text:
‘1-8 interferon inducible gene family’: putative colon carcinoma-associated antigens

Db−/−xβ2 microglobulin (β2m) null mice transgenic for a chimeric HLA-A2.1/Db-β2m single chain (HHD mice) are an effective biological tool to evaluate the antitumour cytotoxic T-lymphocyte response of known major histocompatibility-restricted peptide tumour-associated antigens, and to screen for putative unknown novel peptides. We utilised HHD lymphocytes to identify immunodominant epitopes of colon carcinoma overexpressed genes. We screened with HHD-derived lymphocytes over 500 HLA-A2.1-restricted peptides derived from colon carcinoma overexpressed genes. This procedure culminated in the identification of seven immunogenic peptides, three of these were derived from the ‘human 1-8D gene from interferon inducible gene’ (1-8D). The 1-8D gene was shown to be overexpressed in fresh tumour samples. The three 1-8D peptides were both antigenic and immunogenic in the HHD mice. The peptides induce cytotoxic T lymphocytes that were able to kill a colon carcinoma cell line HCT/HHD, in vitro and retard its growth in vivo. One of the peptides shared by all the 1-8 gene family primed efficiently normal human cytotoxic T lymphocyte precursors. These results highlight the 1-8D gene and its homologues as putative immunodominant tumour-associated antigens of colon carcinoma.

Supplementary approaches to surgery and chemotherapy promise better treatment to metastases. One such approach is specific active immunotherapy aimed at induction of anti-tumour cytotoxic T lymphocytes (CTL) . Upon appropriate vaccination, CTL constitute powerful effectors against tumours that present on their cell-surface major histocompatibility (MHC) class I molecules with bound tumour-associated antigenic (TAA) peptides .
Different types of TAA have been identified. Antigens that are uniquely expressed in tumours (Luftl et al, 2004); antigens derived from differentiation markers (Panelli et al, 2000); antigens arise from mutations that are common in tumours (McArdle et al, 2000); antigens from a viral source (Youde et al, 2004); and selfantigens that are overexpressed in tumours (Bar-Haim et al, 2004). In the latter category, although strong immune reactions against this type of antigens might result in the destruction of normal tissues, experience with peptide immunisation has not demonstrated high toxicity in humans and animals (Gross et al, 2004).
Thus far, little is known about target antigens for CTL in colorectal carcinoma. The few potential targets belong to the family of self overexpressed genes, such as her-2/neu, CEA and Ep-CAM (Melief et al, 2000). Therefore, there is a strong need to expand the armoury against colon carcinoma by discovering new TAAs (Nagorsen et al, 2000). Since the repertoire of peptides eluted from surface MHC class I molecules is highly similar between normal colon and colon tumours (Melief et al, 2000), we focused our efforts to identify novel colon carcinoma TAAs from colon carcinoma overexpressed genes.
We utilised the data of Zhang et al (1997) comparing transcripts of colon tumour tissue samples and normal tissues excised from the same patients. This study yielded a set of 26 overexpressed genes that are expressed in tumours at least five-fold higher than in normal tissue.
We applied the HHD model and showed that from over 500 putative TAA peptides derived from the 26 overexpressed genes in colon carcinoma, seven peptides were antigenic and immunogenic in HHD mice. Three of the seven were derived from 'human 1-8D from interferon inducible gene' (1-8D gene, IFITM2). These peptides elicit a CTL response against a colon carcinoma cell line in vitro and in vivo. One of the peptides shared by all members of the 1-8 interferon inducible gene family was highly immunogenic in human peripheral blood mononuclear cells (PBMCs). These results highlight 1-8D gene and its family as a novel colon carcinoma TAA.

Mice
The derivation of HLA-A2.1/D b -b2 monochain, transgenic, H-2D b xb2m double-knockout mice (named HHD mice) has been described by Pascolo et al (1997). CD1-nude mice were bred in the Weizmann Institute of Science (Rehovot, Israel). All protocols involving animals were in compliance with the Institutional Animal Care and Use Committee and in accordance with the guidelines of the Weizmann Institute of Science institutional animal care and use committee (IACUC). All the experiments, methods and facilities have been accredited by Ministry of Health: The Council for Experimentation on Animals, National Institute of Health: Office of Laboratory Animal Welfare and by Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). These accreditations meet the standards required by the UKCCCR guidelines.

Preparation of tumour extract peptides
Total acid-extracted peptides of colon tumour or of normal adjacent tissues were prepared from a pool of six post-surgical colon cancer specimens. Non-necrotic (1 -2 cm) tumour masses were homogenised in PBS, 0.5% Nonidet P-40, 10 mg ml À1 soybean trypsin inhibitor, 5 mg ml À1 leupeptine, 8 mg ml À1 aprotonin and 0.5 mM PMSF and homogenised using a glass-teflon homogeniser. Following further stirring for 30 min at 41C, the homogenates were titrated with 10% TFA to a final concentration of 0.1% TFA and stirred for another 30 min at 41C. After ultracentrifugation for 30 min at 42K r.p.m., the supernatants were applied to Sephadex G25 columns and fractions were monitored at OD 230 nm. Peptide fractions below 10 kDa were pooled, lyophilised and further fractionated by Centripep 3 centrifugation (Amicon, Beverly, MA, USA). Lyophilised samples were dissolved in sterile double distilled water, freed from TFA by repeated lyophilisation, and the relative concentration was assessed by measuring the OD at 230 nm. Following lyophilisation, the peptide pool was dissolved in optiMEM (Life Technologies, Paisley, UK) at 30 -50 OD at 230 nm per ml for further use.

Peptide synthesis
Peptides were synthesised on an ABIMED AMS 22 multiple peptide synthesiser (Abimed, Langenfeld, Germany), employing Fmoc strategy. Crude peptides were purified to homogeneity by semipreparative RP-HPLC. For the screening experiments, peptides were synthesised by multi-parallel synthesis (MPS) method adapted to 96-well plates, as was developed by Peptor Ltd (Rehovot, Israel). The peptides were purified by solid-phase extraction over C-18 Sep-Pak resin and analysed by VG platform, API-ESI MS (Fisons, UK). The average purity of the MPS peptides was about 80% as was analysed by RP-HPLC.

Vaccination
HHD mice were immunised i.p. three times at 7-day intervals with 2 Â 10 6 irradiated (5000 rad) tumour cells, or irradiated peptide-loaded RMA-S/HHD/B7.1 transfectants. Spleens were removed on day 10 after the last immunisation, and splenocytes were restimulated in vitro, either with irradiated tumour cells or with one-third of the lymphocytes prepulsed with 100 mM synthetic peptides in optiMEM (Invitrogen, Paisley, UK) for 2 h at 371C and 5% CO 2 . Restimulated lymphocytes were cultured in RPMI-HEPES medium containing 10% FCS, 2 mM glutamine, combined antibiotics, 1 mM sodium pyruvate, 25 mM HEPES, 5 Â 10 À5 M bME and 1% NEAA for 5 days. Viable cells (effectors) were separated by Lympholyte-M (Cedarlane, Hornby, ON, Canada) gradient, resuspended and admixed at different ratios with 5000 35 S-methioninelabelled target cells. Cytolytic assays were carried out as described previously (Carmon et al, 2000). Extracts of tumour peptides or normal colon peptides were prepared from a pool of six post-surgical colon carcinoma specimens as described before (Carmon et al, 2000).
To determine the threshold for immunogenicity of the individual peptides, we vaccinated two HHD mice with each of the peptides. CTL preparation was made and a cytolytic assay was performed using either the peptide itself or the tyrosinase epitopeloaded target cells as a negative control. We defined the ratio of the specific lysis between the two targets as the 'immunogenic score', which expressed as percent of the control lysis. The lysis of the tyrosinase-loaded target cells was between 10 -20%. We averaged the control lysis results of all the assays (total 44 measurements in triplicates) and defined the threshold as 95% confidence level above the average. This resulted in an immunogenic score of 125% (a horizontal line at this level was added to Figure 2A). Thus, each peptide that yielded an average immunogenic score of two independent experiments above 125% was rendered immunogenic.

RNA extraction and real-time PCR
Total RNA from human tumour and normal colon tissues was isolated by TriReagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacture's instructions. Colon tissues were obtained from Barzilai Medical Center (Ashkelon, Israel) from colon cancer patients according to the Declaration of Helsinki Principles. Reverse transcription was performed on 5 mg of total RNA with an oligo (dT) primer using Superscriptt II (Invitrogen). Relative expression of human 1-8D gene in human tumour and normal colon tissues was detected using the Roche LightCycler FastStart DNA Master SYBR Green I (Roche, Mannheim, Germany). b-Actin gene was used as loading control. Primers were designed to specifically amplify human 1-8D gene: forward primer for human 1-8D: 5 0 -CCTTGACCTGTATTCCA CT-3 0 , reverse primer for human 1-8D: 5 0 -GCCATTGTAGAAAA GCGT-3 0 , these amplify a 102-bp region of the gene. For b-actin, forward primer was 5 0 -GGCATCCACGAAACTAC-3 0 and reverse primer 5 0 -GCTCAGGAGGAGCAAT-3 0 ; these amplify a 209-bp region of the gene. Each reaction was carried out in triplicates in a total volume of 20 ml in glass capillaries containing 5 ml of cDNA sample, 3 mM MgCl 2 , 0.5 mM each primer and 2 ml of LightCycler -FastStart Reaction Mix SYBR Green I mixed with LightCycler FastStart Enzyme. The thermocycling programme consisted of denaturation at 951C for 10 min, followed by 35 -45 cycles of PCR (951C for 5 s, 601C for 7 s, 721C for 11 s and a single fluorescence detection point at 851C for human 1-8D and at 871C for b-actin, with transitions programmed at 201C s À1 ). Melting curves were generated by first heating to 951C, then cooling to 601C for 15 s, and finally heating at 0.11C s À1 to 951C with continuous fluorescence acquisition. Programme was followed by a 30 s 401C. In each case, product identity was demonstrated by the presence of a single peak on derivate melting curve plots, using the LightCycler software (Roche), and a single band of the appropriate size on gel electrophoresis, which corresponded to the sequence in the database (accession number: X57351).
The PCR fragment was cloned into pcDNA3.1/Hygro( þ ) vector between HindIII and XhoI restriction sites. Myc tag was added at the C terminus by eliminating the stop codon. Positive clones were selected by intracellular staining using Cytofix/Cytoperm Plus kit (Pharmingen, San Diego, CA, USA) using anti-Myc (9E10) mAb (Santa Cruz Biotechnologies, Santa Cruz, CA, USA).

Adoptive transfer
CD1-nude mice were challenged in the footpad with 3 Â 10 6 HCT/ HHD cells. Three days later, 10 7 viable restimulated effector cells were injected i.v. to the tail vein. One thousand units of IL-2 were injected i.p. twice a day for 7 consecutive days post transfer. Tumour growth was monitored twice a week by measuring the diameter of the footpad with calipers. Mice were killed when one of the tumour diameters reached 10 mm.

In vitro priming of human CTL
Leukapheresis products of three healthy donors were obtained from Barzilai Medical Center according to the Declaration of Helsinki Principles. Peripheral blood mononuclear cells were isolated by centrifugation on Ficoll -Plaque Plus gradients (Amersham, Uppsala, Sweden). The procedure was carried out according to Thurner et al (1999) with minor modifications.

HCT/HHD and colon carcinoma tumour extracts elicit cross-reactive CTL responses in HHD mice
Successful screening of antigenic peptides relies upon reproducible screening procedures. The CTL responses obtained to vaccinations of the HHD mice with low molecular weight extracts of colon tumours varied from preparation to preparation (data not shown). Therefore, we decided to vaccinate HHD mice with a cellular preparation. After screening several colon carcinoma cell lines, we found that the HCT-15 cell line does not express any HLA class I molecules and therefore may serve as a convenient platform for introduction of the single-chain HHD ( Figure 1A, lower left panel). We established HCT-15 cells that highly express the HHD and murine B7.1 ( Figure 1A, upper panels) as potent inducers of CTL responses in HHD mice ( Figure 1B, panel b). We tested the ability of CTL raised against HCT/HHD/B7.1 cells to identify antigens extracted from a pool of six colon tumour specimens (tumour extract (TE)). HHD mice were vaccinated with irradiated HCT/ HHD/B7.1 cells, spleens were removed and cytolytic activity was assayed on several targets ( Figure 1B). We performed a reciprocal experiment, in which HHD mice were vaccinated with RMA-S/ HHD/B7.1 loaded with the colon-derived TE and cytolytic activity was measured on the indicated targets ( Figure 1C). HCT/HHD/ B7.1 elicits a powerful CTL response against itself in an HHDrestricted manner (compare HCT/HHD vs HCT). The CTL preferentially identified TE loaded on target cells but not cells loaded with either normal extract (NE) (prepared from a pool of normal colon), or tyrosinase-derived peptide, as a negative control ( Figure 1B, panel a). When RMA-S/HHD/B7.1 cells loaded with TE were used to induce a CTL response, HCT was lysed in an HHDrestricted manner. The response mounted against NE and tyrosinase was approximately 20% lower than the response against TE ( Figure 1C, Po0.05). These experiments demonstrate a significant overlap between HCT antigens and antigens extracted from colon carcinoma tumour specimens. Thus, establishing vaccination with HCT/HHD/B7.1 as an immunological model for human colon carcinoma.
Twenty-two peptides are antigenic to anti-HCT/HHD/B7.1 effector cells We focused on putative non-secreted 26 genes (Table 1), previously reported to be overexpressed at least five-fold in colon tumour tissues (Zhang et al, 1997). The protein sequences were screened for putative HLA-A2.1-restricted peptides using the 'independent binding of individual peptide side-chains' software (Parker et al, 1994). We selected high-and medium-affinity HLA-A2.1-restricted peptides (score over 1). Overall, 503 peptides were synthesised by the MPS synthesis technology and loaded on 35 S-labelled RMA-S/HHD/B7.1 to serve as targets for CTL. Splenocytes of HCT/HHD/B7.1-immunised animals were incubated with the target cells in a 50 : 1 effector to target ratio. Targets that were specifically lysed by more than 10% than the negative control in two consecutive experiments were regarded a positive hit. A list of the antigenic peptides and their position in the protein of their origin is shown in Table 2.
Seven peptides derived from overexpressed genes are immunogenic in HHD mice Next, the antigenic peptides were tested for their specific immunogenicity in HHD mice. HHD mice were immunised with RMA-S/HHD/B7.1 cells loaded with the individual peptides. A cytolytic assay was performed on the peptide itself loaded on labelled RMA-S/HHD or tyrosinase peptide, and the immunogenic score was determined. These experiments were repeated and the average score obtained for each peptide is shown in Figure 2A. Threshold was set as explained in Materials and Methods. According to this criterion, seven peptides showed significant immunogenicity in the HHD mice. Three out of the seven peptides, 1 -6, 3 -5 and 3 -7, The genes were selected according to the following rules: (1) candidate genes with secretion sequences were excluded; (2) candidate gene must be overexpressed in tumours at least five-fold higher than in normal tissue. HLA-A2.1-restricted peptides from the selected genes were selected according to their consensus binding motifs. The 1-8D-derived peptides are bold and underlined.

The peptides of '1-8D gene' induce anti-HCT/HHD CTL responses
Intrigued by this finding, we decided to focus on the 1-8D peptides.
Using an HLA-A2 stabilisation assay on T2 cells (Firat et al, 1999), we verified the binding of the 1-8D peptides to HLA-A2.1 ( Figure 2B), the 1-8D peptides bind to HLA-A2.1, although to a lower extent than for the tyrosinase peptide, a strong HLA-A2.1 binder. We next tested 1-8D peptides for induction of anti-HCT/HHD CTL responses. HHD mice were vaccinated with the 1-8D peptides as previously described (Carmon et al, 2000). As a negative control, we used an HIV-derived peptide, TLNAWVKVV. 1-8D peptides mount more than twice higher CTL responses than the HIV peptide at a 50 : 1 effector to target ratio ( Figure 2C). We conclude that 1-8D peptide elicits anti-HCT CTL responses.
The 1-8D gene is overexpressed in primary colon tumour The relative expression of the human 1-8D gene in colon tumour tissues vs normal tissues was measured by real-time PCR. An internal standard curve was generated by amplification of fourfold dilution of cDNA for each gene in each run. Based on the cycle number, when the fluorescence detected increased over the noise or base line (crossing point), the linear regression curve was calculated. The correlation coefficient r was À1.00, the error was as well minimal, and the slope near to À3.33. Expression level of b-actin was used for normalisation. Results are expressed as 1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 3-4 3-5 3-6 3-7 1-4 1-5 1-6 1-7 1-8 1-9 1-10 1-12 1-11 Figure 2 (A) Seven peptides are immunogenic. Peptides were loaded on RMA-S/HHD/B7.1, washed and irradiated. For each peptide, two HHD mice were immunised three times as described in Materials and Methods. In vitro cytolytic assays were performed on the relevant peptide as target and on an irrelevant peptide, and the immunogenic score was calculated. The positive peptides (above 125%) are marked by arrows. Peptides 1 -6, 3 -5 and 3 -7 are derived from 'human 1-8D gene'. (B) 1-8D peptides stabilise HLA-A2 on T2 cells. T2 cells were acid stripped and incubated overnight with the indicated peptide and recombinant b2 or with b2m alone as a negative control. Cells were stained with BB7.2 mAb and analysed by flow cytometry. The number of positive cells above control level is indicated for each of the peptides. (C) 1-8D peptides can mount anti-HCT/HHD CTL responses. HHD mice were immunised with HCT/HHD/B7.1 and with peptides loaded on RMA-S/HHD/B7.1. In vitro cytolytic assays were performed using HCT/HHD cells as targets. The specific lysis at a 50 : 1 effector to target ratio of a representative experiment out of three is shown. P a t ie n t 1 0 P a t ie n t 2 0 P a t ie n t 1 4 P a t ie n t 1 3 P a t ie n t 1 2 P a t ie n t 1 8 P a t ie n t 8 P a t ie n t 1 6 P a t ie n t 1 7 P a t ie n t 1 9 P a t ie n t 1 5 P a t ie n t 7 P a t ie n t 9 Normal Tumour Relative expression Figure 3 The 1-8D gene is overexpressed in tumour colon tissues. The relative expression of human 1-8D gene in tumour and normal colon tissue was determined using real-time PCR. Expression level of b-actin was considered equal in both normal and tumour samples and was used for normalisation while calculating the expression level of the 1-8D gene. Average±s.d. is presented. relative expression level for human 1-8D. Human 1-8D gene is overexpressed in 9 samples out of 13 that were tested (Figure 3). These findings indicate that the human 1-8D gene is a bona fide overexpressed gene in tumour colon tissues.
Adoptively transferred anti-1-8D peptide lymphocytes inhibit the growth of HCT/HHD in nude mice To check whether activation of lymphocytes induced by the 1-8D peptides harbours an immunotherapeutic potential, HCT/HHD cells were inoculated to the footpad of nude mice. Three days post footpad inoculation, restimulated HHD lymphocytes were transferred i.v. Transfer of anti-1-8D peptide CTL caused significant retardation in tumour growth. Retardation was detected for peptide 3 -5 starting from day 11 (Po0.05), and for peptides 1 -6 and 3 -7 starting from day 13 (Po0.05) applying one-tail Student's t-test ( Figure 4B).

Peptide 3 -7 activates peripheral CTL precursors in normal human PBMCs
Finally, we checked peripheral blood of three healthy individuals for the presence of CTL precursors (CTLp). Peptide 3 -7 primed effectively CTL in lymphocytes from donors A and B and to a  Figure 4 (A) 1-8D peptides mount CTL responses that lyse 1-8D transfectants. Each peptide was loaded on RMA-S/HHD/B7.1 and used to immunise HHD mice. In vitro cytolytic assays were performed using EL4/HHD cells (open circles) and EL4/HHD/1-8D-transfected cells (dark circles) as a target. The specific lysis of a representative experiment out of three is shown. (B) HCT/HHD growth is inhibited by vaccination with 1-8D peptides. Nude mice were challenged in the footpad with HCT/HHD cells. Lymphocytes from vaccinated HHD mice were transferred together with IL-2 to the tumour-bearing mice and the growth of the tumour was monitored twice a week. Statistical significance was achieved for peptides 1 -6 and 3 -7 from day 13 and for 3 -5 treatment from day 11 (Student's t-test, Po0.05). (C) 1-8D peptide primes in vitro normal CTLp. Peripheral blood mononuclear cells of three leukapheresis samples were isolated and in vitro priming was performed with peptide-pulsed autologus DC. Peripheral blood mononuclear cells were supplemented with IL-7, and 2 days later IL-2 was added and renewed every 3 days. The rest of the stimulations were performed every 7 days over peptide-pulsed monocytes. Seven days after the third stimulation, lymphocytes were harvested and a cytolytic assay was performed using peptide-pulsed or non-pulsed T2 cells as targets. ', CTLp from donor A; K, CTLp from donor B; n, CTLp from donor C; and J, non-pulsed T2 cells.
lesser extent in lymphocytes from donor C. Peptide 1 -6 primed CTL in lymphocytes of donor B alone and peptide 3 -5 primed CTL in lymphocytes of donors B and C. Unfortunately due to paucity of PBMCs of donor A, this was not tested with peptide 3 -5 ( Figure 4C).

DISCUSSION
One approach to identify human tumour-associated HLA class Irestricted peptides utilises CTL lines derived from patients (Heidecker et al, 2000). This approach suffers from several caveats: establishment of CTL lines is a complicated procedure; CTL lines derived from cancer patients may represent the repertoire of an anergised immune system; finally, propagation of CTL lines in vitro might reflect sporadic clones surviving culture conditions rather than authentic antitumour clones. In order to bypass these pitfalls and to obtain pure HLA-A2-restricted CTL responses, we used the HHD mice (Pascolo et al, 1997). We combined a reverse immunology approach together with a screening procedure that utilises HHD lymphocytes to identify immunodominant epitopes of colon carcinoma-overexpressed genes.
The HCT-15 cell line that possess high-tumorigenic properties was modified to be a suitable antigen-presenting cell by transfection with the single-chain HHD and the costimulatory B7.1 molecule (Cayeux et al, 1997) ( Figure 1A). These cells were used to immunise HHD mice, as a source of CTL for epitope screening. It was important to demonstrate that HCT/HHD/B7.1 elicits a CTL response that overlaps the response obtained with TE to ensure physiological significance of the screen ( Figure 1B).
We found that HCT/HHD/B7.1 elicits strong anti-self, HHDrestricted CTL response that recognises preferentially the TE ( Figure 1B). Furthermore, CTL of mice, immunised with RMA-S/ HHD/B7.1 cell-loaded TE, show high-specific lysis (over 60%) of HCT/HHD. The lysis was five-fold higher than of parental cells. These results suggest that processing and presentation of HLA-A2.1-restricted and colon-associated peptides are performed by the HCT/HHD colon carcinoma cells. Likewise, the high-specific lysis of HCT/HHD transfectants indicates an overlapping peptide repertoire between the TEs and the cell line.
Two-fold preferential lysis of colon-derived tumour peptide-vs normal tissue peptide-loaded targets was observed upon vaccination with colon TE peptides. This observation supports the existence of a window of specificity, towards which tumour immunotherapy can be directed.
We selected 26 genes of putative non-secreted proteins that are expressed at least five-fold higher in tumours than in normal colon (Table 1). These genes were analysed for HLA-A2.1-restricted peptides. We screened over 500 HLA-A2.1-restricted peptides and we found that in a cytolytic assay lymphocytes recognised 22 peptides, rendering them antigenic to the lymphocytes.
Next, we narrowed down the antigenic peptides to those that are also immunogenic. HHD mice were vaccinated with each of the individual peptides, and lymphocytes were assayed for lysing target cells, loaded with either the peptide itself or an irrelevant peptide. Interestingly, only seven peptides could mount a significant CTL response in HHD mice (Figure 2A). Three of the seven were derived from the 1-8 family of interferon inducible proteins, highlighting these gene products as novel TAA. Binding of the peptides to HLA-A2 was confirmed by performing a stabilisation assay on T2 cells ( Figure 2B).
The 1-8 gene family is inducible by both type I and type II interferons (Lewin et al, 1991). Three members of the family: 1-8D, 1-8U and 9-27 (also called IFITM2, IFITM3 and IFITM1, respectively) are linked on an 18-kb fragment of chromosome 11 and are highly homologous (Lewin et al, 1991). The 9-27 was shown to be an integral membrane protein identical to the Leu-13 antigen. It forms a complex with other proteins and is implicated in cell adhesion and growth inhibitory signals (Deblandre et al, 1995). The 1-8U is expressed in colitis-associated colon cancer and in severely inflamed mucosa in ulcerative colitis (Hisamatsu et al, 1999), as well as in an androgen-independent variant of the prostate carcinoma line LNCaP, but not in an androgen-dependent variant (Vaarala et al, 2000). Interestingly, 1-8D and 9-27 are induced by radiation in a p53-negative line, in an interferonindependent mechanism (Clave et al, 1997). The expression of 1-8 gene family is induced in a human adrenocortical carcinoma cell line in response to angiotensin II treatment, implicating other signalling pathways to control their expression (Daido et al, 2001). Moreover, it has been demonstrated that IFITM1 activity is required for primordial germ cell (PGC) transient from the mesoderm into the endoderm and that IFITM3 is sufficient to confer autonomous PGC-like homing properties to somatic cells (Tanaka et al, 2005).
We further characterised the 1-8D peptides as potential antigens for vaccination against colon cancer. Curiously, peptide 3 -7, the most immunogenic peptide in HHD mice harbours a cysteine residue in its sequence. Moreover, the cysteine residue is situated at the anchor position of HLA-A2.1 restriction pattern, which shows preference to aliphatic amino acids like leucine. In congruent to the study of Chen et al (1999), mutation of the cysteine to leucine enhanced the immunogenicity of the peptide albeit not affecting the binding to HLA-A2.1 (data not shown).
An anti-HCT/HHD CTL response was obtained upon vaccination of HHD mice with either of 1-8D peptides with respect to the HIV-derived peptide ( Figure 2C). The recognition of HCT/HHD by anti-1-8D lymphocytes further supports the hypothesis of 1-8D immunodominance in this model.
We elaborated our analysis to tumour specimens and found 1-8D to be overexpressed in 9 out of 13 fresh tumour samples as compared to adjacent normal tissue using real-time PCR (Figure 3). These results establish 1-8D as a bone fide overexpressed gene in colon cancer.
EL4/HHD cells were transfected with 1-8D. HHD mice were vaccinated with either of the 1-8D-derived peptides and the susceptibility of EL4/HHD and EL4/HHD/1-8D cells for lysis by the active lymphocytes was measured. Despite the high background, the 1-8D transfectant was better lysed than EL4/HHD cells, by CTL restricted to peptides 1 -6 and 3 -7, while peptide 3 -5 did not show 1-8D preference. This fact indicates successful processing and presentation of 1 -6 and 3 -7. Presentation of 3 -5 was not confirmed by this assay ( Figure 4A). Mixing the anti-1-8D peptide lymphocytes prior to incubation with the targets did not improved the lysis of the target cells, but rather resulted in an average lysis (data not shown). This observation suggests that the three peptides are derived from a single protein, and presented on MHC class I at similar levels.
We further examined whether activation of lymphocytes induced by the 1-8D peptides harbours an immunotherapeutic potential. We established an adoptive transfer model in which CTL to 1-8D peptides retarded the growth of HCT/HHD ( Figure 4B). In this experiment, peptide 3 -7 was found to be the least effective, although statistical significance between the various 1-8D peptides was not achieved. Nonetheless, this result is in concordance with the in vitro cytolytic capacity of HCT/HHD that ranked peptide 3 -5 as the most effective vaccine against HCT/HHD ( Figure 2C).
Owing to the strong genetic and familial aetiology of colon cancer, this cancer is appropriate to prophylactic treatment (Severin, 1999;Lindblom, 2001). Hence, we were interested to assess the potential of 1-8D peptides to prime peripheral CTL of normal HLA-A2.1-positive donors. Peripheral blood mononuclear cells of three normal donors were isolated and assayed for the existence of CTLp by in vitro stimulations with autologus DCs. Peptide 3 -7 was found to be highly efficient in activating peripheral CTLp to lyse target cells in a peptide-specific manner for donors A and B and to a lesser extent for donor C. Peptide 1 -6, however, primed CTL in donor B alone and peptide 3 -5 primed CTL in donors B and C, yet with less efficiency than peptide 3 -7 ( Figure 4C). This might reflect specific gaps in the T-cell repertoire in the donors' peripheral blood that might result from expression of additional HLA alleles. CTL precursors for all peptides were found in the peripheral blood of donor B, indicating no tolerance to peptides 1 -6 and 3 -5 ( Figure 4C). Peptide 3 -7 is shared between the three members of the 1-8 gene family, while the other peptides (1 -6 and 3 -5) are unique for the 1-8D gene. The high immunogenicity of peptide 3 -7 in the peripheral blood might be influenced from the contribution from the three distinct genes, as opposed to the two other peptides that derive from a single gene. Additional experiments with PBMCs of different donors are needed to account for immunogenicity of the 1-8D peptides in human.
Our study unravels for the first time immunodominant CTL epitopes of the 1-8D gene family. Based on the interferon regulation of these genes, and the tight connection of colon carcinogenesis to inflammation (Lewin et al, 1991), we propose the 1-8D genes as potential targets for a CTL-mediated immunotherapy of colon carcinoma.


extfieldsofstudy: Biology Medicine

text:
Housekeeping and tissue-specific genes in mouse tissues

Background This study aims to characterize the housekeeping and tissue-specific genes in 15 mouse tissues by using the serial analysis of gene expression (SAGE) strategy which indicates the relative level of expression for each transcript matched to the tag. Results Here, we identified constantly expressed housekeeping genes, such as eukaryotic translation elongation factor 2, which is expressed in all tissues without significant difference in expression levels. Moreover, most of these genes were not regulated by experimental conditions such as steroid hormones, adrenalectomy and gonadectomy. In addition, we report previously postulated housekeeping genes such as peptidyl-prolyl cis-trans isomerase A, glyceraldehyde-3-phosphate dehydrogenase and beta-actin, which are expressed in all the tissues, but with significant difference in their expression levels. We have also identified genes uniquely detected in each of the 15 tissues and other tissues from public databases. Conclusion These identified housekeeping genes could represent appropriate controls for RT-PCR and northern blot when comparing the expression levels of genes in several tissues. The results reveal several tissue-specific genes highly expressed in testis and pituitary gland. Furthermore, the main function of tissue-specific genes expressed in liver, lung and bone is the cell defence, whereas several keratins involved in cell structure function are exclusively detected in skin and vagina. The results from this study can be used for example to target a tissue for agent delivering by using the promoter of tissue-specific genes. Moreover, this study could be used as basis for further researches on physiology and pathology of these tissues.

Background
Housekeeping genes are constitutively expressed in all tissues to maintain cellular functions [1]. Moreover, they are presumed to produce the minimally essential transcripts necessary for normal cellular physiology [2]. In the validation of GeneChips using real-time RT-PCR, a standard curve of a reference housekeeping gene is thus sufficient for the quantification of multiple genes in a tissue. This is especially advantageous for the validation of GeneChip results because multiple genes are quantified in one tissue sample [3]. However, the expression level of the housekeeping genes may vary among tissues or cells and may change under certain circumstances. On the other hand, the highly specific tissue expression of a gene indicates that the gene performs a tissue-specific function. A feature of housekeeping genes is that, in comparison to tissuespecific genes, they evolve on average more slowly [4]. In addition, the methylation is one of the major epigenetic modifications of DNA in mammalian cells, regulating tissue-specific genes as well a housekeeping genes [5]. Furthermore, the differences in the chromatin features between specific genes and housekeeping genes indicate the involvement of chromatin organisation in the control of gene expression [6]. In addition, despite the most fundamental characteristics of housekeeping genes and specific genes, no previous study has quantified the expression level of the housekeeping genes and the tissuespecific genes in a variety of tissues. Using serial analysis of gene expression (SAGE) strategy which indicates the relative level of expression of each transcript matched to a tag, this study represents, to our knowledge, the first characterization of the tissue-specific genes and housekeeping genes in an extensive array of tissues and organs. Using SAGE method, previous studies have characterized the transcriptome of numerous tissues such as uterus [7], adipose tissue [8,9], skeletal muscle [10], hypothalamus, cerebral cortex and pituitary gland [11,12].
The invention and application of SAGE method have paralleled those of microarray/chip technologies. Whereas hybridization-based technologies may allow for shorter detection times and high throughput expression analysis, the SAGE method not only identifies unknown genes but also quantifies the gene expression level relatively to the total mRNA population. Indeed, the SAGE method can be performed to accurately measure the abundance of both known and novel transcripts on global scale [13]. This method is ideal to analyze a large number of transcripts in a given tissue, and it allows the quantitative cataloguing and comparison of expressed genes under various physiological and pathological states [13][14][15].

Number of tags and tag species analyzed
A total count of 1,834,621 SAGE tags were analyzed for the 15 tissues representing 320,624 tag species. For each tissue, approximately 130,000 tags were sequenced, except for testis, ovary, mammary gland, vagina and bone which had approximately 50,000 tags.
This study has identified 1,111 ubiquitously expressed transcripts. These genes are expressed in all the tissues and therefore are likely candidates as the genes responsible for cellular maintenance also known as housekeeping genes. Among the ubiquitous genes identified, 280 genes are constantly expressed in all tissues. The rest (831 transcripts) are not expressed at the same level in all the 15 tissues. The 280 transcripts are detected at similar level in each of the 15 tissues and can be useful as a set of controls.
In the Table 1, we present the expressions levels of the top 10 constantly expressed housekeeping genes, which are expressed in all tissues without significant difference in expression levels and the previously postulated housekeeping genes. To see if the ratio of the housekeeping genes changes in another condition, we have performed biologically relevant experiments. The data presented in the Table 1 have shown that most of these housekeeping genes are not only constant between intact tissues, but also between tissues that have been subjected to hormones such as dihydrotestosterone (DHT) and glucocorticoid (Gcc), as well as gonadectomy (GDX) and adrenalectomy (ADX) conditions. We have investigated the effects of GDX and DHT in prostate, mammary gland, uterus, vagina, adipose tissue and skeletal muscle as well as the regulation of ADX and Gcc in lung, hypothalamus and pituitary gland. Only ribosomal protein L37 and leukocyte receptor cluster (LRC) member 8 were regulated by GDX compared to the intact mice in mammary gland and prostate, respectively. On the other hand, only the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a previously postulated housekeeping gene, was regulated by DHT in adipose tissue and uterus compared to GDX mice. Remarkably, eukaryotic translation elongation factor 2 (eEF-2) was highly and constantly expressed in all the investigated tissues. Despites their moderate levels of expression, proteasome (prosome, macropain) 26 subunit non-ATPase 4, eukaryotic initiation factor 3 (eIF3), and ribosomal protein L38 are constantly expressed in all the tissues. We have also identified three RIKEN cDNAs constantly expressed in all the tissues. The results have also shown that, the previously postulated housekeeping genes namely peptidyl-prolyl cis-trans isomerase A (PPIase), GAPDH, beta-actin are not constantly expressed in all the tissues.

Most expressed tissue-specific genes in the 15 tissues
We have also identified the genes uniquely detected in each of the 15 tissues. These genes are critical for the specific functions that characterize and distinguish the testis, prostate, ovary, mammary gland, uterus, vagina, skin, liver, adipose tissue, lung, bone, skeletal muscle, cerebral cortex, hypothalamus, and pituitary gland. The specific genes are expressed in only one tissue. Table 2 presents the transcripts exclusively detected in the male sexual organs. Obviously, the levels of gene expression in these tissues were statistically different from the other tissues. The current study shows 61 tissue-specific transcripts in the testis. Despite their moderate expression in the testis, some transcripts such as dipeptidase 3, ankyrin repeat domain 5, and ubiquitin-conjugating enzyme E2N are exclusively found in the testis. In contrast, only 7 transcripts are detected in the prostate as specific genes. Noteworthy, seminal vesicle protein secretion 2 is both highly and exclusively detected in the prostate. According to the tran- scripts specifically detected in the female sexual organs (Table 3), most of the tissue-specific genes are expressed at lower quantity in the ovary, mammary gland, uterus and vagina in comparison to the majority of specific genes expressed in the male sexual organs. Remarkably, prolinerich acidic protein 1 and the novel transcript with the sequence tag CTGTATTTGGG are both highly and uniquely detected in the uterus and the vagina, respectively. The Table 4 shows the transcripts exclusive to the skin, liver, adipose tissue, lung, bone and skeletal muscle. The majority of tissue-specific genes expressed in the skin are involved in cell structure. Keratin associated protein 8-1 is more expressed than the other tissue-specific genes detected in the skin. We report 26 transcripts exclusively detected in the liver. Moreover, two tissue-specific genes namely albumin 1 and alpha microglobulin/bikunin are also highly expressed in the liver. Despite their low expression in the liver, major urinary protein 1, solute carrier family 27 (fatty acid transporter) member 2 and silicainduced gene 111 are also specifically detected. Furthermore, the tissue-specific transcripts expressed in the lung and the bone are involved in cell defence and we have observed the high expression of the tissue-specific genes such as proteoglycan 2 bone marrow and solute carrier family 4 (anion exchanger) member 1 in the bone. The adipose tissue and the skeletal muscle expressed less tissue-specific genes than the other tissues. Only two transcripts namely leptin and lectin galactose binding soluble 12 are reported as candidate specific genes in the adipose tissue. The transcripts such as myosin light chain phosphorylatable fast skeletal muscle and tropomyosin 3 gamma are both moderately and uniquely detected in the skeletal muscle. Among the brain areas investigated, the pituitary gland expressed more tissue-specific transcripts (Table 5). In addition, the majority of these genes are also highly expressed in this gland. We found 3 genes expressed in cerebral cortex, which were not detected in any of the other 14 tissues. In the hypothalamus, RIKEN cDNA A230109K23 gene is both exclusively and highly detected. To see if the tissue-specific genes were detected in other tissues, we have compared our results to the public data deposited in Gene Expression Omnibus (GEO, NCIBI). The public data have confirmed the exclusive detection of tissue-specific genes in each tissue. The transcripts which were specifically detected in the 15 intact tissues were not expressed in other mouse tissues such as embryonic fibroblasts, heart, thymus, bladder, kidney, spleen and pancreas ( Table 2 to 5). In addition, using Uni-Gene and EST expression information in the public databases, the normalized expression (%) of intestine is shown and is compared to our results in Table 2 to 5. All the genes mentioned for the uterus, as well as carbonic anhydrase 1, retinol binding protein 2 cellular and keratin complex 1 acidic gene 13 were also expressed in the intestine tissue.

Discussion
The most abundant housekeeping genes Quantitative gene expression data are often normalized to the expression levels of control or so-called housekeeping genes. An inherent assumption in the use of housekeeping genes is that expression of the genes remains constant in the tissues under investigation. Housekeeping genes do not vary in their expression levels during cell development, treatment, or disease state anomalies [16]. About 40 years ago, housekeeping genes were simply defined as those genes that are always expressed [1], also known as ubiquitous genes. However, for many experimental applications, we must make the difference between the housekeeping genes which are constantly expressed in all the tissues and the ubiquitous genes. According to the current study, the ubiquitous genes also previously called housekeeping genes such as PPIase, GAPDH, and beta-actin are expressed in all tissues with significant difference in their expression levels. On the other hand, the housekeeping genes eEF-2, ribosomal protein L37 and L38, proteasome (prosome, macropain) 26 subunit non-ATPase 4, eIF3, hypothetical protein, LRC member 8 also known as interleukin-8 receptor (IL-8R), and the RIKEN cDNA C030036P15, 1500016L11 and 2410104I19 genes are constantly expressed in all the tissues investigated. Our results are consistent with the previous report that ribosomal protein L37, proteasome and hypothetical protein are expressed at the same level in 11 human adult and fetal tissues [17]. In addition, except LRC member 8 and the RIKEN cDNAs, the transcripts reported as housekeeping genes in this study have also been previously presented as maintenance genes in a study investigating 11 human adult and fetal tissues [17]. Using the SAGE method, Velculescu et al also identified the same ubiquitous transcripts among nearly 1000 transcripts expressed at more than or equal to 5 copies par cell from 19 human tissues [18]. However, they did not apply the statistics to identify the constantly expressed genes. In the current study, we report eEF-2 as the housekeeping gene which is abundantly and constantly transcribed in all of the 15 tissues. Indeed, eEF-2 is known to be required for elongation in most eukaryotes [19]. Since eEF-2 does not vary in relative abundance in different tissues, this gene can be used as a standard or internal control. The constitutive expressions of ribosomal proteins L37 and L38 were observed in all the tissues. Our results are also consistent with the report that ribosomal protein L37 is constitutively expressed during transitions from quiescence to active cell proliferation or terminal differentiation in all tissues in rat and human [20]. Another study has shown that ribosomal protein L37 ranks amongst the fifteen most highly expressed housekeeping genes [21]. Furthermore, the present study identified LRC member 8 and eIF3 as housekeeping genes. LRC is known to play an important role in cell defence in all tissues. In mammals, the cell surface          receptor encoded by LRC member 8 regulates the activity of lymphocytes and natural killer cells in order to provide protection against pathogens and parasites [22]. Previous studies have presented interleukin-12 receptor beta 2 [18] and interleukin-1 receptor-associated kinase [17] as ubiquitously expressed genes. No previous study had investigated LCR member 8 as a housekeeping gene or internal control. According to this study, we suggest for the first time interleukin-8 receptor as housekeeping gene or internal control since this transcript is ubiquitously expressed in all the tissues investigated. Proteasome (prosome, macropain) 26 subunit non-ATPase 4, the major proteolytic machinery responsible for degradation of both normal and damaged proteins has been identified as housekeeping gene. Our results are in agreement with the knowledge that proteasome is a multicatalytic complex found in all eukaryotic cells [23], and also with the report that proteasome ranks amongst the fifteen most constant housekeeping genes [21]. Therefore, our results are consistent with previous reports showing that housekeeping genes are generally involved in a variety of basic cellular functions, including intermediary metabolism, transcription, translation, cell signalling/communication and cell structure/ motility [21]. In addition, the current study has identified three EST RIKEN cDNAs (C030036P15, 2410104I19 and 1500016L11) as housekeeping genes. We have also investigated three previously postulated housekeeping genes namely PPIase, beta-actin, and GAPDH [24] which are expressed with significant difference according to the current study. Our study is in agreement with the previous report that PPIase, beta-actin and GAPDH are maintenance genes expressed in 11 human adult and fetal tissues but are not expressed at the same level in these tissues [17]. GAPDH was especially highly expressed in the skeletal muscle. Beta-actin was also differentially expressed between the tissues. According to the present study, PPIase is more constantly expressed in all the tissues than beta-actin and GAPDH. This result can explain the previous report that PPIase is a better internal control than beta-actin and GAPDH [25].

Specific genes expressed in the male sexual organs
According to this study, the tissue-specific genes were observed in higher proportion in testis than any other tissues. The top 8 most abundant tissue-specific transcripts, namely protamine 2, transition protein 1, t-complex-associated testis expressed 3, the novel transcript GTGCCAG-GAGA, tubulin alpha 3, diazepam binding inhibitor-like 5, lactate dehydrogenase 3 C chain sperm specific, and outer dense fiber of spermtails 2 are also the top 8 most abundant transcripts in this organ. These tissue-specific genes are all expressed in the specific stage of spermatogenesis. Other tissue-specific genes such as spermatogenic Zip 1 [26] and glyceraldehydes 3-phosphate dehydrogenase-spermatogenic [27], are expressed only during spermatogenesis. Translin associated factor X (Tsnax) interacting protein 1 is highly expressed in testis and in germ cells, suggesting a possible role in spermatogenesis [28]. Meanwhile, four-and-a-half-Lim-domain 5 is a LIM-only protein expressed exclusively in round spermatids [23], while kinesin family member 2b [29] and dipeptidase 3 [30] are expressed only in testis. The current study also reports the expression of testis specific genes such as ATPase class I type 8b member 3 which has hydrolase and phospholipid-translocating ATPase activity, and ATP-dependent aminophospholipid transporter which is exclusively expressed in the acrosomal region of spermatozoa [31]. In mammals, thioredoxins (trx) are generally ubiquitously expressed in all tissues, with the exception of sperm-specific trx (sptrx) which is exclusively detected in sperm cells [32]. The specific functions of these transcripts involved in spermatogenesis, explain the exclusive expression of these transcripts in the testis.
We have identified some prostate specific genes such as microseminoprotein (beta-MSP), seminal vesicle protein secretion 2, seminal vesicle antigen (SVA) and mucin 10 (MUC10) which are involved in protein secretion, cell signalling and spermatogenesis. In addition, one novel transcript was observed to be solely expressed in the prostate. Beta-MSP, also known as prostate secretory protein of 94 amino acids (PSP94), is an abundant secretory protein of the prostate gland and is generally considered to be prostate tissue-specific [33]. Results from western and northern-blot analyses for various tissues had previously indicated that the seminal vesicle is the sole organ producing SVA which is able to induce autoantibody formation [34]. MUC10, the submandibular salivary gland (SMG) mucin which is the primary histodifferentiation product of submandibular epithelia, was observed as tissue-specific gene to the prostate in our study. MUC10 is a type 1 integral membrane protein with a disintegrin and metalloprotease domain (ADAM29) precursor involved in spermatogenesis and fertilization [35]. Other prostate tissue specific identified are CUB and zona pellucida-like domains 1 (Cuzd1), and serine peptidase inhibitor Kazal type 3. In mouse, the expression of a secretory protease inhibitor is constitutive in the pancreas but stimulated by testosterone in ventral prostate, coagulated gland and seminal vesicle [36]. Cuzd1 was identified as highly and predominantly expressed gene in mouse epididymis using a cDNA microarray [37]. Since prostate-specific antigen (PSA), also known as gamma-seminoprotein, is a serine protease produced and secreted abundantly by prostate cancer cells [38], we did not detect PSA in the current results on normal prostate. Probasin, previously postulated as tissue-specific gene in the prostate [39], was abundantly expressed in the prostate (1925 tags) and was also found in the liver (282 tags) according to the present study. These results may have important repercussion, since prostate tissue-specific genes expression is crucial for driving potentially therapeutic genes to target specifically to the prostate [39].

Specific genes expressed in the female sexual organs
Only two transcripts with unknown function and a novel transcript with sequence tag GTCAACACAGG were specifically detected in ovary. We report two transcripts, namely glycosylation cell adhesion molecule 1 (Glycam1) and the novel transcript with sequence tag TCCGGAGAAAA, which are expressed only in the mammary gland. Glycam1 has a protein binding activity and previous studies have reported that prolactin induced Glycam1 expression in primary mammary epithelial cell of mice [40].
In uterus, three transcripts namely proline-rich acidic protein 1 (Prap1), hydroxysteroid 11-beta dehydrogenase 2 (Hsd11b2) and chloride channel calcium activated 3 (clca3) were characterized as tissue-specific. Northern analyses have demonstrated that Prap1 also known as pregnancy-specific uterine protein expression is limited to the pregnant uterus [41]. In the rat, the enzyme Hsd11b2 converts the glucocorticoid corticosterone into receptorinactive 11 dehydrocorticosterone, thereby allowing preferential access of aldosterone to mineralocorticoid receptors. The effects of glucocorticoids are thus critically regulated by the intracellular enzyme Hsd11b2 which was shown here to be highly expressed in the uterus [42]. In addition, the information from UniGene and EST expression has shown that the transcripts identified in uterus are also expressed in intestine tissue.
Two novel transcripts were observed to be tissue-specific to the vagina. Furthermore, the keratins such as keratin intermediate filament 16a, keratin complex 1 acidic gene 13 and keratin complex 2 basic gene 6 g, members of a family of fibrous structural proteins were also detected in high proportions in the vagina. Surprisingly, retinol binding protein 2, which functions in the intracellular transport of retinol, is detected only in vagina. According to the UniGene and EST databases, retinol binding protein 2 is also expressed in intestine tissue.

Specific genes expressed in the skin, liver, adipose tissue, lung, bone and skeletal muscle
The majority of transcripts specifically detected in the skin represent keratin. Moreover, the S100 calcium binding protein 17 and G protein-coupled receptor family C group 5 member D, both involved in cell signalling, were also specific to the skin. In addition, lymphocyte antigen 6 complex locus G6C, involved in cell defence, was also exclusively observed in the skin.
The transcripts exclusive to the liver are involved in several functions such as cell defence, protein and steroid hor-mone synthesis, as well as transport and amino acid metabolism. We report in the current study that interalpha-trypsin inhibitor-4 (Itih-4) was exclusively detected in the liver. This result is in agreement with the report that Itih-4 is a liver-restricted member of the serine protease inhibitors family, with diverse functions such as antiapoptotic action and matrix stabilization molecule that are important throughout development [43]. The present results also suggest that betaine-homocysteine methyltransferase (BHMT), cytochrome P450 family 2 subfamily d polypeptide 26 (Cyp2d26), microglobulin/bikunin precursor (AMBP) and plasminogen are solely expressed in the liver amongst the included organs. Previous studies have shown that BHMT [44] and Cyp2d26 [45] were expressed only liver and kidney, whereas strong expression of AMBP has been observed in developing hepatocyte, pancreas, kidney and gut [46]. The expression of plasminogen mRNA from hepatocyte is dependent on the cell density and stimulation [47] and is solely expressed in liver. Two mouse plasma proteins involved in cell defence, namely murinoglobulin 2 and kininogen 2 were exclusively expressed in the liver. Murinoglobulin is characterized as a single-chain proteinase inhibitor [48], while kininogen is known as a major acute phase protein whose levels increase 10-20 fold in response to an inflammatory challenge [49]. Traditionally, 4-hydroxyphenylpyruvic acid dioxygenase (HPD) has been considered as an enzyme primarily expressed in liver and to a lesser extent in kidney [50]. This previous study is agreement with the current study in which HPD is exclusively detected in the liver.
Two transcripts, leptin and lectin galactose binding soluble 12 (Lgals12), were exclusively detected in adipose tissue. Leptin is a peptide hormone produced predominantly by white adipose tissue. Beside its key role in the regulation of food intake and energy expenditure, leptin is also involved in the pathogenesis of inflammatory and autoimmune disease [51]. On the other hand, Lgals12 also known as galectin-12 is preferentially expressed in mouse preadipocytes and is up-regulated when preadipocytes undergo cell cycle arrest [52].
The transcripts specific to the lung are involved in cell defence, except claudin 18 which participates in cell signalling. The surfactant-associated proteins A (SP-A) and SP-D are members of a family of collagenous host defence lectins, designated collectins. The lung is the main site of SP-A and SP-D synthesis [53]. They are considered to be molecules of the innate immune system involved in the first line of defence of mucosal surfaces, especially in lung [53]. Another specific transcript is the palate lung and nasal epithelium clone (Plunc), also renamed Splunc1, which is a small secreted protein expressed in the oropharynx and upper airways of humans, mice, rats and cows.
The members of the PLUNC family may be involved in the innate immune responses in regions of the mouth, nose and lung, which are sites of significant bacterial exposure [54]. Another transcript exclusively detected in the lung is secretoglobin family 3A member 1 which is thought to play a role in inflammation and/or epithelial cell differentiation in the lung. The mRNA encoding for this gene is expressed predominantly with low level in terminals bronchioles [55].
The transcripts exclusively detected in the bone are involved in transport, cell structure, signalling and defence. The tissue-specific genes ascribed to proteoglycan 2 bone marrow and solute carrier family 4 (anion exchanger) member1 are highly and solely expressed in the bone. The other exclusive transcripts such as proteinase 3, neutrophil elastase, eosinophil peroxidase, cathepsin G and carbonic anhydrase 1 are involved in cell defence. Evidence from northern analysis has shown that proteinase 3 expression is primarily confined to the promyelocytic/myelocytic stage of bone marrow development [56]. The highly related serine protease known also as neutrophil elastase, proteinase 3 and cathepsin G are exclusively detected in promyelocytes and packaged in azurophil granules [57,58], whereas carbonic anhydrase 1 is expressed in adult human and mouse erythroid cells and colon epithelia, from two distinct promoters [59]. Another transcript exclusively expressed in bone matches to haematopoietic cell-specific transmembrane-4 (HTm4), member of a family of membrane-spanning 4domain proteins. It is known that HTm4 is expressed in hematopoietic tissue and is tightly regulated during the differentiation of hematopoietic stem cells [60].
In the current study, the transcripts exclusively detected in skeletal muscle are novel transcripts except tropomyosin 3 gamma (Tpm3) and myosin light chain phosphorylatable fast skeletal muscle (MLC2) which are involved in cell structure. In the mice, the Tpm3 mRNA is found exclusively in the skeletal muscle but not in the cardiac tissue at any development stage, whereas, in human, Tpm3 is found in both adult heart and skeletal muscle [61,62]. The specificity of MLC2, reported in the current study is in agreement with the previous study showing this gene expressed specifically in skeletal muscles of new-born and adult mice as well as rats [63].

Specific genes expressed in the brain
The current study reports that solute carrier family 17 (sodium-dependent inorganic phosphate cotransporter) member 7, also known as vesicular glutamate transporters (Vglut1), and synaptic vesicle glycoprotein 2 b (SV2B) as well as gene model 748 were exclusively expressed in cerebral cortex. This result is consistent with the report that Vglut1 mRNA is strongly expressed in the hippocampus and cerebellar cortex [64]. Vglut1 has been observed to play an unanticipated role in membrane trafficking at the nerve terminal [64]. SV2B is a protein highly related to a family of transporters. SV2B expression was observed to change during development; it is more widely expressed in the immature brain and is found in cells that have yet to establish synaptic contacts. SV2B is expressed in cerebral cortex, therefore further studies on the expression pattern of SV2B are needed to investigate the consistency with its function as a specific neurotransmitter transporter [65].
We report that cocaine and amphetamine regulated transcript (CART), calbindin 2, hypocretin and RIKEN cDNA A230109K23 gene were exclusively detected in the hypothalamus. The protein calbindin 2 buffers intracellular calcium is speculated to be involved in the integration of neuronal signalling. CART encodes a hypothalamic neuropeptide precursor protein which has been identified and characterized in rat brain and later in human brain [66,67]. Furthermore, hypocretin-1 and -2 (Hcrt-1 and Hcrt-2), also referred to as orexin-A and -B, are neuropeptides synthesized by a few thousand neurons in the lateral hypothalamus. Studies have hinted at a role of hypocretins in driving drug-seeking through activation of stress pathways in the brain [68]. Further investigation should be made in the characterization of RIKEN cDNA A230109K23.
The transcripts exclusively expressed in the pituitary gland were also highly expressed in this gland. Except growth hormone, pro-opiomelanocortin-alpha, prolactin, luteinizing hormone beta (LH beta) and thyroid stimulating hormone beta subunit (TSH beta) also known as thyrotropin beta which are involved in cell signaling, the other specific genes to this gland had no match in public databases and therefore may represent novel transcripts. LH beta is essential for ovulation and reproductive fitness, and is well known to be synthesized specifically in pituitary gonadotropes [69]. In addition, this glycoprotein is essential for ovarian follicular development, maturation of the oocyte, steroidogenesis and ovulation. In males, LH beta is involved in regulation of steroidogenesis in the Leydig cells [70]. TSH is an anterior pituitary glycoprotein hormone which modulates thyroid hormone production by the thyroid gland. TSH is constituted of two subunits: the alpha subunit is held in common with the gonadotropins luteinizing hormone and follicle-stimulating hormone, and the beta subunit which is unique and confers biological specificity to the intact hormone [71].

Conclusion
Using SAGE strategy, this study has shown the housekeeping genes and the tissue-specific genes expressed in 15 intact tissues. The identified housekeeping genes can rep-resent appropriate controls for RT-PCR and northern blot when comparing the expression levels of genes in several tissues. Several transcripts exclusively detected in a tissue are known to be tissue-specific genes according to previous studies. Furthermore, we have identified several new tissue-specific genes. These genes show well the specialty and particularity of each tissue. The tissue-specific genes can be used as a targeting agent in order to reach a particular tissue/organ. In addition, the current data can contribute significantly to comparative genomics in general and gene expression and regulation among different mouse tissues. Further studies will be needed to investigate other tissues to confirm the specific or housekeeping gene expressions. In addition, this study can serve as a basis for future studies on the novel transcripts and the transcripts with unclear functions despite their tissue specificity.

Methods
C57BL6 mice (12)(13)(14)(15) week old) were obtained by Charles River Laboratories (St. Constant, Québec). They were housed in an air-condition room (19-25°C) with controlled lighting from 07:15 to 19:15 h and were given free access to food (Lab Rodent Diet No. 5002) and water. The GDX and ADX groups had surgery 7 days before death. The intact, GDX and ADX groups received vehicle solution (0.4% (w/v) Methocel A15LV Premium/5% ethanol) 24 hours before sacrifice. DHT (0.1 mg) was injected 3 h prior to killing in GDX+DHT groups. ADX mice received sodium chloride (0.9 g/dl) in their drinking water after the surgery. Gcc (corticosterone, 0.1 mg per mouse) was subcutaneously injected into ADX mice, and the tissues were harvested 3 h after the injection. All animal experimentation was conducted in accord with the requirements of the Canadian Council on Animal Care. All the tissues were from male mice except for female sexual tissues. The tissues were dissected from 15-51 mice, frozen in liquid nitrogen and stocked at -80°C until analysis.
Total RNA was isolated from tissues by using the RNA extraction kit (TRIzol Reagent, Invitrogen Canada Inc., Burlington, ON). Approximately 5 μg of mRNA was extracted with Oligotex mRNA Mini Kit (Qiagen Inc., Mississauga, ON). The SAGE method was performed as previously described [13,14]. In brief, double-strand cDNA was synthesized from the mRNA using a biotinylated (T) 18 primer and cDNA synthesis kit (Invitrogen Canada Inc.). The cDNA libraries were digested with the restriction enzyme NlaIII (New England Biolabs Inc., Pickering, ON). The 3'-terminal cDNA fragments were captured using streptavidin-coated magnetic beads (Dynal, Biotech LLC, Brown Deer, WI). After ligation of 2 annealed linker pairs, the cDNA fragments were digested with BsmFI (New England Biolabs Inc.). The blunting kit from Takara Bio Inc. (Otsu, Japan) was used for the blunting and liga-tion of the two tag populations. The resulting ligation products were amplified by PCR and digested with NlaIII. The band containing the ditags was extracted from the 12% polyacrylamid gel. Using T4 ligase (Invitrogen Canada Inc.), the ditags were self-ligated to form concatemers that were cloned into SphI site of pUC19. White colonies were screened by PCR and agarose gel to select long inserts for automated sequencing (Applied Biosystems 3730, Foster City, CA). The data discussed in this publication have been deposited in NCBIs Gene Expression Omnibus [72] and are accessible through GEO Series accession number GSE5915. The sequence and occurrence of each of the transcript tags has been determined using the software SAGEana.pl, an updated version of SAGEparser.pl [73]. To identify the transcripts, we have generated a SAGEmap of 11 bp tags by the script SAGEmap.pl using the NCBI 10 bp tags SAGEmap, as well as the UniGene Clusters and mitochondrial mRNA sequences. The tag sequences must perfectly match at the last NlaIII restriction site (CATG) at the 3' end of a given transcript. To overcome the lower quality of some EST sequences, the tags that did not identify a well-characterized mRNA were required to match at least two ESTs in the same UniGene Cluster including one EST with a known polyA tail. To identify the transcripts, the sequences of 15 bp SAGE tags were matched with public databases. The tag numbers normalized by 100000 are shown in Tables 1, 2, 3, 4, 5.
The SAGE method has very good reproducibility [73]. However, several factors can affect this reproducibility such as the failure to provide relevant matching statistics. When a tag matches multiple genes, it is impossible to know the number of copies which are contributed by each gene since the matching stastitic is giving by the mixture of all contributing genes. However, using a 15 bp (CATG + 11 bp) tag, the SAGEparser program decreases the number of multiple matches and increases the number of tags which uniquely identify a transcript [73]. Therefore, the tags matching multiple genes were excluded from the current report. We used the comparative count display (CCD) test to identify the transcripts that were significantly (p ≤ 0.05) differentially expressed between the groups with more than 2-fold change. CCD test performs a key-by-key comparison of two key-count distributions by generating a probability that the frequency of any key in the distribution differs by more than a given fold factor from the other distribution. This statistical test has already been described elsewhere [74]. We have used the web site source at Stantford [75] to add the comparison of the tissue-specific genes with the UniGene and EST expression database information.

Authors' contributions
KEK has participated in the SAGE analyses including the bioinformatic analysis and wrote the paper with assist-ance from YN and JFCG. MY contributed to the conception and design of the project and drafted the manuscript. JSA directed the study, contributed to the conception and design of the project, analysis and interpretation of the data and drafted the manuscript. All the authors edited, read and approved the final manuscript.


extfieldsofstudy: Medicine Biology

text:
Single nucleotide polymorphisms (SNPs) distinguish Indian-origin and Chinese-origin rhesus macaques (Macaca mulatta)

Background Rhesus macaques serve a critical role in the study of human biomedical research. While both Indian and Chinese rhesus macaques are commonly used, genetic differences between these two subspecies affect aspects of their behavior and physiology, including response to simian immunodeficiency virus (SIV) infection. Single nucleotide polymorphisms (SNPs) can play an important role in both establishing ancestry and in identifying genes involved in complex diseases. We sequenced the 3' end of rhesus macaque genes in an effort to identify gene-based SNPs that could distinguish between Indian and Chinese rhesus macaques and aid in association analysis. Results We surveyed the 3' end of 94 genes in 20 rhesus macaque animals. The study included 10 animals each of Indian and Chinese ancestry. We identified a total of 661 SNPs, 457 of which appeared exclusively in one or the other population. Seventy-nine additional animals were genotyped at 44 of the population-exclusive SNPs. Of those, 38 SNPs were confirmed as being population-specific. Conclusion This study demonstrates that the 3' end of genes is rich in sequence polymorphisms and is suitable for the efficient discovery of gene-linked SNPs. In addition, the results show that the genomic sequences of Indian and Chinese rhesus macaque are remarkably divergent, and include numerous population-specific SNPs. These ancestral SNPs could be used for the rapid scanning of rhesus macaques, both to establish animal ancestry and to identify gene alleles that may contribute to the phenotypic differences observed in these populations.

Background
The rhesus macaque (Macaca mulatta) has served a critical role in the study of human disease for more than half a century. This macaque remains the animal of choice for much of biomedical research and is the primary model for the study of human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS) [1]. Though Indian-origin rhesus were originally used in most research protocols, the 1978 ban on the export of primates from India resulted in reduced availability of these animals. Because the growing demand for rhesus macaques has exceeded the domestic supply, the U.S. breeding colonies have imported large numbers of these animals from China.
In recent years, a variety of studies have investigated the relationship between Indian and Chinese rhesus macaques. Comparisons of Indian and Chinese rhesus mitochondrial DNA (mtDNA) sequences, including the hypervariable sequence I (HVS1), 12S and 16S rRNA loci, have shown that as much as 90% of the mtDNA genetic heterogeneity is accounted for by country of origin differences [2][3][4]. Studies of chromosomal microsatellite loci have also identified marked differences in allele frequencies between Indian and Chinese rhesus macaque populations [5][6][7][8]. Similarly, population-specific differences in the allele distributions within both Class I and II major histocompatibility complex (MHC) loci support the contention that the two populations have distinct genetic characteristics [9,10]. All of these studies support the conclusion that since their geographic separation, Indian and Chinese rhesus macaques have diverged to become two separate subtypes.
The genetic divergence of Indian and Chinese rhesus macaques is thought to underlie the observed phenotypic differences between the two subtypes in experimental protocols. These differences are observed at many levels, including morphology, behavior and physiology [11,12]. Significant differences in host response and disease progression have been observed in Indian and Chinese rhesus macaques exposed to the same simian immunodeficiency virus (SIV) challenge [13,14]. Both viremic peaks and set points were, in general, lower in the Chinese rhesus macaques than in Indian animals exposed to SIV/ DeltaB670, leading to much longer survival in the Chinese animals [14]. The Chinese rhesus macaques are also more resistant to SIVmac239, maintaining both lower acute and chronic viral loads than Indian rhesus macaques infected with the same viral challenge [13].
Because Indian and Chinese rhesus macaques can respond very differently to the same research protocol, consideration of animal ancestry is warranted in both study design and result interpretation. Most breeding facilities in the United States include both Indian and Chinese-origin rhesus. Although efforts are taken not to interbreed the two populations, lack of information or misinformation regarding animal history can result in unrecognized crossing of the two subtypes. The introduction of misclassified animals may compromise research results. Thus, being able to correctly identify rhesus macaque ancestry, including hybrids, would be very valuable to both researchers and animal breeders.
Current approaches for rhesus macaque ancestry determination include: 1. sequence comparison of mtDNA loci, [2,7]; 2. microsatellite analysis [6,7]; and 3. SNP analysis [15]. Since mtDNA is only inherited through the maternal line, this analysis cannot identify Indian-Chinese hybrid animals. The microsatellite analysis involves three loci in which the allele frequencies differ significantly between Indian and Chinese populations. The current literature only reports five SNPs in 3 genes (NDN, H19 and IGF2) that are unique to either Indian or Chinese rhesus macaques, identified by Fujimoto [15]. Unfortunately, though both the reported microsatellite and SNP markers can work for distinguishing purebred Indian and Chinese rhesus, neither offers a sufficient number of markers to reliably detect hybrid animals.
We have developed algorithms for identifying primers to amplify the 3' end of all rhesus macaque genes [16]. Over 5,000 of the sequences we obtained using these primers were used in the design of the Affymetrix rhesus macaque GeneChip [16,17]. We tested whether these primers could also be used to discover gene-based SNPs. The 3' ends of 94 selected genes were analyzed in 20 Indian and Chinese rhesus macaques. This effort identified 661 SNPs. Four hundred and fifty-seven of these SNPsappeared uniquely in either the Indian or Chinese rhesus macaques. A subset of those SNPs was further tested using a SNP genotyping assay against a population of 75 additional Chinese and Indian animals, as well as 4 known Chinese/Indian hybrid rhesus. Eighty-four percent of the SNPs were confirmed to show population-specificity. All of the known hybrids were detected with the Indian/Chinese SNP Assay panel.
The Indian/Chinese SNP Assay panel will be useful for animal ancestry determination, for the identification of hybrid animals and for the genetic analysis of the phenotypic differences that characterize the two populations. Further, this work validates the efficiency of using genebased, 3' primers to discover SNPs in rhesus macaques.

Rhesus macaque SNP discovery
We selected 20 unrelated rhesus macaques for use in SNP discovery, 10 each of alleged Indian or Chinese ancestry.
All of the Chinese rhesus monkeys were imported directly from primate suppliers in China. However, neither records of capture site or breeding history were available to more specifically identify their geographic origins within Asia. Two of the Indian animals had been wildcaught in Northern India, while the remaining 8 rhesus macaques came from U.S. breeders who indicated that the animals were of Indian-descent. To validate the ancestries of all of the animals, we sequenced the 12S rRNA portion of the mitochondrial DNAs and compared them with previously published sequences from Indian and Chinese rhesus macaques [2,4]. We also used the allele sizes of three microsatellite loci with disparate allele frequencies in Indian and Chinese populations (DX18S537, D1S548, DXS2506) to corroborate the animal ancestries [6]. Only rhesus macaques that satisfied each of these criteria for being of pure Indian or Chinese heritage were included in this study.
The 3' ends of 94 different genes were amplified and sequenced in this study, with the average amplicon length being 712 bp/gene (range: 334-905 bp). The 94 genes included at least one from every chromosome with the exceptions of 17 and Y (see Additional file 1 -Gene summary). In total we identified 661 SNPs in the rhesus macaques (see Additional file 2 -SNP identity and NCBI accession numbers). All except for three of the genes (AR, CHRM4, IL7R) identified at least 1 SNP, with the overall average being 7.3 SNPs/gene ( Figure 1). Some genes were very polymorphic: 20 genes contained between 10 and 17 SNPs (Figure 1; see Additional file 1 -Gene summary). The overall minor allele frequency (MAF) of the SNPs was 0.127, with 281 SNPs having a MAF ≥ 0.2 in the sample population.
To assess how the SNPs identified in rhesus macaques compared with those found in the same regions of the human orthologs, we identified 10 genes that were also analyzed in a study of 48 individuals of either European and African decent [18]. The rhesus sequences contained more polymorphisms than did the human sequences, averaging 1 SNP/107 bp in the rhesus compared with 1 SNP/179 bp in humans ( Table 1). None of the common SNPs in these gene regions were detected in both humans and rhesus macaques.

Population comparison of rhesus macaque SNPs
We analyzed the SNPs discovered within each rhesus macaque population. We identified 555 SNPs in the Chinese group, while the Indian animals had a total of 312 SNPs (Table 2; see Additional file 1 -Gene summary). The distribution of the minor allele frequencies of the SNPs within each population ranged from 0.04 to 0.5 ( Figure 2). There were a higher percentage of rare alleles Number of SNPs identified in the 3' end of 91 rhesus macaque genes Figure 1 Number of SNPs identified in the 3' end of 91 rhesus macaque genes. SNPs were identified in 20 Chinese and Indian rhesus macaques. There were no SNPs identified in 3 genes; between one and 18 polymorphisms were found in the other 91 genes.  (Table 2; see Additional file 1 -Gene summary). Almost two thirds of the Chinese-specific SNPs were relatively rare, occurring in less than 10% of the population. However, 61 of the Chinese-specific SNPs and 39 of the Indian-specific SNPs had an allele frequency of ≥ 0.2. In some cases, the population-specific SNP allele was the major allele in that population. (For example, CD40LG:738 -the "G" allele is unique to the Indian population and occurs at 0.65 frequency.) The MAOA gene contained a polymorphism that was fixed in each population.

Genotype assay of population-specific SNPs
To further explore the population-specificity of the The 44 putative population-specific SNPs were interrogated using the Sequenom iPLEX assay [19]. Six of the SNPs (14%) were excluded when they were found to have

Discussion
This study identified a striking difference in the genome composition of Indian and Chinese rhesus macaques. There were 76% more SNPs identified in Chinese than in Indian rhesus macaques, a finding consistent with the greater mtDNA sequence and morphological diversity described for Chinese rhesus macaques [3,10,20,21]. The increased genetic heterogeneity in Chinese rhesus could be a consequence of the Chinese animals inhabiting a large geographic range and evolving more regional population substructure [3]. In addition, the reduced diversity of the Indian rhesus macaques suggests this population may have experienced an evolutionary bottleneck or expanded relatively recently from a small founder population.
A surprising finding was that 69% of the SNPs identified were unique to either Chinese or Indian rhesus macaques. If the initial cohort of animals chosen for sequencing included any misclassified or hybrid animals, then the real number of private SNPs would be even higher. However based upon both the background screening and the genotype analysis of a larger set of animals, the initial cohort appears to have correctly reported ancestries.  [3]. Alternatively, the spurious population exceptions may have resulted from the intermixing of Indian and Chinese rhesus macaques at some breeding colonies. Future studies with native populations may clarify this point.
Based upon the results of an expanded genotype assay, we predict that as many as 85% of the population-specific SNPs identified in this study will remain largely exclusive to one ancestral group when examined in additional studies. We therefore estimate that 80-85 of the SNPs identified in this study are both population-specific and occur at high frequency (MAF ≥ 0.2). These SNPs will be useful for validating rhesus macaque ancestry in an efficient manor. The overall high frequency of population-specific SNPs also suggests that a large-scale genome array of either Indian or Chinese-specific SNPs could be generated. Such an array would enable admixture studies of population-associated phenotypes.
Some of the alleles queried in the 38 SNP genotyping panel (ADBRK2:109, CD40LG:738, CD4:558) were found to deviate from Hardy-Weinberg equilibrium. The apparent under representation of particular genotypes may reflect the small number of animals sampled, population substructure, or possibly selective pressure against these, or linked, genotypes.
Based upon a comparison of 10 gene sequences that were analyzed in similar studies of rhesus macaque and human populations, it appears that the Indian and Chinese rhesus has about 1.6 times as many SNPs as the African and European human populations. Given that only 20 animals were included in this study, that estimate is likely to be conservative. The higher level of heterogeneity is not surprising, since the evolutionary history of the macaques is longer, affording more time for sequence variations to accumulate.
Some of the SNPs identified in this study may be functional, disrupting microRNA target sequences or affecting gene expression through another mechanism. Indeed, 3'UTR polymorphisms in the rhesus macaque DAT [22] and TPH2 genes [23] were shown to be associated with altered gene expression, and in the case of TPH2, with hypothalamic-pituitary-adrenal (HPA) axis activity. A finding from this study was that the MAOA gene contains alleles that significantly differ in Indian and Chinese rhesus macaques. The MAOA gene encodes an enzyme that contributes to the breakdown of serotonin; thus we speculate that disparate MAOA expression or activity may contribute to the different levels of 5-HIAA (serotonin metabolite) and the aggressive behavior that distinguish Chinese and Indian rhesus macaques [12,24].

Conclusion
This work demonstrates that the 3' end of rhesus macaque genes is a rich source for identifying sequence polymorphisms. This study also demonstrates that there is a high frequency of SNPs that are unique to either Indian or Chinese-origin rhesus macaques. These population-specific SNPs can be used to rapidly screen and establish rhesus macaque ancestry and to identify genes that underlie phenotypes characteristic of each population.  Indian animals and hybrid animals being from the ONPRC. Three of the latter animals were captured and imported directly from Northern India in 1976.

DNA extraction and validation of animal ancestry
Ten ml of blood was drawn from each animal in accordance with the Oregon Health and Sciences University Institutional Animal Care and Use Committee (0492-09), using universal precautions. DNA was extracted from each blood sample (QiAMP Blood Midi Kit, Qiagen, Inc.).
Two genetic analyses were used to corroborate the ancestry of each animal. First, a portion of the mtDNA 12S rRNA locus was PCR amplified (primers F-5' ACT-GGGATTAGATACCCCACT-3' and R-5'AGGGTGACG-GGCGGTGTGT-3'), sequenced and compared to published Indian and Chinese rhesus macaque sequences [4]. Second, the allele sizes of three microsatellite markers (DX18S537, D1S548, DXS2506), which have differing allele frequencies in Chinese and Indian rhesus macaques, were established for each animal (Veterinary Genetics Laboratory, Davis, CA). Only rhesus macaques with mtDNA and microsatellite results supporting their documented ancestry were included in this study.

Amplification and sequencing of DNAs
The genomics extracted from the 10 Chinese and 10 Indian rhesus macaques were used as templates for the PCR amplifications. PCR was performed as described in previously [16]. PCR products were sequenced in both directions either at Agencourt Biosciences, Inc. (Beverly, MA), or at the ONPRC using a Genetic Analyzer 3130 (Applied Biosystems, Inc.) using the amplification primers.

SNP analysis
The workflow for the DNA sequence analysis and SNP identification is outlined in Figure 4. After receipt of raw data from sequencing source, files were compiled on a Macintosh XServer at the OHSU Advanced Computing Center cluster. The Phred/Phrap/Consed package was used to perform sequence alignment [25,26]. The output sequences were aligned directly against the publish reference sequences [27], rather than being assembled into contigs first. Identification of polymorphisms on the aligned sequences was done using PolyPhred [25]. Control of these procedures was implemented in a Perl program designed for this work. Outputs from PolyPhred were then processed and loaded into a local MS-SQLbased genetics database, using a Java-based program developed for this project. In most cases, both the forward and reverse sequence reads for an individual were in agreement; in the rare case that the two sequence reads contained a difference in a single base assignment, the read with the higher PolyPhred quality score was automatically identified and entered into the database as the correct genotype for that individual. In cases where identical quality scores were attributed to a divergent base, a manual identification of the correct genotype was determined using Sequencher software (Gene Codes Corporation, Ann Arbor, MI) and visual inspection. A visual review of the sequence quality of each gene contig was also carried out before inclusion of SNPs into the final data set. A second Java program written for this project facilitated the statistical genetic calculations. It was used to calculate allele frequencies for animals in the Indian and Chinese populations, both individually and when combined; this program can also compare individuals to pre-defined populations. The verified SNPs were deposited into dbSNP [28] and the rhesus-specific database, Monkey SNP [29]. Figure 4 SNP analysis workflow. The DNA sequence output was analyzed using PolyPhred software to identify SNPs and to report the relative quality score of the SNP calls. SNPs with low quality scores or with inconsistent reports were reviewed manually. The approved SNP data was deposited into a local database for population, frequency and genotype analysis. Based upon the statistical results, some SNPs were selected for inclusion in Sequenom iPlex SNP panel.

SNP genotyping
Genotyping was performed using iPLEX reagents and protocols for multiplex PCR, single base primer extension (SBE) and generation of mass spectra, as per the manufacturer's instructions (for complete details see iPLEX Application Note, Sequenom, San Diego). Three multiplexed assays contained 28, 17, and 7 SNPs, for a total of 52 SNPs queried, of which 50 successfully generated genotyping data. Briefly, initial multiplexed PCR was performed in 5 μl reactions on 384-well plates containing 5 ng of genomic DNA. Reactions contained 0.5 U HotStar Taq polymerase (QIAGEN), 100 nM primers, 1.25× HotStar Taq buffer, 1.625 mM MgCl 2 , and 500 μM dNTPs. Following enzyme activation at 94°C for 15 min, DNA was amplified with 45 cycles of 94°C × 20 sec, 56°C × 30 sec, 72°C × 1 min, followed by a 3 minute extension at 72°C. Unincorporated dNTPs were removed using shrimp alkaline phosphatase (0.3 U, Sequenom). Single-base extension was carried out by addition of SBE primers at concentrations from 0.625 μM (low MW primers) to 1.25 μM (high MW primers) using iPLEX enzyme and buffers (Sequenom, San Diego) in 9 μl reactions. Reactions were desalted and SBE products measured using the MassAR-RAY Compact system, and mass spectra were analyzed using TYPER software (Sequenom, San Diego), in order to generate genotype calls and allele frequencies.