Introduction
============

The major problem with the use of nonsteroidal anti-inflammatory drugs (NSAIDs) is the high prevalence of their gastrointestinal toxicity \[[@B1]\]. Accordingly 40% of patients have non-ulcer gastric lesions, 10--25% gastric ulcer and 65% NSAID enteropathy \[[@B2]\]. The development of safer NSAIDs is dependent on knowledge of the pathogenesis of damage. In general, two distinct phases (topical and systemic) are suggested to be involved in the pathogenesis of enteropathy \[[@B3]-[@B5]\]. The topical phase of damage may be due to the effect of NSAIDs to uncouple mitochondrial oxidative phosphorylation or inhibit electron transfer \[[@B4]-[@B6]\] either of which would result in a decrease in cellular ATP levels\[[@B7]\] and a disrupted intestinal barrier function. Consequently, it is suggested, increased intestinal permeability transforms the cellular damage into a tissue reaction where inflammation and ulcers occur because of mucosal exposure of luminal aggressive factors (such as acid, pepsin, bile, pancreatic secretions and bacteria) and the effects of decreased mucosal prostaglandins on the microvasculature.

In respect of early pathogenic events in the development of NSAID damage it has been shown that uncoupling of oxidative phosphorylation and inhibition of electron transport occurs with all commonly available acidic-NSAIDs. Our earlier studies indicate \[[@B8],[@B9]\] that indomethacin, aspirin and naproxen uncouple mitochondrial oxidative phosphorylation at concentration between 30 and 500 μM and inhibit the electron transport chain at higher concentrations. The issue of inhibition of electron transport by NSAIDs in the pathogenesis of damage and its pathophysiological consequences is complicated by the fact that nitric oxide (NO•), which plays a pathogenic role in the damage, may bind to metalloproteins \[Fe-S\] of the respiratory chain and by itself increase intestinal permeability \[[@B10]\]. Thus NSAIDs may inhibit electron transport directly or indirectly by induction of nitric oxide (NO•) \[[@B11]-[@B16]\]. The aim of the current study was to assess and discriminate between the direct and indirect effects of NSAIDs on electron transport of the rat small intestine using electron paramagnetic resonance (EPR) spectroscopy.

 mitochondrial electron transfer system plays a significant role in the pathogenesis of NSAID enteropathy. In the mitochondria electron transfer occur in the iron sulphur proteins arranged sequentially. Loss (oxidation) and gain of electrons (reduction) in this iron sulphur proteins can be visualized as a spectrum in the EPR machine. It has been hypothesized that NSAID might have an affinity towards these iron sulphur proteins and alter the redox state.

Materials and methods
=====================

Experimental design
-------------------

### Electron paramagnetic studies

Male Sprague-Dawley rats (200--250 g) were used throughout EPR studies. 12 rats received indomethacin 20 mg/kg, which is a commonly used dose to induce consistent small intestinal damage in the rat (characterized by mitochondrial damage on electron microscopy within an hour,\[[@B17]\] neutrophil infiltration of mucosa on light microscopy at 3--5 hours and macroscopic lesions (ulcers) at 12--18 hours \[[@B18]\]); . A separate group of 12 rats received the non-acidic pro-NSAID nabumetone (500 mg/kg). The maximum human doses of indomethacin in man is 150 mg/day and 2000 mg for nabumetone so that the equivalent doses given here are at least 8 and 17 fold greater than the recommended doses for indomethacin and nabumetone, respectively and many times higher than this required for anti-inflammatory activity.

Nabumetone is unusual amongst NSAIDs in that it does not produce any small intestinal lesions in the rat, presumably because it is non-acidic (and hence not a proton translocator or an inhibitor of the electron transport chain) and its active component 6-methoxy-2-naphthylacetic acid (6-MNA) is not subjected to excretion in bile \[[@B9],[@B19]\]. Incorporation of nabumetone therefore allows assessment of the systemic effects of cyclooxygenase inhibition in isolation from its topical effects.

In separate studies the inhibitor of inducible nitric oxide synthase, 2,4 diamino-6-hydroxy pyrimidine (DAHP; a potent inhibitor of tetrahydrobiopterin synthesis, which is a cofactor for the inducible nitric oxide synthase activity) 1.0 gm /kg b.w. was administered to 12 rats intra-peritonealy 1 hr before indomethacin (20 mg/kg) gavages.

Preparation of drugs: Indomethacin and nabumetone were dissolved in 10% dimethyl sulfoxide and then diluted to the final concentration of 5% and administered orally by a gastric gavage without anaesthesia. Control rats were received the same volume and concentrations of dimethylsulfoxide.

### Intestinal permeability assessment

Rats, 12 in each group, were fasted overnight and received solvent, 20 mg/kg indomethacin or nabumetone (500 mg/kg) by gavage. 12 rats received DAHP i.p. (1.0 g/kg) 1 hr before indomethacin (20 mg/kg). Rats were fed 2 hrs later and fasted the following night. Twenty hrs after NSAID administration these rats received the intestinal permeability marker 51Cr- EDTA (10 uCi / rat in 0.5 ml followed, by 0.5 ml water) by gavage and were placed in metabolic cages. Urine was collected for 5 hrs and the samples were counted in Wallace 1842-LKB gamma counter with appropriate standard. The percentage of the orally administered radioactive chromium excreted in urine was calculated.

### Morphological assessment

20 hours after administration of these drugs rat small intestinal mucosa (n = 12) was exposed by a cut through the anti- mesenteric side and laid out each group on a piece of cork for assessment of macroscopic damage.\[[@B24]\] The assessments were carried out by an independent person unaware of the drug treatments. An ulcer count was made to distinguish between pointed (\<5 mm) and longitudinal (\>5 mm) ulcers.

### Isolation of mitochondrial fractions

Bovine heart mitochondria were isolated based on a modification of Schneider & Hogeboom method (1950) \[[@B20]\], diluted to 10 mg protein/ml and sonicated in 3 ml aliquots on ice with eight 15 sec bursts, interrupted by 15 sec intervals, at 18 amps using a micro tip probe. The sonicated suspension was centrifuged at 26,000 g for 10 min at 4\'C. The supernatant fraction was centrifuged at 130,000 g for 1 hr, and the sub mitochondrial particles pellet resuspended for EPR analysis after checking the purity of this fraction (by studying the electron transfer reactions using potassium ferricyanide as an electron acceptor and succinate as a substrate). This experiment was repeated for three times at various intervals. It has been attempted to isolate submitochondrial fraction of jejunal mitochondria from rats. In most of the cases we could not able to achieve a good yield of submitochodrial particles. Hence we used bovine heart miotochondria to understand the basic phenomena of NSAID binding sites.

### EPR spectroscopy

1 hr and 24 hr after administration of the drugs, the animals were anaesthetized (Hypnovil and Hypnorm) and underwent laparotomy. Segments of jejunum 10 cm distal to the ligament of Trietz were snap frozen with liquid nitrogen and stored for a maximum of 2 weeks. Samples were thawed and homogenized in 10% (w/v) of sucrose (74 mM), mannitol (225 mM), homogenising buffer (0.25 M) containing EDTA (1 mM), 5 mg/ml BSA solution in MOPS-NaOH buffer (10 mM), pH 7.4, by 15 strokes of a tight fitting teflon pestle. 100 ul samples (1 mg protein) were placed in quartz EPR tubes (3 mm-internal diameter) and immediately frozen in liquid nitrogen. X-band EPR spectra were recorded on a Bruker ESP 300 spectrometer fitted with an Oxford Instruments ESR900 liquid helium flow cryostat at 8 K and 20 K or 30 K. The effect of 10 ul of 250 mM indomethacin added to 90 ul (1 mg protein) of submitochondrial particles in a quartz EPR tube and read the EPR spectra at 8 K was assessed with appropriate controls. Spectra were an average of six scans and normalized for direct comparison. The experiments were repeated for three different times with separate group of animals.

EPR conditions: Temperature 8 K or 20 K or 30 K: microwave power 20 mW, microwave frequency 9.35 GHz, modulation frequency 100 Khs, modulation amplitude 1 mT, receiver gain 1.25 × 10^5^, time constant 0.33 sec, sweep time 2.4 mT/sec. Spectra displayed are average of 6--8 scans.

Statistics
----------

Data are presented as mean ±SEM. Statistical comparison between groups were made by Mann-Whitney U test.

Results
=======

Morphology
----------

No macroscopic changes were observed in controls or with indomethacin and nabumetone 1 hr after drug administration. At 24 hr the indomethacin-treated group exhibited severe intestinal ulceration (pointed ulcers: mean = 55 ± 10, longitudinal ulcers: mean = 15 ± 9). No ulcers were seen in controls, nabumetone or DAHP treated indomethacin gavaged rats. \[Table [1](#T1){ref-type="table"}\]

::: {#T1 .table-wrap}
::: {.caption}
######

The ulcerogenic and intestinal permeability (51Cr-EDTA excretion) effect of DMSO (Control), indomethacin, DAHP pretreated indomethacin and nabumetone gavaged rat small intestine.
:::

  Group   Treatment                      Pointed ulcers   Longitudinal ulcers   \% Excretion of ^51^Cr-EDTA in Urine
  ------- ------------------------------ ---------------- --------------------- --------------------------------------
  1       Control(DMSO)                  0                0                     2.3 ± 1.2
  2       Indomethacin                   55 ± 10          15 ± 9                ^\*^ 10.5 ± 2.5
  3       DAHP pretreated indomethacin   0                0                     ^\*^ 11.5 ± 0.8
  4       Nabumetone                     0                0                     2.7 ± 1.5

^\*^ P \< 0.01
:::

Intestinal permeability
-----------------------

Controls excreted 2.3 ± 1.2% of the orally administered 51Cr-EDTA in 5 h urine. Intestinal permeability was not significantly (p \> 0.5) increased following nabumetone (mean 2.7 ± 1.5%). Indomethacin treated rats and those pre-treated with DAHP (+indomethacin) had significantly increased (p \< 0.01) intestinal permeability to 51Cr-EDTA (10.5 ± 2.5% and 11.5 ± 1.8% respectively) \[Table [1](#T1){ref-type="table"}\]

EPR spectroscopic study of NSAIDs on sub-mitochondrial particles
----------------------------------------------------------------

At 8 K, the EPR spectrum of bovine heart sub mitochondrial particles administered DMSO alone (Fig. [1](#F1){ref-type="fig"}) displayed a prominent resonance at g \~ 2.014. This signal was only observed in the oxidized state at temperatures below 25 K, and is characteristic of the \[3Fe-4S\]ox cluster of mitochondrial Complex II Centre S3. Extra features at g \~ 2.04, 1.99 are present, and are assigned to interactions between ubisemiquinone radicals \[[@B23]\].

![The EPR spectra of (I) DMSO-control and (ii) indomethacin (25 mM final concentration) treated bovine heart submitochondrial particles at 8 K.](1471-230X-2-8-1){#F1}

Indomethacin but not nabumetone or DAHP added to the sub mitchondrial particles significantly decreased the intensity of the latter featrures (Fig. [1](#F1){ref-type="fig"}) (g \~ 2.04 and 1.99) suggesting formation of radical species which is indicative of binding of indomethacin to and the inhibition of the respiratory chain direct to this site.

EPR spectroscopic studies of NSAIDs on rat jejunal homogenates
--------------------------------------------------------------

At 20 K, control rat intestinal homogenates also displayed a signal indicative of Centre S3, albeit weaker (Fig. [2a](#F2){ref-type="fig"}). A similar signal could be detected 1 hr and 24 hr after nabumetone administration. \[Fig. [2b](#F2){ref-type="fig"} and Fig [3b](#F3){ref-type="fig"})\] However, with indomethacin the \[3Fe-4S\]ox signal intensity was decreased at 1 hr, and by 24 hr the EPR spectrum of intestinal homogenates displayed a prominent new resonance centered at g \~ 2.0, with a peak at g \~ 2.07 and a triplet splitting centered at g \~ 2.01, indicative of nitrosyl-haem (haem-NO) formation. \[Fig. [2c](#F2){ref-type="fig"} and Fig [3c](#F3){ref-type="fig"}\]. DAHP pre treated indomethacin group significantly decreased the haem-NO intensity at 24 hr after administration \[Fig. [4](#F4){ref-type="fig"}\], thereby allowing detection of the Centre S3 signal at 8 K \[Fig. [5](#F5){ref-type="fig"}\]. DAHP pretreated indomethacin group displayed the similar signals at 1 hr, when compared to indomethacin alone treated group \[.i.e., decreased in the \[3Fe-4S\]ox signal at 1 hr, Data not shown\]. The haem-NO signal observed in Fig. [3c](#F3){ref-type="fig"} shows a splitting of A (14N) \~ 330 mT \[Fig. [4](#F4){ref-type="fig"} at 30 K\]. Similar signals have been observed for the NO-adducts of type II haem proteins such as cytochrome c oxidase and hemoglobin \[[@B21]\] Figure [3](#F3){ref-type="fig"} explains the unique features of nitrosyl signals (g \~ 2.04) at 20 K only in indomethacin treated group and not in the DMSO-control or nabumetone group. Figure [4](#F4){ref-type="fig"} displays the differential pattern of the nitrosyl signals (i)-(ii) at 30K after 24 hr of indomethacin (i) and DAHP treated indomethacin group(ii) when compare to the DMSO-control (iii) spectra. This prominent nitrosyl signals at 30 K is the characteristics of the free radical species and temperature sensitive when compare to the signals at 8 K, displayed in Figure [5](#F5){ref-type="fig"}. In indomethacin alone treated group (Figure [4](#F4){ref-type="fig"}) this broad signal displayed a peak at g \~ 2.04, a distinct trough at g \~ 1.98 and complex splitting (triplet) centered at g \~ 2.01 at temperature above 25 K, indicative of haem-nitrosyl formation.\[[@B21]\]

![The EPR spectra of rat jejunal homogenates at 20 K after 1 hr oral gavage of (a) DMSO, (b) nabumetone (500 mg / kg b.wt) and (c) indomethacin (20 mg/kg b.wt)](1471-230X-2-8-2){#F2}

![The EPR spectra of rat jejunal homogenates at 20 K after 24 hr oral gavage of (a) DMSO, (b) nabumetone (500 mg / kg b.wt) and (c) indomethacin (20 mg/kg b.wt)](1471-230X-2-8-3){#F3}

![Comparison of EPR spectra of indomethacin and DAHP pretreated indomethacin group at 30 K after 24 hr treatment.](1471-230X-2-8-4){#F4}

![Comparison of EPR spectra of indomethacin and DAHP pretreated indomethacin group at 8 K after 24 hr treatment.](1471-230X-2-8-5){#F5}

Discussion
==========

The present results show that indomethacin treated rats and not those pretreated with DAHP exhibited small intestinal ulcers at 20 hr. There was increased intestinal permeability and an early (1 hr) direct effect of indomethacin consistent with inhibition of electron transport in both groups but only the indomethacin treated group exhbited a haem-nitrosyl complex EPR signal at g \~ 2.07. Thus the present result suggests that the nitric oxide radical may play a role in the development of indomethacin-induced ulcers. Within the current pathogenic framework \[[@B5]\] where it is proposed that NSAIDs affect mitochondrial energy metabolism leading to increased intestinal permeability and where the micro vascular alterations, caused by changes in prostaglandin and nitric oxide metabolism are the driving force in the development of ulcers. The involvement of nitric oxide occurs temporarily later or at a different pathophysiological step than the intestinal permeability changes. The non-acidic Pro-NSAID nabumetone caused no intestinal lesion as previously described \[[@B22]\] and was not associated with changes in the EPR spectra, despite being effective inhibitors of cyclooxygenase and associated with decreased mucosal prostaglandins at much smaller doses\[[@B22]\]. These findings are consistent with previous in vitro and in vivo work showing that nabumetone itself does not alter mitochondrial energy metabolism whilst its active metabolite 6MNA does. However it would appear that as 6MNA is formed following absorption of nabumetone, over 99% of 6MNA is protein bound within the circulation and it is not excreted in bile, insufficient intestinal concentrations of 6MNA are achieved to affect enterocyte mitochondria.

NSAIDs uncouple oxidative phosphorylation at 30--500 uM and inhibit mitochondrial electron transport at higher concentration in vitro \[[@B8]\] The relative importance and contribution of uncoupling and inhibition in the pathogenesis of the \"topical\" Phase of damage is uncertain, but either mechanism would lead to decreased cellular ATP and hence increased intestinal permeability. In this study we assessed the characteristics of NSAID modulation of electron transport in vivo in rats and in vitro in bovine heart submitochondrial particles. The EPR spectra observed at 1 hr would appear to be the result of a direct effect of indomethacin on electron transport unrelated to NO\', since pretreatment with DAHP did not alter the indomethacin spectra. The EPR spectra at 24 hr post indomethacin \[Fig. [2B (c)](#F2){ref-type="fig"}\] indicated formation of NO\' which is further substantiated by abolition of this signal when rats were pretreated with DAHP \[Fig. [3](#F3){ref-type="fig"}\].

The activity of iNOS is controlled mainly at the levels of transcription and translation and is dependent on the availability of co-factors including tetrahydrobiopterin (BH4). A recent study has demonstrated that inhibition of BH4 synthesis reduces NO• production in vivo in experimental endotoxic shock\[[@B12]\]. The same strategy of inhibition of BH4 synthesis has been applied in this present attempt to investigate ulcer formation in indomethacin-induced intestinal ulcer. We found that injection of DAHP an inhibitor of BH4 synthesis prevented ulcer formation, although it did not alter the increased intestinal permeability induced by indomethacin. This suggests that it does not modulate the direct biochemical actions of NSAIDs but interferes at a later stage in the pathogenesis of the damage. One possibility is that DAHP suppresses NO formation in neutrophils (iNOS), which infiltrate the mucosa in response to increased intestinal permeability. In order to study the specificity of the nitric oxide synthesis the experiments with N(G)-nitro-L-argenine methyl ester (L-NAME-10 mg /kg b wt s.c 1 hr prior to indomethacin) was carried out to prevent nitrosyl signals and ulcers induced by indomethacin. We did not find the inhibition of intestinal ulcers.

This observation is further confirmed by the recent reports \[[@B25]\] stating the worsening effect of L-NAME (5--20 mg/kg) on these lesions was dose-dependently observed in association with further enhancement of the bacterial translocation and intestinal hypermotility following indomethacin. Thus it has been concluded that this effect may be due to the nonspecific inhibition of L-NAME and it is not a specific inhibitor of iNOS cofactor biopterin complex and hence excluded from EPR study.

Conclusions
===========

In summary, it is interesting that in spite of the increased intestinal permeability, there were no ulcers seen in the DAHP treated group indicating that inhibition of inducible nitric oxide synthase (in neutrophils and macrophages) could become a targeted therapeutic approach to reduce the intestinal toxicity of NSAIDs, and nabumetone is comparatively safer with neither inducing ulcer nor formation of nitrosyl complex in this study.

Competing interests
===================

None declared

Authors\' contributions
=======================

SS carried out the planning, animal experimentations, EPR measurements and drafted the manuscript. RS contributed for drafting the manuscript. SR carried out submitochondrial particle isolation and *In vitro* NSAIDs inhibition study. JKS helped for the computation of EPR spectra and contributed in the interpretation of the spectra. JW and IB conceived of the study and participated in its design, coordination, corrections and helping to draft this manuscript.

Grant Support
=============

This work was supported by an unconditional grant from Smith Kline Beecham, USA to Dr. S. Somasundaram.

Abbreviations
=============

EPR, electron paramagnetic resonance; NSAIDs, nonsteroidal anti-inflammatory drugs; DAHP, 2,4-diamino-6-hydroxy pyrimidine; DMSO, dimethyl sulfoxide

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

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Acknowledgements
================

The authors thank Professor. R. Cammack for providing electron paramagnetic resonance spectroscope for the above study.


Background
==========

Vacuolar-type H^+^-ATPases are enzymes responsible for the energization of membranes and the acidification of compartments within the eukaryotic cell via the establishment of proton and electrochemical gradients at the expense of ATP. The vacuolar type H^+^-ATPase in plants is a large multimeric enzyme complex whose function is to pump protons across a membrane via primary active transport. Vacuolar type ATPases are homologs of the F-type ATP synthases and probably convert the free energy of hydrolysis of the high-energy phosphate bond into rotational motion \[[@B1]-[@B9]\]. Vacuolar type H^+^-ATPases are critical for the maintenance of homeostasis in eukaryotic cells \[[@B10],[@B11]\].

In plant cells V-ATPases are responsible for the deacidification of the cytosol and the energization of secondary transport processes across the tonoplast, as well as in the endocytotic and secretory pathways \[[@B12]\]. In addition, the vacuolar type ATPase is thought to be primarily responsible for the acidification and expansion of the large central vacuole \[[@B12]-[@B15]\]. In plants a critical event in growth and development is the maturation and expansion of the central vacuole. Upon maturation, the vacuolar contents can comprise more than eighty percent of the total cell volume \[[@B16]\]. Primarily filled with water, the vacuole also is a repository for a wide variety of solutes fulfilling numerous important metabolic functions. The influx of water and metabolites into the vacuole is dependent in part on the generation of a proton motive force across the tonoplast.

Vacuolar type H^+^-ATPases are large, multimeric enzyme complexes of 500--750 kDa consisting of 15 or more different proteins. Structurally, they can be divided into integral (V~0~) and peripheral (V~1~) membrane sectors \[[@B17]\]. The peripheral membrane sector is located on the cytoplasmic side of the membrane and is composed of at least five different subunits including the catalytic subunit, subunit A. The V~1~ peripheral sector is further divided into a head group and stalk region. The V~0~ integral sector is embedded in the membrane and consists of at least 4 different subunits.

Subunit A of the vacuolar-type H^+^-ATPase is the catalytic subunit. The subunit A protein is approximately 70 kDa in most organisms studied and is a hydrophilic peptide located in the head group of the V~1~ peripheral sector in three copies per holoenzyme \[[@B18]\]. This subunit contains a nucleotide binding motif and functions to bind and hydrolyze ATP \[[@B19]\]. In addition, subunit A contains a highly conserved cysteine residue located within the enzymes\' catalytic center that may be involved in regulation of the holoenzyme \[[@B20]-[@B23]\]. Cloning, sequencing and characteristics of subunit A of the V-Type ATPase from *Arabidopsis thaliana* have been previously reported \[[@B24]\].

Small multigene families of V-ATPase subunit A are known to exist in flowering plants and algae. The catalytic subunit exists as two distinct genes with highly conserved exons and intron boundaries in twelve species of plants \[[@B25]\]. In the flowering plant *Daucus carota* (carrot) evidence exists for two distinct isoforms for the catalytic subunit, one of which is tonoplast specific while the other is localized to the Golgi \[[@B26]\]. In addition, two distinct mRNAs have been isolated from cotton (*Gossypium hirsutum*) \[[@B27]\]. Two subunit A mRNAs are expressed in the unicellular alga *Acetabularia acetabulum*\[[@B28],[@B29]\]. Isoforms of other V-ATPase subunits are also known from plants including subunit B \[[@B27],[@B30],[@B32]\], the proteolipid subunit \[[@B33]-[@B37]\] and subunits D and E \[[@B38]\].

What is lacking in our knowledge of vacuolar type ATPases is a sense of what purpose these multigene family proteins may serve in the cellular milieu and how they participate in the growth, development, and metabolism of higher plants. It has been proposed that multigene families might be encoding functionally distinct isoforms of V-ATPase subunits. These isoforms may be required to provide unique functions or regulatory constraints within the varied micro-compartments requiring V-ATPases \[[@B26],[@B27],[@B34],[@B39],[@B43]\]. In addition, different isoforms may be responsible for targeting to varied subcellular compartments \[[@B10],[@B26],[@B28],[@B36],[@B41],[@B44],[@B47]\]. Furthermore, it is possible that gene family members may behave in a differential manner with respect to environmental stress \[[@B36]\].

Three enzymes are primarily responsible for the maintenance of proton flux across the tonoplast in plants, the vacuolar H^+^-ATPase, the H^+^ pumping pyrophosphatase \[[@B48]\] and the Na^+^/H^+^ antiporter \[[@B49],[@B50]\]. All three enzymes have been implicated in allowing plants to cope with environmental stress conditions.

Several studies have been conducted in an attempt to evaluate the role vacuolar type ATPases play in allowing plants to cope with environmental stress. These endeavors have primarily concentrated on salt and cold stress. Cold tolerant *Brassica napus,* a close relative of *Arabidopsis thaliana,* demonstrated an increase in both subunit A mRNA and protein in response to chilling at 2°C \[[@B50]\]. This chilling also resulted in a concomitant increase in cell sap osmotic pressure and endogenous ABA accumulation. These researchers indicated that *Arabidopsis* also was a cold tolerant plant which exhibited a similar response with respect to V-ATPase subunit A mRNA, however they provided no data. In rice (*Oryza sativa*) chilling plants at 10°C resulted in a two fold increase in vacuolar type ATPase activity evaluated by in vitro assay of isolated membranes \[[@B51]\]. This study did not analyze changes in protein level in the isolated membranes in response to chilling. Cold sensitive mung bean seedlings (*Vigna radiata*) subjected to chilling at 4°C for 48 hours contrastingly showed no increase in vacuolar type ATPase activity in isolated membranes \[[@B52]\]. In addition, these researchers found no change in the amount of V-ATPase protein levels in response to chilling.

The facultative CAM plant *Mesembryanthemum crystallinum* displays a differential organ level response to salt stress while still engaged in C~3~ photosynthesis \[[@B53]\]. Subjecting plants to 400 mM NaCl resulted in an approximately two fold increase in the transcript levels of subunits A, B, and the proteolipid in roots prior to the shift to Crassulacean acid metabolism. Fully expanded leaves exhibited an increase only for the proteolipid mRNA, an increase of approximately three fold over non salt stressed plants \[[@B54]\]. Cell suspension cultures when subjected to salt stress exhibited increase in activity of both P-Type and V-Type ATPases without concomitant increase in protein levels \[[@B55]\]. Transcript levels of subunit E of the V-ATPase in *Mesembryanthemum crystallinum* were recently shown to increase in leaves but not roots of salt stressed plants with a concomitant increase in protein levels in leaves \[[@B56]\]. On the other hand, subunit E in *Hordeum vulgare* showed no effect on mRNA level in response to salt stress \[[@B57]\]. The effects of salt stress on V-ATPases has also been investigated in tobacco (*Nicotiana tabacum*) suspension cells. Subjecting NaCl adapted cells to salt stress resulted in an approximately four fold increase in both proton transport and ATP hydrolysis on isolated membranes compared to unadapted cells \[[@B58]\]. Remarkably this increase in overall V-ATPase activity coincided with an apparent reduction in the amount of enzyme on the tonoplast. This apparently contradictory finding may be the result of physical changes in the enzyme itself or changes in the lipid environment \[[@B58]\]. Especially confounding is the finding that mRNA levels for subunit A increase two to four fold in response to the same levels of salt stress in tobacco suspension cells \[[@B59]\]. Experiments with tomato (*Lycopersicon esculentum*) showed similar results with respect to induction of mRNA for subunit A with an approximately two to four fold increase seen with respect to salt stress \[[@B60]\]. Recent work in the resurrection plant *Tortula ruralis* indicated that transcript levels of the proteolipid (subunit c) increased in response to salt stress but that there was no corresponding increase in protein levels \[[@B61]\].

The limited work done to evaluate the role of V-ATPases in drought stress has provided conflicting results. Drought stress in winter *Brassica napus* resulted in a very similar response as cold stress, that is a dramatic increase in subunit A mRNA \[[@B50]\]. However, drought stress in *Lycopersicon esculentum* resulted in no apparent increase in mRNA levels for subunit A \[[@B60]\].

None of the previously mentioned efforts into evaluating the role of vacuolar type ATPases in environmental stress acclimation take into account the fact that subunits A, B, and the proteolipid are most certainly present as small gene families in the plants studied. Furthermore, it is possible that these gene family members may behave in a differential manner with respect to stress. The only work published previously on V-ATPase subunit isoforms and stress is concerned with the proteolipid gene family in *Arabidopsis thaliana.* Three proteolipid genes were evaluated for their response to salt stress as well as etiolation \[[@B36]\]. All three genes exhibited an approximately two fold increase in mRNA levels in response to salt stress (50 mM). However, with respect to etiolation two isoforms showed no response while the third demonstrated an increase in mRNA levels.

A number of genes in plants have been found to contain multiple poly (A) attachment sites yielding different transcripts from a single gene. Several examples have recently emerged where differentially polyadenylated genes have transcripts that are post transcriptionally regulated based on sugar sensing. In rice (*Oryza sativa*), two transcripts generated off an α amylase gene exhibit differential stability based on sucrose availability \[[@B65]\]. A cell wall invertase gene (*Incw-1*) in Maize (*Zea mays*) is expressed as two transcripts differing in 3\' ends. The two transcripts are differentially expressed in response to sugars \[[@B66]\]. In this study we explore the possibility that differentially polyadenylated transcripts of the V-ATPase subunit A encoding gene show different responses with respect to etiolation, salt, or chilling stress.

Results and discussion
======================

A vacuolar type H^+^-ATPase subunit A cDNA has been previously identified and sequenced (GenBank accession number U65638) \[[@B24]\]. The cDNA was identified as subunit A of the vacuolar type H^+^-ATPase via identity with other previously sequenced subunit A genes from other organisms. The primary nucleotide and amino acid sequence of the *A. thaliana* subunit A cDNA showed a high degree of identity with subunit A cDNA sequenced from other organisms. This cDNA encoded a putative open reading frame consistent with that of other subunit A genes and was shown to have all the motifs common to subunit A \[[@B67]\]. This high degree of conservation was evident across taxa as diverse as animals, plants, fungi, and protozoa \[[@B68]\].

Genome blot analysis was utilized to determine the number of genes in the *A. thaliana* genome corresponding to subunit A of the vacuolar type ATPase. Genomic DNA was isolated as described, subjected to restriction enzyme digestion, and immobilized on a positively charged nylon membrane. Ten single restriction enzyme digestions were performed. Hybridization was conducted at moderate stringency with a 300 base pair homologous *A. thaliana* cDNA probe corresponding to subunit A of the vacuolar type ATPase. Results indicated hybridization to only a single band for nine out of the ten enzymes (Fig. [1](#F1){ref-type="fig"}). The *Hae*III digest showed faint hybridization to a second band that was later shown to be the result of a *Hae*III site located in the region of the probe (results not shown).

![**Whole genome blot analysis of vacuolar type H^+^-ATPase subunit A gene in *A. thaliana.*** Each lane represents 5 μg genomic DNA digested with the indicated restriction enzyme. Restriction enzymes used were; H, *Hind*III, B, *Bg*lII, D, *Dra*I, K, *Kpn*I, P, *Pvu*II, E, *Eco*RI, Ps, *Pst*I, Ev, *Eco*Rv, Bc, *Bc*lI, and Ha, *Hae*III. Hybridization was to a single band in each lane except for *Hae*III due to the presence of a recognition site in the region corresponding to the probe. Positive hybridization control was 20 pg of unlabeled probe DNA generated via PCR. *Hind*III digested lambda DNA (2 μg) was used as negative control. Numbers to the left of the blot indicate the approximate size of DNA fragments (in kilobases) as determined by *Hind*III digestion of Lambda DNA.](1471-2229-2-3-1){#F1}

All evidences acquired to date failed to indicate the presence of a second V-ATPase subunit A gene in *A. thaliana.* Whole genome blot analysis detected a single gene in the *A. thaliana* genome. Extensive screening of a cDNA library derived from all organs and multiple developmental stages of the plant revealed only a single subunit A cDNA. A polymerase chain reaction based screen failed to detect an intron length polymorphism (results not shown) between two subunit A genes found to be present in other plants \[[@B25]\]. Lastly, extensive database searches of GenBank, the *Arabidopsis* EST database, and the *Arabidopsis* Genome Initiative (TAIR, <>) report only a single subunit A gene in the current version of the *Arabidopsis* genome (chromosome 1 BAC F9K20).

This situation of a single subunit A gene appears to be unusual in plants. Admittedly only a small subset of plants has been examined in detail. Of the species examined to date three have only a single subunit A gene detected, the alga *Coleochaete scutata,* sugar beet (*Beta vulgarise*\[[@B63],[@B64]\], and *A. thaliana*\[[@B25]\]. Flowering plants shown to have at least two subunit A genes include oat (*Avena sativa*), carrot (*Daucus carota*), tobacco (*Nicotiana tabacum*), tomato (*Lycopersicon esculentum*), cotton (*Gossypium hirsutum*), magnolia (*Magnolia virginana*), *Hydrastis canadensis, Chenopodium rubrum,* and clematis (*Clematis liguisticfolio*). In addition, the unicellular alga *Acetabularia acetabulum* has two V-ATPase subunit A genes.

RNA blot analysis was performed to determine the organ level expression pattern of the subunit A gene. Vacuolar type ATPases are housekeeping enzymes critical for the maintenance of homeostasis. Therefore, it is expected that all living plant cells require V-ATPase function. Since *A. thaliana* seemed to possess only a single gene for subunit A of the V-ATPase it was expected that this gene should be expressed in all organs tested. Whole RNA was harvested from leaves, bolts, siliques, flowers, axenically grown seedlings, and axenically grown roots. RNA was denatured with glyoxal, fractionated via agarose gel electrophoresis, and immobilized on a positively charged nylon membrane. Hybridization was conducted at high stringency with a 1700 base pair homologous cDNA probe labeled with digoxigenin. In agreement with our expectations, results indicated high levels of expression in all organs tested (Fig. [2](#F2){ref-type="fig"}) with slightly higher levels of subunit A mRNA present in seedlings. This is most likely due to the fact that seedlings are the most rapidly growing and metabolizing stage of the plant tested. Seedlings are undergoing dramatic and rapid vacuole biogenesis to allow for rapid growth at this stage of development and therefore would be expected to require a higher level of subunit A expression than other more terminally differentiated organs. Roots, leaves, bolts, and flowers showed approximately equivalent amounts of signal with somewhat lower levels in siliques.

![**RNA blot analysis of subunit A in *A. thaliana.*** The probe utilized was approximately 1700 base pairs in length corresponding to the vacuolar type H^+^-ATPase subunit A cDNA from *Arabidopsis thaliana.* Probe DNA was labeled with digoxigenin via the Polymerase Chain Reaction. Each lane contained 20 μg of whole RNA. Lanes correspond to the following organs: S-seedlings, R-roots, L-leaves, B-bolts, Si-siliques, and F-flowers. EF-Tu is a nuclear encoded chloroplast gene transcribed in all organs of the plant. Hybridization to a digoxigenin labeled probe corresponding to EF-Tu was performed to evaluate the RNA isolated from each organ to determine its suitability for RNA blot analysis.](1471-2229-2-3-2){#F2}

Most eukaryotic mRNAs are characterized by the presence of a poly (A) tail, a series of adenine residues post transcriptionally attached to the 3\' untranslated region. The primary function of the poly (A) tail is thought to be in message stability assisted by a series of poly (A) binding proteins \[[@B69]\]. In addition, the poly (A) tail in conjunction with the 5\' cap may be required for efficient translation of plant mRNAs \[[@B70]\].

Subunit A of the vacuolar type H^+^-ATPase in *A. thaliana* was shown to exhibit multi site polyadenylation. Sequencing the 3\' ends of seven independent clones isolated from the PRL-2 cDNA library and one EST obtained through the *Arabidopsis* EST database revealed four different length transcripts produced from the single subunit A gene based on different poly (A) tail attachment sites (Fig. [3](#F3){ref-type="fig"}). Multiple polyadenylation sites are fairly common in plant mRNAs but this phenomenon is rare in animal genes \[[@B71],[@B72]\]. Since the *A. thaliana* V-ATPase subunit A gene produced at least four different transcripts (all producing the same protein) and is one of two flowering plants observed to date not to have a multigene family for this subunit, experiments were designed to determine if muiti site polyadenylation might be a mechanism for gene regulation in plants.

![**Vacuolar type H^+^-ATPase subunit A 3\' untranslated region nucleotide sequence.** Nucleotide sequence is depicted in the 5\' to 3\' direction. The diamond (♦) indicates the coding region stop (TAA). Arrows (↓) indicate sites of Poly (A) tail attachment for the four different transcripts amplified. A total of eight cDNA clones were sequenced from the 3\' end, in each case the poly (A) tail was added at an adenine residue in the DNA sequence. Bold underlined motifs correspond to putative polyadenylation signals.](1471-2229-2-3-3){#F3}

V-ATPase proteolipid isoforms were shown to exhibit differential transcript expression levels in response to environmental stresses \[[@B36]\]. This fact implies a possible role for these isoforms in allowing the plant to deal with stress. Experiments were designed to determine if, in *A. thaliana,* differentially polyadenylated transcripts could be regulated in response to stress.

First, it was critical to confirm that subunit A transcript levels were at all responsive to environmental stress conditions. Three environmental stresses were chosen including 100 mM NaCl, etiolation for four days, and chilling at 6 degrees for four days. In all three situations axenic, seven-day-old seedlings grown on agar were evaluated. Seven day old seedlings were chosen for their short growth time, limited space requirements, and ease in culturing. In addition, the facts that they are rapidly growing and metabolizing, were shown to produce ample quantities of subunit A transcript, and possess several organs including roots, hypocotyls, and cotyledons made them an attractive choice for analysis.

Whole RNA blot analysis was used first to evaluate the overall response of subunit A to stress conditions. RNA was extracted from stress treated seedlings and non-treated control plants. Hybridization was performed as previously described. Results indicated a subunit A transcript response to stress. An approximate 2--4 fold increase in transcript level was detected in seedlings subjected to salt and chilling stress compared to control plants (Fig. [4](#F4){ref-type="fig"}). Etiolated seedlings showed essentially equivalent amounts of subunit A transcript compared to controls. Since etiolation results in rapid elongation of seedlings it was expected that there would be a concomitant increase in subunit A messenger RNA as seen with proteolipid subunit mRNA in *A. thaliana*\[[@B36]\] and subunit A message levels in rapidly expanding cotton (*Gossypium hirsutum*) fibers \[[@B73]\]. Equivalent loading of whole RNA was evaluated via spectrophotometry measuring absorption at 260 nm using a Perkin-Elmer Lambda-3 spectrophotometer and by visual confirmation using non denaturing ethidium bromide stained agarose gel electrophoresis (results not shown).

![**Subunit A transcriptional response to environmental stress conditions in *Arabidopsis* seedlings.** Each lane contained 20 μg whole RNA. The probe used was the same for organ level RNA blot analysis (1700 base pair subunit A cDNA labeled with digoxigenin). Lanes correspond to the following: N-normal, untreated control plants grown at 23°C with 16 hour photoperiod, S-sodium chloride treated plants subjected to 100 mM sodium chloride in the medium, E-etiolation, where plants were germinated in light for three days then transferred to total darkness for four days, C-cold, where plants were germinated under normal conditions for three days and then transferred to a cold room at 6°C for four days.](1471-2229-2-3-4){#F4}

Having established that overall subunit A transcript levels were responsive to environmental stress conditions an assay was designed to examine the response of the four individual lengthed transcripts to the same stress conditions. The goal was to determine if any of the four were differentially expressed in response to stress. A PCR based approach was taken to evaluate transcript levels for the four different subunit A mRNAs. Primer pairs were designed (Table [1](#T1){ref-type="table"}) that would only amplify one of the four transcripts. This was accomplished by anchoring the downstream primer in the poly (A) tail. The upstream primer was located in the coding region of the subunit A cDNA. Anchoring the downstream primer in the poly (A) tail prevented that oligonucleotide from priming at any other site since 10 out of 18 nucleotides were T\'s at the 5\' end. The primer pairs were designed such that the four amplification products would differ in size by approximately 100 base pairs for ease of visualization on agarose gels and blots (Fig. [5](#F5){ref-type="fig"} and Fig. [6](#F6){ref-type="fig"}). Primer pair T-11 and T-12 corresponds to transcript-1 and produce an amplification product of 579 base pairs. Primer pair T-21 and T-22 corresponds to transcript-2 and amplify a band of 455 base pairs. Primers T-31 and T-32 correspond to transcript-3 and amplify a 362 base pair product. Lastly, primer pair T-41 and T-42 corresponds to transcript-4 and produce a 238 base pair amplification product. The transcript-4 amplification product, which is the smallest and nested within the other three, acted as probe to detect all four transcripts. This nesting was accomplished to avoid differences in signal during hybridization based on different probe lengths and labeling efficiency.

![**Subunit A 3\' end transcript control amplification.** Whole RNA was extracted from seedlings and cDNA was generated as described in materials and methods. Polymerase Chain Reaction amplifications were performed with the appropriate primer pairs. Amplification products were fractionated by agarose gel electrophoresis, transferred to solid support and hybridized with a digoxigenin labeled probe corresponding to the transcript-4 amplification product. All four transcripts were successfully amplified. Bands produced from the PCR reaction were all of the appropriate, expected size. In addition, the transcript-4 probe successfully detected all four amplification products.](1471-2229-2-3-5){#F5}

![**Overlapping nature of the four transcript amplification products.** Transcript-4 is the smallest amplification product nested within the other three to be used as a probe to equally detect all four transcripts. Downstream primers are anchored in the poly (A) tail of the message by a series of ten T residues. Thus only the first 8 nucleotides of each downstream primer correspond to nucleotides in the 3\' end of the subunit A gene. Primer pair T-11 and T-12 amplifies a 579 base pair fragment corresponding to transcript-1. Primer pair T-21 and T-22 amplifies a 455 base pair fragment corresponding to transcript-2. Primer pair T-31 and T-32 amplifies a 362 base pair fragment corresponding to transcript-3. Primer pair T-41 and T-42 amplifies a 238 base pair fragment corresponding to transcript-4.](1471-2229-2-3-6){#F6}

::: {#T1 .table-wrap}
::: {.caption}
######

Polymerase Chain Reaction primers. Primers SM-2, SM-3 and VA-1, VA-9 were utilized in the generation of digoxigenin labeled probes for hybridization. Primers T-11 through T-42 were utilized to amplify subunit A transcripts based on the position of the poly A tail.
:::

  **Primer**   **5\' to 3\' sequence**    **Amplification product**         **5\' nucleotide position in cDNA**
  ------------ -------------------------- --------------------------------- -------------------------------------
  SM-2         CCTGGTGCATTTGGTTGTGGAAA    Subunit A cDNA (300 bp probe)     779
  SM-3         CATCATACTAACATTGTAACCCAT   Subunit A cDNA (300 bp probe)     1055
  VA-1         TCTTCGAACACACAAGCCACTTT    Subunit A cDNA (1700 bp probe)    274
  VA-9         AGAGACCATGAATGAAACCCTCT    Subunit A cDNA (1700 bp probe)    1959
  T-11         TTTTTTTTTTACAAAATG         Subunit A, 3\' end transcript-1   2264
  T-12         TGAGAGAGCAGCTGGAAT         Subunit A, 3\' end transcript-1   1702
  T-21         TTTTTTTTTTATCCCCAA         Subunit A, 3\' end transcript-2   2227
  T-22         TCAGAAGTTCGAAGACCC         Subunit A, 3\' end transcript-2   1789
  T-31         TTTTTTTTTTGGAGATAA         Subunit A, 3\' transcript-3       2210
  T-32         CTGGATTCCGTGCTTTGG         Subunit A, 3\' transcript-3       1866
  T-41         TTTTTTTTTTCAAATCAA         Subunit A, 3\' transcript-4       2173
  T-42         GGAGAAAGAGGGTTTCAT         Subunit A, 3\' transcript-4       1952
:::

A control experiment was performed prior to the actual stress analysis to ensure that the primer pairs amplified the correct transcript only and that the transcript-4 probe would detect all four amplification products. Whole RNA was extracted from seedlings and cDNA was generated using oligo dT as described in materials and methods. Subunit A transcripts were amplified from the generated cDNA with the appropriate primer pairs and transferred to solid support. Hybridization was performed at high stringency with the transcript-4 amplification product as probe. The results of this experiment indicated that all four primer pairs amplified only a single band of the appropriate and expected size and that the transcript-4 probe adequately hybridized to all four amplification products (Fig. [5](#F5){ref-type="fig"}).

The differential expression of subunit A transcripts was evaluated in response to salt, cold stress, and etiolation. Complementary DNA\'s were generated from seven-day-old seedlings subjected to individual stress conditions. Transcripts 1--4 were evaluated via RT-PCR using transcript specific primers. Amplification products were immobilized on solid support and hybridized with the transcript-4 amplification product. The results of this experiment indicate that all four transcripts behaved identically with respect to individual stress conditions (Fig. [7](#F7){ref-type="fig"}). All four transcripts showed approximately 2--4 fold increases in response to 100 mM sodium chloride stress compared to untreated controls. Similarly, treatment of plants at six degrees for four days resulted in a similar increase in signal for all four transcripts. Etiolation resulted in slightly higher message levels for transcripts -1, -2, and -3 compared to controls. The lesser degree of increase for transcript-3 might be due to near saturation of the signal. The latter might be due to the higher abundance of the transcript, or due to more efficient amplification of the transcript-3 primer set. Transcript-4 seems to show slightly less signal than control plants but the autoradiograph is somewhat under developed. These results are in agreement with the whole RNA stress blot analysis. Apparent quantitative differences in upregulation between transcripts shown in (Fig. [7](#F7){ref-type="fig"}) might be due to differential amplification efficiency of the four different primer sets and do not necessarily represent true differential upregulation of the four transcripts. The data can only be evaluated quantitatively between samples amplified with the same primer set. In summation, no significant differential regulation of the four subunit A transcripts was detected in seedlings in response to the three stress conditions evaluated.

![**Differential expression of subunit A transcripts in response to environmental stress conditions.** Whole RNA was extracted from stress treated seedlings and control plants. Complementary DNA\'s were generated and transcripts amplified by PCR as described in materials and methods. All sixteen amplification products were fractionated on a single agarose gel, transferred to a solid support and hybridized with a digoxigenin labeled probe corresponding to the transcript-4 amplification product. Multiple time exposures were taken and the best was used for each panel, thus this figure does not represent equivalent time exposures for all four transcripts. These results are of a single experiment which has not been replicated. Lanes correspond to the following: N-normal, untreated control plants, S-sodium chloride treated plants, C-cold, E-etiolation.](1471-2229-2-3-7){#F7}

Conclusions
===========

Evidence gathered to date indicates the presence of a single gene encoding subunit A of the vacuolar type H^+^-ATPase in *A. thaliana.* The search for additional subunit A genes was conducted using whole genome blot analysis, screening a lambda PRL-2 cDNA library, intron length polymorphism analysis, and extensive computer searches of the *Arabidopsis* Expressed Sequence Tag database, GenBank, and the *Arabidopsis* Genome Initiative. Most flowering plants evaluated to date have a small multigene family encoding subunit A of the vacuolar type H^+^-ATPase. The small size of the *A. thaliana* genome may be indicative of a trend toward streamlining of DNA in this plant, which may have eliminated a redundant subunit A gene over evolutionary time. Subunit A genes and subsequently proteins are highly conserved. Therefore, it is not likely that a subunit A gene would have escaped the screen by having a low degree of nucleotide identity.

Vacuolar type ATPases are housekeeping enzymes probably needed in all living plant cells. The single *A. thaliana* subunit A gene was shown by whole RNA blot analysis to be transcribed in all organs of the plant tested. The organs analyzed included roots, leaves, bolts, siliques, and flowers. In addition, relatively high levels of transcript were found in whole seedlings. These results support the notion of a single subunit A gene in *A. thaliana* that is transcribed in all cells of the plant.

Numerous examples are present in the literature \[see \[[@B14]\] and references therein\] of changes in transcript, protein, or activity levels of vacuolar type H^+^-ATPases in response to environmental stress. These results indicate a potential role for the vacuolar type H^+^-ATPase in maintenance of homeostasis during times of environmental stress. Whole RNA blot analysis performed on seedlings exposed to environmental stress conditions indicated that transcript levels of subunit A were responsive to salt and cold stress treatments. Transcript levels were shown to increase approximately 2--4 fold in seedlings treated with cold and salt stress. Etiolation did not produce as significant a change in transcript level as salt and cold stress.

The single vacuolar type H^+^-ATPase subunit A gene detected in *A. thaliana* can produce at least four different length transcripts by using different polyadenylation sites. These transcripts differ only in their 3\' untranslated region and produce identical proteins. The presence of regulatory elements in the 3\'end of the subunit A gene could be a potential mechanism by which this protein is post transcriptionally regulated.

However, the evidence gathered in this work indicates that the four different transcripts generated off the single subunit A gene are not significantly differentially regulated in response to salt, chilling, or etiolation in axenically grown seedlings. Multi site polyadenylation does not appear to be a mechanism for posttranscriptional control of gene expression for subunit A of the vacuolar type H^+^-ATPase in *A. thaliana* for the stress conditions and developmental stage tested.

Materials and methods
=====================

Plant materials
---------------

*Arabidopsis thaliana* ecotype Columbia seeds were obtained from the *Arabidopsis* Biological Resource Center, Ohio State University, Columbus OH. Seeds were germinated in 4-inch pots according to standard protocols. Axenic seedlings, and soil grown plants were maintained in a growth chamber at 23°C under a 16-hour photoperiod until appropriate organs were mature. Axenic seedling cultures were prepared by placing surface sterilized seeds on 90 mm germination plates containing 1% agar and 4.4 g/L MS salts with minimal organics (Sigma Chemical Co. St. Louis, MO), pH 6.0. Seedlings were harvested when 7 days old. Axenic roots were obtained by plating surface sterilized seeds on Nunc square Bio-Assay dishes (Fisher Scientific, Pittsburgh, PA). Plates were allowed to dry under a laminar flow hood, sealed with paraflim and incubated vertically as above. Roots were harvested 7--14 days later.

Growth effects of salt stress were evaluated by germinating surface sterilized seeds on agar containing various concentrations of sodium chloride. The optimal concentration chosen to evaluate the effects of salt stress on V-ATPase subunit A transcript levels was 100 mM. Higher concentrations resulted in failure to germinate or extremely poor growth. At 100 mM salt most seeds germinated but required more time to do so than controls. Plantlets did grow but were less robust than non-treated controls (results not shown). Seedlings were harvested after seven days of growth.

In the case of etiolation stress, surface sterilized seeds were germinated on agar for three days under light and temperature conditions described above. After three days the plates were placed in a light sealed box at the same temperature conditions. Seedlings were harvested four days later. Plantlets exhibited the typical characteristics of etiolation, the hypocotyls were lengthened and the cotyledons reduced compared to light grown controls.

Cold stress was evaluated by germinating surface sterilized seeds as described for three days under normal light and temperature. Agar plates containing seedlings were then transferred to a cold room at 6°C. Seedlings were maintained at this temperature for four days before being harvested.

Isolation of nucleic acids
--------------------------

Genomic DNA was extracted from mature leaves using a miniprep protocol \[[@B74]\] modified by the addition of a CTAB extraction \[[@B75]\]. *A. thaliana* leaves were harvested, hand washed and then ground to a fine powder in liquid nitrogen. Ground material was used immediately for DNA extraction or stored at -80°C. Post extraction samples were treated with RNAse A at 37°C for 15 minutes to remove contaminating RNA.

Organ specific whole RNA extraction was performed as described \[[@B75]\]. Specific organs were harvested and immediately ground to a fine powder in liquid nitrogen. Special care was taken in the case of siliques and flowers to harvest the organs directly into liquid nitrogen to prevent degradation of the RNA. Standard precautions were taken to eliminate ribonuclease contamination in solutions and glassware including baking all glassware at 200°C for six hours, and treating all solutions (except those containing Tris) with 0.1% diethylpyrocarbonate for 12 hours followed by autoclaving.

Nucleic acids were quantitated by spectrophotometry and via inspection of ethidium bromide stained agarose gels.

Genome blot analysis
--------------------

Genomic DNA (5--10 μg) was digested with restriction enzymes according to the manufacturers recommendations. Digested DNA was size fractionated via gel electrophoresis in 12 cm gels containing 0.8% agarose run in IX TAE or TBE at 25 volts for 12 hours. Fractionated DNA in gels was depurinated, denatured, and neutralized according to standard protocols \[[@B76]\]. DNA was transferred to positively charged nylon membranes (Hybond N^+^) (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) by the wick transfer method using 20X SSC overnight. Membranes were baked at 80°C for 1.5 hours.

Hybridization was performed according to a procedure from \[[@B77]\] modified as described below. Membranes were first washed for 5 minutes in 5X SSC at room temperature followed by 1 hour at 65°C in 1X SSC/0.1% SDS. Membranes were then pre-wet for 10 minutes in wash buffer (20 mM Na~2~HPO~4~ pH-7.2, 1 mM EDTA, 1% SDS). Prehybridization was performed at 67°C for two hours in a seal-a-meal bag (Dazey Corporation, Industrial Airport, KS) with hybridization solution (0.25 M Na~2~HPO~4~ pH-7.2, 1 mM EDTA, 20% SDS with 0.5% blocking reagent-Boehringer Mannheim, Biochemical Products, Indianapolis IN) at a volume of 125 μl/sq. cm. of membrane and with formamide added to a final concentration of 25% and 15 μg/ml boiled salmon sperm DNA. Hybridization was performed overnight at 67°C in a sealed bag with 125 μl/sq cm fresh hybridization solution containing formamide and salmon sperm DNA as above with the addition of 40 ng/ml digoxigenin labeled probe denatured by boiling for 5 minutes. The probe utilized was a 300 base pair digoxigenin labeled PCR product.

Membranes were washed after hybridization four times at 65°C for 30 minutes each with vigorous agitation in wash buffer. Membranes were then soaked for 10 minutes in maleate buffer (0.1 M maleic acid, 3 M NaCl, 0.3% Tween 20, pH 8) at room temperature followed by blocking for two hours at room temperature in maleate buffer with 0.5% blocking reagent (Boehringer Mannheim, Biochemical Products, Indianapolis IN). Blocked membranes were sealed in a bag with 125 μl/sq. cm. of maleate buffer with 0.5% blocking reagent to which was added anti digoxigenin Fab fragment antibody (diluted 20,000:1) conjugated with alkaline phosphatase (Boehringer Mannheim, Biochemical Products, Indianapolis IN). After incubation for 30 minutes at room temperature membranes were washed in maleate buffer four times for 15 minutes each at room temperature. Next, membranes were soaked for 10 minutes in substrate buffer (100 mM TRIS, 100 mM NaCl, 50 mM MgCl~2~ pH 9.5) at room temperature. Incubation was then performed for ten minutes at room temperature with CDP-Star chemiluminescent substrate (Boehringer Mannheim, Biochemical Products, Indianapolis IN) diluted 1:100 in substrate buffer. Membranes were then exposed to X-ray film.

Whole RNA blot analysis
-----------------------

RNA was fractionated via denaturing agarose gel electrophoresis using glyoxal according to standard protocols \[[@B76]\]. Immediately before use the glyoxal was deionized by vortexing in the presence of mixed bed resin beads (Sigma Chemical Co. St. Louis, MO) until the pH was greater than 5. Determination of pH was made using Hydrion pH sensitive paper (Fisher Scientific, Pittsburgh, PA).

Agarose gels (0.8%, 12 cm) cast in 10 mM sodium phosphate buffer were run for 2 hours at 100 volts with the buffer recirculated by a peristaltic pump. RNA gels were blotted using the same procedure as DNA gels omitting the depurination, denaturation, and neutralization steps. In addition, hybridization and chemiluminescent immunodetection were also performed using the same protocol for genome blot analysis except for the following. Just prior to prewetting in wash buffer for prehybridization RNA membranes were given two deglyoxylation washes in 20 mM Tris pH 8 for twenty minutes each at 65°C. The probe used for whole RNA blot analysis was a 1700 base pair PCR product.

Generation of complementary DNA
-------------------------------

Synthesis of cDNA was accomplished using MMLV reverse transcriptase (Gibco BRL Life Technologies, Bethesda, MA) and oligo dT according to the manufacturers instructions. Whole RNA was diluted to a concentration of 0.67 μg/μl in a total volume of 10--15 μl. The sample was heated at 90°C for 5 minutes and chilled on ice. The reverse transcription reaction mixture included the following components in a total volume of 20 μl. Nucleotides (dNTP\'s) (Amersham Pharmacia Biotech, Piscataway, NJ) were added to a final concentration of 0.5 mM. Forty units of a ribonuclease inhibitor (RNAsin) (Promega, Madison, WI) were added. Denatured, whole RNA (2 μg) was added in a volume of 3 μl. Oligo dT primer (1.1 μl) was added to a final concentration of 1.5 μM. Dithiothreitol was added to a final concentration of 10 mM. Dilution buffer (5X concentration, provided by the manufacturer) was added to a final concentration of 1X. After all the components were assembled, 200 units of MMLV reverse transcriptase (Gibco BRL Life Technologies, Bethesda, MA) were added. The sample was incubated at 42°C for 1 hour followed by heat denaturation of the enzyme at 95°C for 10 minutes. Samples were stored at -20°C.

Polymerase Chain Reaction amplifications
----------------------------------------

In general, PCR reactions were performed in a volume of 50 μl with 50 picomoles of each primer, dNTP\'s (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) at a final concentration of 0.5 mM, 1 μl of template, 1X PCR buffer (10X PCR buffer contained 700 mM Tris pH 8.8, 20 mM MgCl~2~, 1% Triton X-100, and 0.1% Tween 20) and Taq polymerase (Perkin-Elmer Inc., Wellesley MA) diluted to the manufacturers recommendations. The reaction components were assembled except Taq polymerase and heated at 95°C for 5 minutes. Polymerase was added and the PCR allowed to cycle for 1 minute at 94°C, 1 minute at 55°C and 1.5 minutes at 72°C for 30 cycles followed by 10 minutes at 72°C.

All digoxigenin labeled probes for non-radioactive detection were generated using the Polymerase Chain Reaction. Probes were labeled using 10X PCR labeling mix (Boehringer Mannheim, Biochemical Products, Indianapolis IN) at a concentration of 1X replacing the normal dNTP mix. In each case 1 μl of template was added to the reaction mix. Template was created by amplifying a band off an Expressed Sequence Tag (EST G~5~B~3~T~7~) from the *Arabidopsis* expressed sequence tag database \[[@B78]\], fractionating the product on a low melting point agarose gel, and excising the band. The excised, diluted band was re-amplified to ensure fidelity of the PCR. After successful re-amplification the template was utilized for probe generation.

A 300 base pair cDNA probe corresponding to nucleotide residues 779--1078 of the *A. thaliana* vacuolar type H^+^-ATPase subunit A gene was generated using primers SM-2 and SM-3 (Table [1](#T1){ref-type="table"}). This probe was used for whole genome blot analysis. An approximately 1700 base pair cDNA probe corresponding to nucleotide residues 274--1981 of subunit A was generated using primers VA-1 and VA-9. This probe was used for multi organ RNA blot analysis and stress response northerns. In both cases template was generated off EST G~5~B~3~T~7~. Cycling parameters for both probes were identical to those described above.

Probes were precipitated by adding 1 μl of tRNA (10 mg/ml) or 1/50^th^ volume of glycogen (10 mg/ml in sterile distilled water) to the PCR reaction followed by 1/10^th^ volume of sterile 4 M lithium chloride. Next, 3 volumes of ice-cold absolute ethanol were added and the DNA precipitated for 30 minutes at -80°C. DNA was pelleted by centrifugation at 4°C for 15 minutes. Pellets were washed with ice cold 70% ethanol, air dried for 15 minutes and resuspended in 50 μl TE buffer/0.1 % SDS. Probes were quantitated by fractionation of 1/10^th^ volume in a 0.8% agarose gel compared to a known quantity of *Hind*III digested lambda DNA. Probes were stored at -20°C until use.

Four different subunit A transcripts were amplified using RT-PCR. Primer pairs were designed with one upstream coding region primer and one primer anchored in the poly (A) tail of the transcript. In all four cases template was 1 μl of seedling cDNA. Amplification reaction mixtures were assembled as described, with PCR buffer modified to contain only 1 mM magnesium chloride. Transcript 1 was amplified using primers T-11 and T-12 (Table [1](#T1){ref-type="table"}). Transcript 2 was amplified using primers T-21 and T-22. Transcript 3 was amplified using primers T-31 and T-32. Transcript 4 was amplified using primers T-41 and T-42. The PCR was allowed to proceed in each case for 30 cycles of 1 minute at 94°C, 1 minute at the annealing temperature, and 1 minute at 72°C. Primers amplifying transcripts 1 and 2 were annealed at 35°C, primers amplifying transcripts 3 and 4 were annealed at 40°C. Following 30 cycles, primer extensions were completed at 72°C for 10 minutes. PCR products were then fractionated on 2.5% agarose gels.

The transcript 4 amplification product was used as template for the generation of a digoxigenin labeled probe for use in the subunit A transcript stress response experiment. The transcript 4 amplification product was purified via low melting point agarose gel electrophoresis and used as template for probe generation. The digoxigenin labeled probe was generated as described using the transcript 4 cycling parameters and primers T-41 and T-42.

DNA sequence of all PCR primers used in this research can be found in Table [1](#T1){ref-type="table"}. A diagrammatic representation of the PCR amplification products generated from transcript specific primers is shown in fig. [6](#F6){ref-type="fig"}.

Abbreviations
=============

H^+^-ATPase-proton pumping adenosine triphosphatase, V-ATPase-vacuolar type proton pumping adenosine triphosphatase, EST-expressed sequence tag, PCR-Polymerase Chain Reaction, RT-PCR-reverse transcriptase-Polymerase Chain Reaction.

Acknowledgments
===============

The authors wish to thank Shenell Antrobus and Alex Delcampo for their excellent technical assistance. This research was supported through NSF grant BSR 9020868 and by the University of Connecticut Research Foundation.


Background
==========

Sequencing of Expressed Sequence Tags (ESTs) has resulted in the rapid identification of expressed genes \[[@B1]\]. ESTs are single-pass, partial sequences of cDNA clones from a large number of disease and normal tissue libraries. ESTs have been used extensively for gene discovery and for transcript mapping of genes from a wide number of organisms \[[@B2]-[@B4]\]. Even with the finished working draft of the human genome, the generation of a complete and non-redundant catalog of human genes is still a big challenge facing the genome research community. Full-length cDNA data are currently available for only 10,000 human genes \[[@B5]\], less than one-third of the total using the most conservative recent estimates of human gene numbers \[[@B6],[@B7]\]. Evidence of differential expression is one of the most important criteria in prioritizing the exploitation of genes in both academic and pharmaceutical research \[[@B8]-[@B10]\].

While identifying individual differentially expressed genes attracts most of the interest, a genome wide transcriptome map may not only provide a tool to identify candidate genes that are over-expressed or silenced in certain disease tissue, but may also help to understand the structure and organization of the genome. Genomes are the blueprints of life and they should not be considered as a simple collection of genes. In fact, the organization of genes into operons, complex regulons \[[@B11]\], or pathogenicity islands \[[@B12]\] suggests that related functions usually share physical proximity. Different types of transcriptome maps can help to identify different types of transcription domains. Those domains can now be analyzed as to how they relate to known nuclear substructures, such as nuclear speckles, PML bodies and coiled bodies \[[@B13]-[@B15]\].

Two strategies have been commonly used to evaluate large-scale gene expression: experimental and computational. The former is represented by DNA microarray technology \[[@B16]\]. Computational methods consist of generating a large number of random ESTs from non-normalized cDNA libraries. The variation in the relative frequency of those tags, stored in databases, are then used to point out the differential expression of the corresponding genes: this is the so called \"digital Northern\" comparison. Digital Northern data can be used to provide quantitative assessment of differential expression within a certain limit \[[@B17]\]. Velculescu et al. \[[@B18]\] introduced another digital method called serial analysis of gene expression (SAGE). The SAGE method requires only nine nucleotides, therefore allowing a larger throughput. In both protocols, the number of tags is reported to be proportional to the abundance of cognate transcripts in the tissue or cell type used to make the cDNA library.

The recently announced first draft of the human genome \[[@B19],[@B20]\] holds in it an unprecedented wealth of information, available for public study and scrutiny. How are genes organized in the human genome? Is there any distribution pattern of tissue specific genes in terms of chromosomal location? In this study, we combined the concept of digital Northern and transcript mapping for all public and Incyte LifeSeq ESTs to evaluate the tissue specific transcriptome. The goal of this paper is not to evaluate the digital expression of individual genes; instead we are looking at the tissue enriched digital expression level for a given chromosomal region. Particularly, we looked at the distribution pattern of brain-enriched genes in the genome and how that pattern changes in brain tumor tissues. We are well aware of the fact that this method and associated approaches are quite primitive. However, the tissue specific transcriptome data strongly suggest that human genome organization is correlated to the tissue type and its dynamics.

Results
=======

Distribution of brain-enriched genes along the chromosomes
----------------------------------------------------------

With the unavailability of the complete annotated human gene catalog, it is not practical to document each individual gene that is expressed in brain within one chromosome region. Since the number of sequence tags is reported to be proportional to the abundance of cognate transcripts in the tissue or cell type used to make a given cDNA library, the number of ESTs within a chromosome region should reflect the abundance of the cognate transcripts in that region. Therefore comparison of the abundance of brain tissue derived transcripts relative to those from other tissues within the same chromosome region can highlight regions that have more brain-enriched gene expression.

We performed digital expression analysis of brain-enriched genes across the human genome with a window size of 5 Mbp and an interval of 1 Mbp. The transcript density factor for normal (non-tumor) brain libraries (TDF~NB~) was calculated as described in Methods. Figure [1](#F1){ref-type="fig"} is an example of the distribution of TDF~NB~ over chromosome 1 using publicly available EST sequences from dbEST and reveals a number of \"peaks\" that represent transcripts that appear to be preferentially expressed in brain tissues. To check the validity of these peak regions and make sure that the difference is not due to random picking or partial sequencing of cDNA libraries (which is the common random fluctuation caused by digital Northern approach) \[[@B17]\], the analysis was repeated using ESTs and the associated library information from the Incyte Genomics LifeSeq database. The distribution pattern of TDF~NB~ shows an overall correlation coefficient of 0.658 for the whole genome between these two data sources. If we only analyze the region with Z-score \>= 2 (i.e. peak regions), the correlation coefficient is 0.935 which suggests that the peak regions resulting from the analysis of public data are most likely not artifacts. Figure [2](#F2){ref-type="fig"} shows the comparison of the distributions of TDF on chromosome 1 calculated from ESTs derived from brain tissue libraries vs. ESTs derived from breast tissue libraries. The overall pearson correlation coefficient for these two tissues is 0.113 which suggests that the peak regions observed in Figure [1](#F1){ref-type="fig"} are brain specific.

![Comparison of distribution of TDF~NB~ calculated from data derived from dbEST and Incyte Genomics LifeSeq for chromosome 1. Pearson Correlation Coefficient = 0.658.](1471-2164-3-10-1){#F1}

![Comparison of distribution of TDF on chromosome 1 calculated from ESTs derived from brain tissue libraries vs. ESTs derived from breast tissue libraries. Pearson Correlation Coefficient = 0.113.](1471-2164-3-10-2){#F2}

There are 16 high TDF~NB~ regions (enriched with brain specific expression) with Z-score \>= 2 (Table [1](#T1){ref-type="table"}). To assess the validity of our finding using public data, similar analysis using Incyte LifeSeq data shows that the same peaks can be derived (data not shown). Table [2](#T2){ref-type="table"} summarizes all the low TDF~NB~ regions (lack of brain specific expression) over the whole genome with Z-score \>= 2. It\'s interesting to note that the majority of high TDF~NB~ or low TDF~NB~ regions have close to average gene density indicating that those regions are not biased toward extremely high or low gene density.

::: {#T1 .table-wrap}
::: {.caption}
######

Summary of chromosome regions with significant brain-enriched gene expression (high TDF~NB~ region) (window size 5 Mbp, Z-score \>= 2.0). All regions are confirmed by separate analysis using Incyte LifeSeq ESTs. Pearson Correlation Coefficient = 0.935.
:::

  Chromosome   Region(Mbp)   TDF~NB~   Gene Density Ratio
  ------------ ------------- --------- --------------------
  1            208--212      0.68      0.33
  2            14--18        0.46      0.76
  3            90--96        0.39      0.58
  5            184--188      0.30      1.0
  6            82--86        0.32      0.79
  6            138--142      0.28      0.67
  7            73--77        0.56      0.59
  10           145--149      0.43      0.1
  12           85--89        0.36      0.75
  13           65--69        0.35      0.71
  15           21--26        0.69      0.78
  18           80--87        0.88      0.83
  20           8--15         0.54      0.52
  22           46--50        0.30      0.17
  X            103--109      0.43      0.58
  X            150--155      0.57      0.25
:::

::: {#T2 .table-wrap}
::: {.caption}
######

Summary of chromosome regions lacking in brain-enriched gene expression (low TDF~NB~ regions) (window size 5 Mbp, Z-score \>= 2.0). All regions are confirmed by separate analysis using Incyte LifeSeq ESTs. Pearson Correlation Coefficient = 0.935.
:::

  Chromosome   Region (Mbp)   TDF~NB~   Gene Density Ratio
  ------------ -------------- --------- --------------------
  1            117--111       -0.80     1.11
  1            218--222       -0.56     0.50
  1            229--233       -0.62     0.79
  2            92--99         -0.99     1.79
  3            175--179       -0.76     1.0
  3            217--211       -0.97     0.76
  4            75--81         -1.31     2.78
  7            152--159       -0.80     1.26
  9            117--122       -0.76     0.99
  10           57--61         -0.82     0.62
  11           55--59         -1.24     0.25
  11           111--117       -0.92     0.94
  12           55--60         -0.71     4.15
  14           111--116       -1.08     0.81
  19           30--35         -0.60     0.41
  19           53--57         -0.62     1.58
  21           7--11          -1.83     0.03
  22           10--14         -0.43     0.10
:::

Expression profile change of some chromosome regions with extreme TDF~NB~ in brain tumor
----------------------------------------------------------------------------------------

Our analysis strongly suggests that brain-enriched genes are distributed throughout the genome in a non-random fashion. Some regions are dense with brain-enriched genes or brain specific expression. It would be interesting to know if any of these patterns change during tumorgenesis. A similar analysis was performed using ESTs generated from brain tumor libraries and their digital expression profile relative to the pooled tissue was plotted against the genome. The chromosomal distributions of these putative brain tumor enriched transcripts and the normal brain enriched transcripts are quite different. Table [3](#T3){ref-type="table"} lists all the chromosome regions with high TDF in non-tumor brain libraries (TDF~NB~) which become low TDF or neutral TDF in brain tumor (TDF~TB~). Chr15, 21--25 Mbp, Chr12, 85--89 Mbp, and Chr18, 45--52 Mbp (Figure [3](#F3){ref-type="fig"}) are some of the examples. While most of the low TDF regions in normal brain remain low in brain tumor, a few regions did become high TDF regions in brain tumor tissues (Table [3](#T3){ref-type="table"}). Chr2, 93--99 Mbp and Chr19, 53--58 Mbp (Figure [4](#F4){ref-type="fig"}) are two examples. The digital expression profile in those regions was further confirmed by using data from Incyte LifeSeq (data not shown).

![Differential expression between tumor tissues vs. non-tumor tissues (chromosome 18). a) An example of transition from a brain high TDF region on chromosome 18 (45 Mbp--52 Mbp) in normal tissue libraries to low TDF region in brain tumor libraries; b) Corresponding non-tumor breast tissue vs. tumor breast tissue transcriptome map on the same chromosome. The brain high TDF region observed that changed to low TDF region in a) was not observed in b).](1471-2164-3-10-3){#F3}

![Differential expression between tumor tissues vs. non-tumor tissues (chromosome 19). a) An example of transition from a brain low TDF region on chromosome 19 (53 Mbp--58 Mbp) in normal tissue libraries to high TDF region in brain tumor libraries; b) Corresponding non-tumor breast tissue vs. tumor breast tissue transcriptome map on the same chromosome. The brain low TDF region observed that changed to high TDF region in a) was not observed in b).](1471-2164-3-10-4){#F4}

::: {#T3 .table-wrap}
::: {.caption}
######

High TDF~NB~ and low TDF~NB~ (Z-score \>= 2) regions which show significant differential expression between tumor brain tissues and non-tumor brain tissues.
:::

  Chromosome   Region (Mbp)   TDF~NB~   TDF~TB~
  ------------ -------------- --------- ---------
  2            14--18         0.45      0.06
  2            91--99         -0.93     0.11
  10           58--62         -0.74     0.92
  12           85--89         0.36      -0.56
  13           54--61         -0.78     0.19
  15           21--25         0.74      0.21
  18           45--52         1.0       -1.63
  19           53--58         -0.64     1.8
  20           10--15         0.64      -0.62
  22           30--34         -0.48     0.15
  X            103--109       0.43      0.09
  X            24--28         -0.72     -0.11
:::

Discussion
==========

A genome is not a simple collection of genes. It has been reported that significant correlation exists between the distribution of genes along the chromosome and the physical architecture of the cell in bacteria \[[@B21]\]. The human genome is much more complex and a complete understanding of its organization awaits completion of the finished sequence as well as a definitive annotation of the human gene catalog. Considerable evidence has already shown that related genes tend to exist as clusters in the genome. For example, 80% of the over 900 olfactory genes are found in clusters of 6--138 genes \[[@B22]\]. The 3.6 Mbp human major histocompatibility complex (MHC) on chromosome 6p21.3 is a critical repository for the immune response genes \[[@B23]\]. Extensive analysis of the genomic organization of the MHC region has revealed that at least 27 of its resident genes possess duplicated copies in at least one of the three other restricted chromosomal regions 1q21-q25, 9q33-q34 and 19q13.1-p13.4. For another example, ABC transporter gene family members are located on 6p21.3, 1q25 and 9q34 as clusters.

The development of distinct tissue and cell types is a fundamental characteristic of growth in higher organisms. Tissue and cellular differentiation, in turn, is highly dependent on specific patterns of gene expression and transcript accumulation. Many studies have been successfully used to pinpoint genes exhibiting tissue or disease specific expression. This study suggests another approach that focuses on the tissue specific transcriptome map study and attempts to study the genomic proximity of tissue specific genes. We show here that some regions on specific chromosomes are enriched with brain-enriched gene expression. Given the chromosome window size (5 Mbp) used to calculate the TDF and the average size of the gene (\~30 Kbp), each 5 Mbp region should contain on average about 170 genes. In addition, we report here only regions that have a normal level of gene density. Therefore the very high TDF~NB~ regions are most likely contributed by the high level expression of brain-enriched gene clusters or the regions containing a high density of brain-enriched genes.

We are aware of the importance of those brain-enriched genes that scatter across the genome whose brain specific or differential expression is diluted by the large volume of neighbor genes within the 5 Mbp window region. Another limitation of this approach is the inability to reveal some regions with equal up-down differential expression. The goal of this study is to pinpoint the chromosomal regions with the most significant brain-enriched gene expression and to elucidate the non-randomness of the tissue specific expression over the chromosome. Naturally, gene proximity within chromosomes is already known to be significant. Finding neighbors of a given gene can shed light on that gene, especially when the neighbors contain objects with similar features. Therefore, this tissue specific transcriptome map study may not only help us to understand the genome organization in the future, but may also provide means to gain leads to the functions of many genes for which this information is not currently available.

Methods
=======

Resources for databases and computer programs
---------------------------------------------

GenBank release 120 was downloaded from <>. ESTs and their associated tissue library information and clone information were extracted and organized in a relational database (Sybase, SQL Server Release 11.0, CA, Sybase Inc.). The EST cDNA libraries were manually curated and catalogued into non-tumor brain libraries and brain tumor libraries. We obtained 208604 dbEST ESTs from 369 non-tumor brain cDNA libraries, 67351 ESTs from 148 brain tumor cDNA libraries. We also extracted 363473 ESTs from 100 Incyte LifeSeq non-tumor brain cDNA libraries and 126538 ESTs from their 23 brain tumor libraries. All non-commercial software used in this study was written in PERL 5.0.

Transcript mapping
------------------

Transcript mapping was done based on the October 2000 Freeze of the University of California at Santa Cruz\'s working draft sequence <>, which presents a tentative assembly of the finished and draft human genomic sequence based on the Washington University-Saint Louis clone map <>. We mapped all the public ESTs (2.5 millions ESTs) from dbEST as well as the ESTs from Incyte Genomics\' LifeSeq database (5.1 millions ESTs) using a local alignment software package AAT. AAT is a local alignment software which extended the BLAST algorithm by assigning fixed penalty to long gaps \[[@B24]\]. To reduce the number of undesirable matches due to interspersed repeats, the DNA sequence is screened for interspersed repeats using the RepeatMasker program (Smit, AFA & Green, P et al <>). Only those ESTs that have over 95% identity to the genomic counterpart over half the length of ESTs\' length or 50 bp whichever is longer are included.

Calculation of the transcript density factor (TDF)
--------------------------------------------------

The TDF for normal brain-enriched gene expression (TDF~NB~) is calculated using 5 Mbp window moving along the chromosome with 1 Mbp interval and defined as:

TDF~NB~ = ln(R~NB~/R)/(T~NB~/T)

Where

R~NB~ is the number of EST clones derived from non-tumor brain tissue within a window of 5 Mbp.

R is the number of EST clones derived from all non-tumor tissue pooled libraries within a window of 5 Mbp.

T~NB~ is the number of total mapped EST clones derived from non-tumor brain tissues.

T is the number of total mapped EST clones derived from all non-tumor tissue pooled libraries.

The distribution of TDF~NB~ of all the 5 Mbp regions should approximate a Gaussian distribution. Theoretically, TDF~NB~ should approach 0 if the expression level of genes in brain tissues has no difference from that in pooled tissues within a 5 Mbp chromosomal region. We define all the regions with high TDF~NB~ (Z-score \>= 2) as brain-enriched regions. Those chromosome regions that have high brain-enriched gene expression are referred as high TDF~NB~ region. Those chromosome regions that have low brain-enriched gene expression are referred as low TDF~NB~ region.

The calculation of transcript density factor for brain tumor (TDF~TB~) is similar to the calculation of TDF~NB~.

TDF~TB~ = ln(R~TB~/R)/(T~TB~/T)

Where

R~TB~ is the number of EST clones derived from tumor brain tissue within a window of 5 Mbp.

R is the number of EST clones derived from all non-tumor tissue pooled libraries within a window of 5 Mbp.

T~TB~ is the number of total mapped EST clones derived from tumor brain tissue libraries.

T is the number of total mapped EST clones derived from all non-tumor tissue libraries.

Correlation analysis of distribution pattern of TDF using public and Incyte data
--------------------------------------------------------------------------------

The Pearson correlation coefficient (r) represents the degree of similarity (strength of correlation) between two sets of data. The correlation coefficient is calculated as follows:

![](1471-2164-3-10-i1.gif)

where X~i~ is TDF values derived from public data and Y~i~ is the TDF values derived from the Incyte data. Values for the Pearson correlation coefficient range from -1 to 1 where zero indicates no correlation, -1 indicates a perfect negative correlation and 1 indicates a perfect positive correlation.

The calculation of gene density
-------------------------------

Using 5 Mbp window moving along the chromosome with 1 Mbp interval, the gene density ratio is defined as UniGene (Version 5.002) \[[@B4],[@B25]\] count divided by the average UniGene count in each 5 Mbp region. Average gene density ratio is equal to 1.0.