC06680epsin 1Rattus norvegicus0.758E-3810
RBC07890probable Pol polyproteinRattus norvegicus0.715E-4310
RBC08283G protein-coupled receptor 150Mus musculus0.814E-8910
RBC08942Egl nine homolog 2Mus musculus0.752E-0720
RBC09487hypothetical protein PP2447 homologMus musculus0.754E-1911
RBC09605putative NF-kappa-B activating proteinRattus norvegicus0.641E-4310
RBC10109polyposis locus protein 1Rattus norvegicus0.792E-3410
RBC11646immunoglobulin light chain variable regionMus musculus0.582E-2710
RBC12591chromosome 10 open reading frame 45Homo sapiens0.624E-4120
RBC12830pORF2Mus musculus0.641E-3410
RBC12840methyl-accepting chemotaxis proteinPseudomonas syringae0.649E-3410
RBC13044ribonuclease P protein subunit p29Homo sapiens0.642E-1920
RBC13272hypothetical protein KIAA0174Rattus norvegicus0.765E-1330
RBC13530histone deacetylase 4 (HD4)Rattus norvegicus0.732E-1810
RBC14441lactoylglutathione lyaseRattus norvegicus0.717E-4920
RBC14577lymphocyte antigen Ly-6D precursorRattus norvegicus0.794E-3820
RBC15029protein C21orf5Homo sapiens0.857E-3310
RBC15417POL polyproteinTrichosurus vulpecula0.719E-2520
RBC15535amino-acid ABC transporter ATP-binding proteinThermobifida fusca0.833E-8010
RBC15661zinedinRattus norvegicus0.761E-2211
RBC15731collagen alpha 1(X) chainMus musculus0.663E-6440
RBC17834probable transcriptional regulatory protein ygiXsynthetic construct0.722E-5310
RBC18842nectin 4Mus musculus0.758E-7310
RBC19899probable 3-hydroxybutyryl-CoA dehydrogenaseBrucella melitensis0.813E-9930
RBC20402hypothetical protein CGI-143Rattus norvegicus0.644E-2810
RIN00207S-adenosylmethionine-dependent methyltransferaseMycoplasma mobile0.683E-5402
RIN00228heat inducible transcriptional repressor proteinMycoplasma mobile0.593E-3201
RIN00249cell cycle control protein cwf15Mus musculus0.564E-1201
RIN00488hypothetical protein C5D6.06cMus musculus0.863E-8501
RIN00804zinc finger protein 35 (Zfp-35)Rattus norvegicus0.625E-2201
RIN01128tyrosine-protein kinase FLKRattus norvegicus0.771E-4201
RIN01130trigger factor (TF)Mycoplasma pulmonis0.657E-1101
RIN01171WD-repeat protein CGI-48Rattus norvegicus0.69E-2801
RIN01386probable cation-transporting ATPase 1Rattus norvegicus0.793E-8201
RIN01389L-lactate dehydrogenaseMesoplasma florum L10.582E-2001
RIN01548THAP domain protein 11Rattus norvegicus0.519E-2201
RIN01784hypothetical protein KIAA0233Rattus norvegicus0.865E-2301
RIN01794cell division protein ftsH homologMycoplasma pulmonis0.644E-3201
RIN01840hypothetical protein MG061Mycoplasma pulmonis0.551E-3404
RIN01932condensin complex subunit 2 (p105)Mus musculus0.883E-1401
RIN02139protein FAM3A precursorMus musculus0.875E-3601
RIN02177ABC transporter ATP-binding proteinMycoplasma gallisepticum0.747E-6003
RIN02445translation initiation factor IF3Mycoplasma fermentans0.628E-1202
RIN02483splicing factor, arginine/serine-rich 2Mus musculus0.662E-5902
RIN02532ABC transporter permease protein MG188 homologMycoplasma gallisepticum0.657E-1102
RIN02603protein c20orf172 homologMus musculus0.892E-4401
RIN03035probable nicotinate-nucleotide adenylyltransferaseMycoplasma mobile0.653E-2601
RIN03110phospholipase A2 inhibitor gamma subunit BRattus norvegicus0.842E-0703
RIN03339hypothetical lipoprotein MPN288Mycoplasma gallisepticum0.564E-1002
RIN03427putative Pol polyproteinRattus norvegicus0.772E-1902
RIN04062hypothetical protein MG148Ureaplasma parvum0.693E-2201
RIN04617probable cation-transporting P-type ATPaseUreaplasma parvum0.551E-3101
RIN05010testis-specific protein PBS13Rattus norvegicus0.785E-8801
RIN05122hypothetical protein KIAA0036Rattus norvegicus0.866E-7601
RIN05229E3 ubiquitin-protein ligase Nedd-4Cricetulus griseus0.864E-4801
RIN05576TLM proteinMus musculus0.641E-2621
RIN05605RRS1 ribosome biogenesis regulator homologMus musculus0.673E-3501
RIN05994eukaryotic translation initiation factor 4E transporterRattus norvegicus0.73E-1301
RIN06079brain protein 14Mus musculus0.618E-1201
RIN06155slingshot 3Rattus norvegicus0.796E-4701
RIN06262mitochondrial respiratory chain complexes assembly proteinMycoplasma pulmonis0.733E-3701
RIN06288SCO2 protein homolog, mitochondrial precursorMus musculus0.764E-4503
RIN06497methionine aminopeptidaseMycoplasma pulmonis0.71E-3701
RIN06650retrovirus-related proteaseHomo sapiens0.542E-1201
RIN07055TGF-beta induced apoptosis protein 2Rattus norvegicus0.622E-5101
RIN07707methyl-CpG binding domain protein 6Rattus norvegicus0.611E-2401
RIN07765splicing factor 3 subunit 1Mus musculus0.552E-3501
RIN07973NADH-ubiquinone oxidoreductase chain 4LRattus norvegicus0.727E-2301
RIN08083signal recognition particle proteinMycoplasma mobile0.721E-2101
RIN08132forkhead box protein K1Homo sapiens0.661E-2601
RIN08211meningioma-expressed antigen 6/11Rattus norvegicus0.712E-2721
RIN08412hypothetical 45.0 kDa protein in NOT1-MATAL2 regionRattus norvegicus0.853E-7901
RIN09109TED proteinMus musculus0.875E-8503
RIN09205SRR1-like proteinHomo sapiens0.832E-4204
RIN0934130S ribosomal protein S6Mycoplasma pulmonis0.552E-1707
RIN09690valyl-tRNA synthetaseMycoplasma pulmonis0.875E-4005
RIN10182protein transport protein SEC61 gamma subunitRattus norvegicus0.75E-2004
RIN10530hypothetical protein KIAA0893Mus musculus0.819E-9313
RIN10621splicing factor, arginine/serine-rich 8Mus musculus0.515E-1501
RIN10814G protein-coupled receptor family C group 5 member CRattus norvegicus0.738E-4302
RIN11008tRNA-splicing endonuclease subunit SEN54Rattus norvegicus0.866E-9803
RIN11027protein yhgFHomo sapiens0.899E-9311
RIN11030p53-associated parkin-like cytoplasmic proteinRattus norvegicus0.849E-4401
RIN11408protein disulfide isomerase precursorRattus norvegicus0.747E-3501
RIN11741polyhomeotic-like protein 1Rattus norvegicus0.823E-5402
RIN11820translation initiation factor IF-1Leptospira interrogans0.749E-1201
RIN11856fructose-bisphosphate aldolaseMycoplasma mobile0.86E-5201
RIN11899nuclear protein Hcc-1Mus musculus0.722E-6702
RIN1202760S ribosomal protein L22Mus musculus0.812E-3901
RIN12420putative ATP-dependent RNA helicase T26G10Rattus norvegicus0.84E-8302
RIN12461synaptic vesicle membrane protein VAT-1 homologRattus norvegicus0.738E-6503
RIN12488glutaminyl-peptide cyclotransferaseRattus norvegicus0.787E-9002
RIN12608probable RNA-dependent helicase p72Rattus norvegicus0.648E-6202
RIN12715oligoendopeptidase F homologMycoplasma pulmonis0.688E-5801
RIN12733PSL10 proteinMus musculus0.777E-8001
RIN12794hypothetical protein ORF-1137Rattus norvegicus0.722E-3201
RIN12806protein HI1455Mycoplasma pulmonis0.747E-5003
RIN12897dimethyladenosine transferaseMycoplasma gallisepticum0.675E-3602
RIN12933zinc finger protein 23Mus musculus0.794E-6312
RIN13293serine hydroxymethyltransferaseRattus norvegicus0.654E-9301
RIN13306peripherinMus musculus0.71E-4201
RIN13714seryl-tRNA synthetaseMycoplasma pulmonis0.823E-6201
RIN13766UBA/UBX 33.3 kDa proteinRattus norvegicus0.651E-5401
RIN13865splicing factor 1Mus musculus0.736E-2302
RIN14137condensin subunit 1Mus musculus0.622E-3301
RIN14394Csr1Cricetulus griseus0.828E-1201
RIN14494hypothetical protein KIAA0117Rattus norvegicus0.777E-8101
RIN14758general transcription factor 3C polypeptide 5Rattus norvegicus0.731E-4811
RIN15622Crumbs protein homolog 1Mus musculus0.67E-0801
RIN16005hypothetical protein C19A8.09Rattus norvegicus0.894E-1102
RIN16184single-strand binding protein 1Rattus norvegicus0.676E-1311
RIN16330zinc finger protein 510Mus musculus0.832E-1902
RIN1639860S ribosomal protein L7Rattus norvegicus0.831E-3213
RIN16446single-strand binding proteinRattus norvegicus0.873E-1301
RIN16588putative adenosylhomocysteinase 2Homo sapiens0.892E-0702
RIN16592thymidine phosphoryleMycoplasma pulmonis0.663E-2001
Table 5

Genes abundantly identified either in islet or in RINm5F cells

IsletRINm5F
Genes with high (> 15 times) expression in either islet and RINm5F and none of the other, are listed.
Gene
Islet amyloid polypeptide (amylin)3210
CD74280
Follistatin-like protein (mac25)240
Mesothelin230
Major alpha-globin230
Insulin receptor-related receptor (IRR)180
Osteonectin170
Interferon-related developmental regulator 1053
DNA topoisomerase II alpha043
Proliferating cell nuclear antigen026
Glycosyl-phosphatidyl-inositol-anchored protein homolog024
Casein kinase 1 gamma 2 isoform022
Ran-GTPase activating protein 1021
Heat shock 70kD protein 5020
High mobility group protein 17017
Phosphoglycerate mutase type B subunit017
Synaptic regulatory protein RIM2beta017
Heat shock protein 60017
HLA-B associated transcript 2016
Secretory carrier membrane protein 3015
Figure 1
Figure 1

Expression profiles of the representative genes abundant either in islet (from Wistar rats) or in RINm5F cells. The expression levels of the genes were estimated by real-time quantitative RT-PCR using the TaqMan system. The mRNA for major α-globin could not be efficiently detected by the TaqMan system in its standard range for unknown reasons.

Citation: Journal of Molecular Endocrinology 35, 1; 10.1677/jme.1.01738

Figure 2
Figure 2Figure 2

(A) In situ hybridization of genes with high expression in islet and none in RINm5F cells (A). The representative results of seven highly expressed genes (> 15 times) and the insulin gene are shown. (B) In situ hybridization of genes with high expression in RINm5F cells and none in islet.

Citation: Journal of Molecular Endocrinology 35, 1; 10.1677/jme.1.01738

We thank S Oike, R Kawakami, Y Yaginuma, I Uda, Y Ibe, and T Takahashi for excellent assistance. This study was supported by Grant-in-Aid for Scientific Research and for Scientific Research on Priority Areas (C) “Medical Genome Science” from the Japanese Ministry of Science, Education, Sports, Culture and Technology and by a Health and Labor Science Research Grant for Research on Human Genome and Tissue Engineering from the Japanese Ministry of Health, Labor and Welfare, the Naito Foundation and the Yamanouchi Foundation. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

References

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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Export Citation

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  • View in gallery

    Expression profiles of the representative genes abundant either in islet (from Wistar rats) or in RINm5F cells. The expression levels of the genes were estimated by real-time quantitative RT-PCR using the TaqMan system. The mRNA for major α-globin could not be efficiently detected by the TaqMan system in its standard range for unknown reasons.

  • View in gallery View in gallery

    (A) In situ hybridization of genes with high expression in islet and none in RINm5F cells (A). The representative results of seven highly expressed genes (> 15 times) and the insulin gene are shown. (B) In situ hybridization of genes with high expression in RINm5F cells and none in islet.

  • AlbertsB Bray D Lewis J Raff M Roberts K & Watson JD 1994 The cell nucleus. In: Molecular Biology of the Cell. pp 335–400. New York: Garland.

  • AltschulSF Madden TL Schaffer AA Zhang J Zhang Z Miller W & Lipman DJ 1997 Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research253389–3402.

    • Search Google Scholar
    • Export Citation
  • Bernal-MizrachiE Cras-Meneur C Ohsugi M & Permutt MA 2003 Gene expression profiling in islet biology and diabetes research. Diabetes Metabolism Research Review1932–42.

    • Search Google Scholar
    • Export Citation
  • Bonner-WeirS & Sharma A 2002 Pancreatic stem cells. Journal of Pathology197519–526.

  • BradshawAD Graves DC Motamed K & Sage EH 2003 SPARC-null mice exhibit increased adiposity without significant differences in overall body weight. PNAS1006045–6050.

    • Search Google Scholar
    • Export Citation
  • ClaessonL Larhammar D Rask L & Peterson PA 1983 cDNA clone for the human invariant gamma chain of class II histocompatibility antigens and its implications for the protein structure. PNAS807395–7399.

    • Search Google Scholar
    • Export Citation
  • DorY Brown J Martinez OI & Melton DA 2004 Adult pancreatic β-cells are formed by self-duplication rather than stem-cell differentiation. Nature42941–46.

    • Search Google Scholar
    • Export Citation
  • EdlundH2002 Pancreatic organogenesis – Developmental mechanisms and implications for therapy. Nature Genetics Review3524–532.

  • FajansSS Bell GI & Polonsky KS 2001 Molecular mechanisms and clinical pathophysiology of maturity-onset diabetes of the young. New England Journal of Medicine345971–980.

    • Search Google Scholar
    • Export Citation
  • GazdarAF Chick WL Oie HK Sims HL King DL Weir GC & Lauris V 1980 Continuous clonal insulin- and somatostatin-secreting cell lines established from a transplantable rat islet cell tumor. PNAS773519–3523.

    • Search Google Scholar
    • Export Citation
  • HirayamaI Tamemoto H Yokota H Kubo SK Wang J Kuwano H Nagamachi Y Takeuchi T & Izumi T 1999 Insulin receptor-related receptor is expressed in pancreatic beta-cells and stimulates tyrosine phosphorylation of insulin receptor substrate-1 and -2. Diabetes481237–1244.

    • Search Google Scholar
    • Export Citation
  • HogeneschJB Ching KA Batalov S Su AI Walker JR Zhou Y Kay SA Schultz PG & Cooke MP 2001 A comparison of the Celera and Ensembl predicted gene sets reveals little overlap in novel genes. Cell106413–415.

    • Search Google Scholar
    • Export Citation
  • International Human Genome Sequencing Consortium 2001 Initial sequencing and analysis of the human genome. Nature409860–921.

  • JinL Wang H Narita T Kikuno R Ohara O Shihara N Nishigori T Horikawa Y & Takeda J 2003 Expression profile of mRNAs from human pancreatic islet tumors. Journal of Molecular Endocrinology31519–528.

    • Search Google Scholar
    • Export Citation
  • KayoT Sawada Y Suda M Konda Y Izumi T Tanaka S Shibata H & Takeuchi T 1997 Proprotein-processing endoprotease furin controls growth of pancreatic β-cells. Diabetes461296–1304.

    • Search Google Scholar
    • Export Citation
  • KitamuraT Kido Y Nef S Merenmies J Parada LF & Accili D 2001 Preserved pancreatic β-cell development and function in mice lacking the insulin receptor-related receptor. Molecular and Cellular Biology215624–5630.

    • Search Google Scholar
    • Export Citation
  • LeeNH Weinstock KG kirkness EF Earle-Hughes JA Fuldner RA Marmaros S Glodek A Gocayne JD Adams MD Kerlavage AR Fraser CM & Venter JC 1995 Comparative expressed-sequence-tag analysis of differential gene expression profiles in PC-12 cells before and after nerve growth factor treatment. PNAS928303–8307.

    • Search Google Scholar
    • Export Citation
  • MaH-T Kato M & Tatemoto K 1996 Effects of pancreastatin and somatostatin on secretagogues-induced rise in intracellular free calcium in single rat pancreatic islet cells. Regulatory Peptide61143–148.

    • Search Google Scholar
    • Export Citation
  • Mouse Genome Sequencing Consortium 2002 Initial sequencing and comparative analysis of the mouse genome. Nature420520–562.

  • PhilippeJ Chick WL & Habener JF 1987 Multipotential phenotypic expression of genes encoding peptide hormones in rat insulinoma cell lines. Journal of Clinical Investigation79351–358.

    • Search Google Scholar
    • Export Citation
  • Rat Genome Sequencing Project Consortium 2004 Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature428493–521.

    • Search Google Scholar
    • Export Citation
  • ScearceLM Brestelli JE McWeeney SK Lee CS Mazzarelli J Pinney DF Pizarro A Stoeckert CJ Jr Clifton SW Permutt MA Brown J Melton DA & Kaestner KH 2002 Functional genomics of the endocrine pancreas. The pancreas clone set and PanChip new resources for diabetes research. Diabetes511997–2004

    • Search Google Scholar
    • Export Citation
  • SchollerN Fu N Yang Y Ye Z Goodman GE Hellstrom KE & Hellstrom I 1999 Soluble member(s) of the mesothelin/ megakaryocyte potentiating factor family are detectable in sera from patients with ovarian carcinoma. PNAS9611531–11536.

    • Search Google Scholar
    • Export Citation
  • TakedaJ Yano H Eng S & Bell GI 1993 A molecular inventory of human pancreatic islets: sequence analysis of 1000 cDNA clones. Human Molecular Genetics21793–1798.

    • Search Google Scholar
    • Export Citation
  • VenterJC Adams MD Myers EW Li PW Mural RJ Sutton GG Smith HO Yandell M Evans CA Holt RA et al.2001 The sequence of the human genome. Science2911304–1351.

    • Search Google Scholar
    • Export Citation