Journal of Cell Science 102, 307-314 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

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Mitochondrial gene expression in the human gastrointestinal tract

ANDREW J. S. MACPHERSON1'2'3*, TIMOTHY P. MAYALL1, KERRY A. CHESTER2, ATTA

ABBASI2, IAN FORGACS3, ALAN D. B. MALCOLM2 and TIMOTHY J. PETERS1

'Department of Clinical Biochemistry, King's College School of Medicine, Bessemer Road, London SE5 9PJ, UK2Department of Biochemistry, Charing Cross and Westminster Medical School, Fulham Palace Road, London W6 8RF, UK3 Department of Gastroenterology, Dulwich Hospital, East Dulwich Grove, London SE22, UK

*Author for correspondence at: Department of Medicine, King's College School of Medicine, Bessemer Road, London SE5 9PJ, UK

Summary

In the human gastrointestinal epithelium, in situhybridisation demonstrates that 12 S and 16 S mitochon-drial ribosomal RNAs show maximal steady-state levelson the surface epithelial cells of the normal smallintestine and colon. The mitochondrial mRNAs, cyto-chrome b and NADH dehydrogenase (TV) have auniform distribution throughout the crypt and surface(villus) epithelial cells of the small intestine and colon.Histochemical stains for the activity of the mitochondrialrespiratory chain enzymes succinate dehydrogenase andcytochrome oxidase also show almost uniform activitiesthroughout the crypt-surface epithelial cell axis in thesmall and large intestines. In sections of normal human

oesophagus the levels of mitochondrial ribosomal RNAs,mitochondrial mRNAs and the activities of mitochon-drial respiratory chain enzymes are maximal over thebasal cells of the stratified squamous epithelium. Theseresults show a relative increase in mitochondrial ribo-somal RNA expression compared with mitochondrialmRNAs in surface cells of simple intestinal epithelia.

Key words: mitochondria, gene expression, oesophagus,small intestine, large intestine, 12 S ribosomal RNA, 16 Sribosomal RNA, messenger RNA, NADH dehydrogenase,succinate dehydrogenase, cytochrome oxidase.

Introduction

The human mitochondrial chromosome is a smallcircular (16.5 kb) double-stranded DNA molecule(Anderson et al., 1981; Kaput et al., 1982). Thisencodes some of the enzymes of the mitochondrialelectron transport chain; the mRNAs for these aretranslated into proteins within the mitochondria (Kaputet al., 1982). The mitochondrial chromosome alsocontains the genes for special ribosomal and transferRNAs required for intramitochondrial protein syn-thesis. Other mitochondrial proteins are coded in thenucleus and synthesised on the cytoplasmic ribosomeswith an N-terminal amino acid signal sequence to targetthem for import into mitochondria (Kaput et al., 1982;Viebrock et al., 1982; Pain et al., 1990).

In humans the mitochondrial genes are closelypacked, and there are no introns (Attardi and Schatz,1988). Each of the two DNA strands is transcribed intoa long RNA precursor, which is then processed intosmaller individual RNA molecules (Ojala et al., 1981).Mitochondrial ribosomal RNAs are synthesised as partof the larger RNA transcript and processed into 12 Sand 16 S molecules, which each form part of the twomitochondrial ribosomal subunits (Eperon et al., 1980).The mitochondrial rRNAs are smaller than the 18 S and

28 S cytoplasmic rRNAs (coded in the nucleus); theyalso have quite distinct sequences (Eperon et al., 1980).Protein synthesis in the mitochondria also showsdifferences in the 'universal' genetic code betweenmRNA and protein; for example, in mitochondria thetriplet AUA codes for methionine whereas in thecytoplasm it would denote isoleucine (Fearnley andWalker, 1987).

Mutations and deletions of mitochondrial DNA havebeen demonstrated in the pathogenesis of a number ofneuromuscular diseases including: (i) the syndrome ofmitochondrial myopathy, encephalopathy, lactic aci-dosis and stroke-like episodes (MELAS) (Goto et al.,1990); (ii) myoclonic epilepsy and ragged red fibredisease (Shih et al., 1991; Shoffer et al., 1990);(iii) chronic progressive external opthalmoplegia in-cluding Kearns-Sayre syndrome (Larsson et al., 1990);(iv) Parkinson's disease (Shapira et al., 1990).

The expression of mitochondrial genes has largelybeen studied in cell culture systems (Attardi and Schatz,1988), where mitochondrial ribosomal RNAs are tran-scribed at a rate 15-60 times higher than mitochondrialmRNAs (Attardi and Schatz, 1988; Kruse et al., 1989).Relatively little is known about mitochondrial geneexpression in differentiated tissues, but in both rathepatocytes (Cantatore et al., 1984) and cerebellar cells

308 A. J. S. Macpherson and others

(Renis et al., 1989) there is an order of magnitude moremitochondrial mRNA compared with mitochondrialrRNA than is observed in HeLa cells. Moreover, mito-chondrial RNA levels decline relative to mitochondrialDNA during development of rat neural cells, and thenrise again in senescence (Polosa and Attardi, 1991).

In this work we have studied mitochondrial geneexpression in biopsies from the human gastrointestinaltract using in situ hybridisation. The levels of mitochon-drial mRNAs and rRNAs have been assessed inintestinal epithelial cells, which are derived frompluripotential stem cells near the base of the crypts anddifferentiate as they migrate towards the luminalsurface (Gordon, 1989). There are abundant mitochon-dria in crypt and villus epithelial cells in both the smalland large intestines (Pittman and Pittman, 1966; Toner,1968; Kaye et al., 1973). The levels of differentmitochondrial transcripts have distinct distributionsalong the crypt-villus axis, which have been comparedwith mitochondrial respiratory chain enzyme activities.

Materials and methods

Intestinal biopsiesFour biopsies of the mid oesophagus and low duodenum weretaken from 18 patients in whom no abnormality was detected,during endoscopy with an Olympus IT20 wide-channelendoscope. These were fixed in neutral buffered formalin for18 hours before dehydration and mounting in paraffin wax; nohistopathological abnormality was detected in any of thesespecimens, and a final clinical diagnosis of non-ulcer dyspep-sia was reached in each case. In 7 patients, 4 biopsies at eachsite were flash frozen in isopentane cooled in liquid nitrogen,and stored dry mounted on card at —70°C.

Colonic biopsies were taken with an Olympus CF20Lcolonoscope from 28 patients, and fixed in neutral bufferedformalin or flash frozen (6 patients) as described above.Rectal biopsies were taken from 9 patients using St. Marksforceps via a rigid sigmoidoscope. No histological abnormalitywas observed in any of the colonic biopsies and nobiochemical or haematological abnormalities were found inany patient; a final clinical diagnosis of irritable bowelsyndrome was made in each case.

Informed consent was obtained from all patients, and thestudy was approved by the ethical committee of theCamberwell Health Authority.

In situ hybridisationTissue sections (4 /«n) were cut from formalin-fixed paraffinwax embedded tissue blocks onto 3-aminopropyltriethoxy-silane-treated slides. Rigorous precautions were takenthroughout the experimental procedure to eliminate exogen-ous ribonuclease (Sambrook et al., 1989); all glassware wasbaked overnight at 250°C, and solutions (apart from Trisbuffers) were treated with 0.1%(v/v) diethylpyrocarbonate(DEPC) overnight and autoclaved. The sections were rehy-drated into DEPC-treated water, and pretreated with 0.2 MHCI at room temperature for 20 minutes, followed by 2xSSC(Sambrook et al., 1989) at 70°C for 10 minutes beforedigestion with proteinase K (Sigma) at 4-6 ^g/ml in 50 mMTris-HCl (pH 7.6) in a humid chamber for 1 hour at 37°C. Thisreaction was stopped by immersion in 0.2% (w/v) glycine atroom temperature for 1 minute. Separate sections from each

Fig. 1. In situ hybridisation of formalin-fixed paraffin-embedded tissue sections of normal human colonic mucosawith cDNA probes to mitochondrial ribosomal RNA, 18 Scytoplasmic ribosomal RNA and cytochrome bmitochondrial mRNA. (A) 16 S mitochondrial rRNAdetected by autoradiography of 32P-labelled probe showinggrains predominantly over the surface epithelial cells(arrow) (x40); (B) 12 S mitochondrial rRNA detected byautoradiography (arrow) (x40); (C) Control sectionpretreated with DNase-free ribonuclease and hybridisedwith ^P-labelled probe for 16 S mitochondrial rRNA(x60); (D) 16 S mitochondrial rRNA detected bydigoxigenin-labelled probe and anti-digoxigenin antibodyconjugate showing dark nitroblue tetrazolium staining overthe surface epithelial cells (arrow) (x40); (E) digoxigenin-labelled probe to IgA heavy-chain mRNA showinghybridisation to plasma cells in the lamina propria (arrow,dark signal), with complete blocking of endogenousalkaline phosphatase activity, and staining reaction onepithelial cells (x40); (F) ^P-labelled probe to IgA heavy-chain mRNA showing hybridisation to plasma cells in thelamina propria (xl50); (G) 32P-labelled probe for 18 Scytoplasmic rRNA detected by autoradiography showingstrong hybridisation to epithelial cells throughout the cryptsand over the plasma cells of the lamina propria (Gj (x36);G,i high-power view of the base of colonic crypt; x96); (H)autoradiography of ^P-labelled probe for cytochrome bmitochondrial mRNA showing hybridisation to epithelialcells throughout the crypt-surface axis (x40); (I) stain forcytochrome oxidase on cryostat section of colonic mucosashowing activity predominantly in colonic epithelial cellswith almost uniform distribution of the crypt surface axis(no counterstain was used) (x40).

block were incubated in 20 jig/ml DNase-free ribonuclease Aat 37°C for 1 hour. All slides were subsequently fixed infreshly made 0.4%(w/v) paraformaldehyde at 4°C for 20minutes.

Prehybridisation was carried out in a humid chamber at37°C for 2 hours with 300 fi\ of 50%(v/v) formamide, 600 mMNaCl, 50 mM Tris-HCl (pH 7.6), 10 mM dithiothreitol,0.02%(w/v) Ficoll 400, 0.02%(w/v) polyvinylpyrrolidone,10% (w/v) dextran sulphate and 100 /tig/ml of sheareddenatured salmon sperm DNA.

Each slide was hybridised at 37°C overnight using 80 fAprehybridisation buffer containing 50 ng/ml probe DNA,which had been labelled with [a<- P]dCTP or digoxigenin-dUTP by the random hexamer priming method (Feinberg andVogelstein, 1983). Dichlorosilane-treated coverslips wereused to cover the sections with hybridisation mixture andavoid evaporation.

After hybridisation, coverslips were removed by immersingthe slides in 2xSSC (Sambrook et al., 1989). Unboundradioactivity was removed by washing in two changes of2xSSC at 37°C for 30 minutes, followed by three changes of0.2XSSC, 50%(v/v) formamide for 30 minutes at 37°C.

To detect radioactively labelled probes, slides were dippedin Ilford K5 (Gel-form) emulsion diluted 1:1 in 2% (v/v)glycerol at 45°C. The slides were allowed to dry at roomtemperature for approximately 2 hours, and then placed in alight-tight box at 4 C for 10-21 days. They were developed byimmersion in distilled water at room temperature for 5minutes, followed by Kodak D19 developer for 5 minutes,l%(v/v) acetic acid for 3 minutes, and fixed in Ilford Hypamsolution (1:10 dilution in distilled water) for 8 minutes. Slideswere then washed in distilled water and counterstained with

Intestinal mitochondrial gene expression 309

AT: 1.

.̂ V i 5 •

310 A. J. S. Macpherson and others

quarter-strength haematoxylin and eosin, cleared andmounted in DPX.

To detect digoxigenin-labelled probes, tissue sections werewashed in 100 mM Tris-HCl, 150 mM NaCl, pH 7.5 (buffer I),followed by incubation in buffer II (100 mM Tris-HCl, 150mM NaCl, pH 7.5, 0.5%(w/v) Boehringer blocking reagent)for 30 minutes at room temperature. After a 5 minute wash inbuffer I, the tissue sections were covered with 1:500 anti-digoxigenin-alkaline phosphatase conjugate for 2 hours atroom temperature. Sections were then washed with buffer Itwice and immersed in buffer III (100 mM Tris-HCl, 100 mMNaCl, 50 mM MgCl2, pH 9.5). After 5 minutes at roomtemperature the sections were removed from buffer 111 andcovered with colour development solution freshly made with35 jil of 5-bromo-4-chloro-3-indoyl phosphate (50 mg/ml indimethylformamide) and 45 fi\ of nitroblue tetrazolium (75mg/ml in 70% (v/v) dimethylformamide) in 10 ml of buffer III.Slides were kept in a dark room for 4 hours to allow colourdevelopment. The reaction was stopped by washing in 10 mMTris-HCl, 1 mM EDTA, pH 8.0. The sections were counter-stained with 0.5%(w/v) neutral red and mounted with DPXmountant (BDH).

Mitochondrial enzyme stainsThese were performed on 6 pan cryostat sections cut fromflash-frozen endoscopic biopsy samples. For cytochromeoxidase activity the slides were incubated at 37°C for 1 hour in50 mM sodium phosphate (pH 7.4), 0.5 mg/ml 3,3'-diaminobenzidine tetrahydrochloride, 1 mg/ml cytochrome c,2 /ig/ml catalase. In control experiments the cytochrome c wasomitted from the incubation mixture or 1 mM sodium cyanidewas added to inhibit the reaction. The slides were sub-sequently dehydrated through serial ethanols, cleared andmounted in DPX. For succinate dehydrogenase determi-nation, the slides were incubated at 37°C for 1 hour in 0.15 Msodium succinate, 0.15 M sodium phosphate (pH 7.2), 40/ig/ml nitroblue tetrazolium. Controls were performed eitherwithout the addition of sodium succinate or with the additionof 10 mM sodium malonate. At the end of the incubation theslides were washed in water, then in 30%(v/v), 60%, 90%,60% and 30% acetone, with a final wash in water beforemounting in glycerine/gelatine. No counterstain was used inexperimental sections, so colour development was due to theenzyme activity; to check the tissue morphology serialsections were fixed and stained with haematoxylin and eosin.

Results

Mitochondrial gene expression in the intestinal mucosawas investigated by in situ hybridisation with radio-labelled cDNA probes on human endoscopic biopsies.Hybridisation to mitochondrial 16 S ribosomal RNAwas detected by autoradiography; the silver grainsshowed that this sequence was predominantly ex-pressed on the surface epithelium (Fig. 1A). The samepattern was seen with sections hybridised using a probeto the 12 S mitochondrial ribosomal RNA (Fig. IB).Control sections showed no hybridisation over thesurface epithelium after pretreatment with DNase-freeribonuclease (Fig. 1C), indicating that the signal wasspecific for RNA. The high steady-state levels ofmitochondrial ribosomal RNA in surface epithelial cellswas confirmed using digoxigenin-labelled cDNAprobes, and detected using anti-digoxigenin antibody

conjugated to alkaline phosphatase. Despite the lowersensitivity of this detection system, high levels of 16 SrRNA were seen, solely in the surface epithelial cells(Fig. ID). In these experiments, serial sections werealso hybridised with a probe for IgA heavy chainlabelled with digoxigenin or 32P, to act as a positivecontrol and detect a different cell type within theintestinal mucosa. The colour reaction or autoradio-graphic signal identified plasma cells within the laminapropria (Fig. 1E,F). No non-specific binding of probewas detected over the epithelial cells, and endogenousepithelial alkaline phosphatase activity was completelyblocked (compare Fig. ID and E). Probes labelled with^P were used for these experiments, since they did notshow the non-specific background binding (particularlyover eosinophils) that we observed with S labelling;sufficient resolution was obtained to detect individualplasma cells in the control hybridisation to IgA mRNA(Fig. IF).

There was also a progressive increase in 12 S and 16 Smitochondrial ribosomal RNA levels from the cryptcells to the absorptive villus enterocytes, with thehighest autoradiographic signal at the villus tip insections of low duodenum and jejunum (16 S rRNA,Fig. 2A; 12 S rRNA, Fig. 2B). As in the colon, specifichybridisation was eliminated in control sections thathad been pretreated with ribonuclease (Fig. 2C).

In contrast to the colon and the small intestine, thedistribution of mitochondrial ribosomal RNAs wascompletely different in the stratified squamous epi-thelium of normal human oesophagus. Fig. 3A showsthat the highest levels of 12 S and 16 S rRNAs were seenover the basal cell layer of the epithelium, withprogressively lower signal over the squamous cellstowards the luminal surface.

Compared with the distribution of the mitochondrialribosomal species, a probe to the 18 S cytoplasmicribosomal RNA hybridised uniformly throughout thecolonic epithelial cells (Fig. 1G). As expected for such aubiquitous species, high levels of signal were alsodetected over plasma cells of the lamina propria, whichare known to have a high content of rough endoplasmicreticulum (Rhodin, 1974).

The distal duodenum and colon were also hybridisedwith probes for mitochondrial mRNAs (cytochrome band NADH dehydrogenase 4). In contrast to themitochondrial ribosomal RNAs, these showed analmost uniform pattern of expression extending fromthe base of the crypts to the luminal surface (forexample, see Figs 1H and 2D for cytochrome bmRNA). However, in the stratified squamous oeso-phageal epithelium, mitochondrial mRNA and rRNAprobes had an identical distribution (as shown in Fig.3 A and B for 16 S rRNA and cytochrome b).

To determine the relationship between the mitochon-drial gene expression and mitochondrial enzyme func-tion, we investigated the activities of cytochromeoxidase and succinate dehydrogenase on flash-frozencryostat sections. Cytochrome oxidase contains 3polypeptides encoded on the mitochondrial chromo-some and synthesised within the organelle, and 10

Intestinal mitochondrial gene expression 311

polypeptides coded in the nucleus and imported fromthe cytoplasm, succinate dehydrogenase is entirelycoded in the nucleus (Kaput et al., 1982). The colourreaction was obtained in these experiments by histo-chemical enzyme activity, and no counterstain wasused. Both enzymes showed almost uniform staining ofepithelial cells, from the crypts to the luminal surface,in the colon (cytochrome oxidase, Fig. II; succinate

dehydrogenase, not shown) and the small intestine(Fig. 2E, F). In comparison with the epithelial celllayer, very little cytochrome oxidase or succinatedehydrogenase activity was seen in other cells of thelamina propria. Control sections demonstrated that thereaction was entirely dependent on the provision ofadded substrate (cytochrome c for cytochrome oxidaseand sodium succinate for succinate dehydrogenase).

ft&i •:>D. ~~.* • Mfia ^."JU.- —Fig. 2. In situ hybridisation of formalin-fixed paraffin-embedded tissue sections of normal human small intestinal mucosawith 32P-labelled cDNA probes to mitochondrial ribosomal RNA and cytochrome b mitochondrial mRNA, compared withactivities of mitochondrial respiratory chain enzymes cytochrome oxidase and succinate dehydrogenase on cryostat sections.(A) Autoradiography of distal duodenal biopsy hybridised with probe for 16 S mitochondrial rRNA showing strong signalover epithelial villus tip cells (arrow) (x40); (B) autoradiography of distal duodenal biopsy hybridised with probe for 12 Smitochondrial rRNA (arrow) (x60); (C) control section pretreated with DNase-free ribonuclease and hybridised with 32P-labelled probe for 16 S mitochondrial rRNA (x40); (D) autoradiography of distal duodenal biopsy hybridised with probefor cytochrome b mitochondrial mRNA showing signal over epithelial cells throughout the crypt-villus axis (x38); (E) stainfor cytochrome oxidase on cryostat section of distal duodenal mucosa showing activity predominantly in epithelial cells withalmost uniform distribution of the crypt-villus axis (no counterstain used) (x60); (F) stain for succinate dehydrogenase oncryostat section of distal duodenal mucosa showing activity predominantly in epithelial cells with almost uniformdistribution of the crypt-villus axis (no counterstain used) (x60).

312 A. J. S. MacDherson and others

., *

Fig. 3. In situ hybridisation of formalin-fixed paraffin-embedded tissue sections of normal human oesophageal mucosa with32P-labelled cDNA probes to mitochondrial ribosomal RNA and cytochrome b mitochondrial mRNA, compared withactivity of mitochondrial respiratory chain enzyme cytochome oxidase on cryostat section. (A) Autoradiography ofoesophageal biopsy hybridised with probe for 16 S mitochondrial rRNA showing signal over the basal epithelial cells(marked with arrow) (x40); (B) autoradiography of oesophageal biopsy hybridised with probe for cytochrome bmitochondrial mRNA showing similar pattern to A (x40); (C) stain for cytochrome oxidase on cryostat section ofoesophageal biopsy showing activity predominantly over basal epithelial cells (marked with arrow). No counterstain wasused (x38).

Furthermore, the cytochrome oxidase was inhibited bythe addition of 1 mM sodium cyanide, and succinatedehydrogenase activity was inhibited by the addition of10 mM sodium malonate (no colour reaction whateverdeveloped in the presence of these inhibitors).

In sections of the oesophagus, strong cytochromeoxidase and succinate dehydrogenase activity was seenover the basal epithelial cells with low levels immedi-ately below the luminal surface (cytochrome oxidase,Fig. 3C). These reactions were also dependent on theprovision of added substrate, and were subject tospecific inhibition as described above.

Discussion

We have shown that in the epithelia of the human colonand small intestine there is a marked differencebetween the levels of mitochondrial ribosomal andmessenger RNA along the crypt-surface axis. Mito-chondrial 12 S and 16 S ribosomal RNAs werepredominantly detected in the surface epithelial cells,with much lower levels in the epithelial cells of thecrypts. The signal was abolished by pretreatment withDNase-free ribonuclease, indicating that hybridisationto RNA species was being detected. We have pre-viously reported a surface distribution of 16 S rRNA inthe superficial epithelial cells of differentiated colonicadenocarcinomas and in adjacent normal colonic mu-cosa (Chester et al., 1991). These results have beenobtained using both radioactive and non-radioactive(digoxigenin) methods of detection. The specificity of

the in situ hybridisation experiments is demonstrated byhybridisation of IgA mRNA in plasma cells.

In contrast to the mitochondrial rRNA distributions,a uniform pattern of activity of the respiratory chainenzymes succinate dehydrogenase and cytochromeoxidase was observed over the crypt-surface epithelialaxis with a minor increase in surface columnar cells,suggesting that the mitochondrial rRNAs are not solelyreflecting differences in mitochondrial content betweencells in different positions in the simple epithelia.Furthermore, the pattern of expression of the mito-chondrial mRNAs cytochrome b and NADH dehydro-genase (ND4) was also uniform throughout the epi-thelial cells. This is consistent with the results forrespiratory chain enzyme activity, since coordinateexpression of the nuclear and mitochondrial mRNAs isrequired for protein synthesis of respiratory chainenzymes (Hood, 1990), and some of these (e.g.cytochrome oxidase) are composed of polypeptidesubunits from both sources (Attardi and Schatz, 1988).

There is no difference in the distribution of mito-chondrial rRNAs and mRNAs in the stratifiedsquamous epithelium of the oesophagus. High levels ofboth are detected over the metabolically active basalcells, which also have high mitochondrial respiratorychain enzyme activity. The low respiratory enzymeactivity (and mitochondrial rRNA and mRNA hybridis-ation signals) higher up the oesophageal epitheliumprobably reflects reduced metabolic activity of the cellsas they pass towards the luminal surface.

The biopsies for these studies were taken fromhuman patients during endoscopy in which no macro-

scopic abnormality was detected. Furthermore, theresults of independent histological examination, fullclinical, biochemical and haematological workup wereall completely normal. These biopsies are thereforetaken to represent the normal human intestinal mucosa.

The results of these in situ experiments reflect steady-state levels of the mitochondrial messenger and ribo-somal RNA species. We have not yet thereforedetermined the kinetics of their synthesis and/ordegradation, and the mechanism that gives rise to anincrease in mitochondrial rRNA in the mature surfaceepithelial cells of the small intestine and colon remainsunclear. In rat cerebellar cells it has also been foundthat mitochondrial mRNAs for cytochrome oxidasesubunit 1 and NADH dehydrogenase (ND4) arerelatively higher (compared with 16 S mitochondrialrRNA) during development than in the mature animals(Reniset al., 1989).

In cell culture the kinetics of RNA expression can bedetermined, and in HeLa cells the mitochondrial rRNAgenes (which are situated in a promoter proximal regionof the heavy-strand transcript) are transcribed at a rate15-60 times higher than the more distally located heavy-strand protein coding genes (Gelfund and Attardi,1981). Using a transcription system in a HeLa mito-chondrial lysate, a protein factor was identified thatprotects a 28 base-pair region of the mitochondrialDNA adjacent to, and downstream from the 16 Smitochondrial rRNA/leucyl tRNA boundary (Kruse etal., 1989). This factor also stimulated H strandtranscription termination when added to a mitochon-drial lysate programmed by an exogenous mitochon-drial DNA template (Kruse et al., 1989). It is possiblethat the relative excess of mitochondrial rRNAs overmRNAs that we have shown in surface epithelial cellsreflects increased activity of the mitochondrial tran-scription termination protein.

In this paper we have demonstrated that mitochon-drial gene expression and respiratory chain enzymeactivity are predominantly detected in the epithelialcells of the intestinal lamina propria. High levels ofmitochondrial ribosomal RNAs are expressed in thesurface cells of epithelia of the small and largeintestines, in contrast to mitochondrial mRNAs, whichare uniformly expressed throughout the intestinalcrypts. In the stratified squamous epithelium of theoesophagus both mitochondrial rRNAs and mRNAsare predominantly expressed in the basal regenerativecells.

We thank Dr P. O'Donnell for the histological assessmentsand the staff of the endoscopy unit at Dulwich Hospital forinvaluable practical assistance. Dr. H. Pringle kindly advisedus on in situ hybridisation and Dr. Erica Hagelburg providedthe cytochrome b, NADH dehydrogenase (4) and 12 Smitochondrial rRNA probes. We are also grateful toProfessor A. Shapira and Drs. T. Bjarnason and I. Talbot forhelpful discussion. This work was supported by the award ofan MRC Training Fellowship to A.J.S.M. and grant no.SP1850 to A.D.B.M. and K.A.C.; T.M. was supported by theJoint Research Council of King's College School of Medicineand Dentistry.

Intestinal mitochondrial gene expression 313

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