©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a Sixth Promoter That Directs the Transcription of -Glutamyl Transpeptidase Type III RNA in Mouse (*)

Geetha M. Habib , Bing Z. Carter , Antonia R. Sepulveda , Zheng-Zheng Shi , Da-Fang Wan , Russell M. Lebovitz , Michael W. Lieberman (§)

From the (1) Department of Pathology, Baylor College of Medicine, Houston, Texas 77030

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We have previously identified five promoters in the 5`-flanking region of the mouse -glutamyl transpeptidase (GT) gene. We now report the localization of a sixth promoter that supports the transcription of type III RNA, the major GT RNA in fetal liver. We made a fetal liver cDNA library enriched for GT RNA and obtained 12 GT type III-specific clones. The longest clone is consistent with a transcription start site for type III RNA at a position 5` to the type IV promoter and about 5 kilobase(s) (kb) 5` to the first coding exon. We estimated by ribonuclease protection assay that about 80% of the GT mRNA in fetal liver was type III. Primer extension and nuclease protection analyses mapped the 5` end of type III mRNA in fetal liver and kidney to a single cluster of potential major and minor transcription start sites. Deletion analysis using transient expression of chloramphenicol acetyltransferase constructs of the type III promoter region revealed the greatest activity with a 1-kb 5`-flanking fragment in mouse kidney proximal tubular cells and no detectable activity in NIH-3T3 fibroblasts. These studies demonstrate that the type III 5` region of the mouse GT gene is organized into two distinct exons (IIIa and IIIb) and that type III RNA is expressed under the control of its own promoter.


INTRODUCTION

-GT() (5-glutamyl peptide:amino-acid 5-glutamyltransferase; EC 2.3.2.2) is a plasma membrane-associated enzyme that plays a key role in the degradation of extracellular glutathione into a -glutamyl moiety and cysteinyl glycine (1) . In rodents and humans, the highest levels of GT are found in the proximal renal tubules, small intestine, epididymis, seminal vesicles, fetal liver, and other organs that are active in secretion and absorption (2) . Studies indicate that GT expression is regulated developmentally and in a tissue-specific manner (3, 4, 5) . To learn more about the regulation of GT and its function, we have cloned the mouse gene and begun to study its expression (6, 7, 8) .

Our previous studies have demonstrated that in mouse there is a single copy gene that encodes seven mRNAs (6, 7, 8) . These have unique 5`-untranslated regions that splice to a common untranslated exon, which in turn splices to another common exon from which translation is initiated (6) . We have identified promoters and mapped transcription start sites for five of these (7) . Of these seven RNAs, only type III is expressed in fetal liver (Ref. 6 and present work).

In the past we were unsuccessful in characterizing type III promoter. The regulation of this RNA has proven difficult to study because of the close spatial organization of type III and IV exons. Our initial cDNA cloning by anchored polymerase chain reaction (PCR) from mouse kidney (6) produced only one type III-specific clone, which was incomplete at the 5` terminus. It was not clear if type III RNA expression results from transcription from one of the five characterized promoters with alternative splicing or from transcription from an upstream promoter. In order to distinguish between these possibilities, we have investigated GT expression in mouse fetal liver and found a previously undescribed unique 5` exon for type III, which splices to a previously identified downstream type III exon (6, 9) . We have also mapped the transcription start of type III mRNA to sequences at the 5` end of this newly described exon.


EXPERIMENTAL PROCEDURES

Materials

Restriction endonucleases, T4 DNA ligase, T4 polynucleotide kinase, Taq DNA polymerase, pUC/M13 forward primer, and T7 promoter primer were purchased from Promega Corp. (Madison, WI) or Boehringer Mannheim. All other primers were synthesized using an Applied Biosystems model 381A synthesizer or a Beckman Oligo 1000 DNA synthesizer. All the radioisotopes were obtained from DuPont NEN.

RNA Extraction

Total RNAs were prepared from adult albino Friend leukemia virus strain B (FVB) mice and mouse proximal tubular cells using the guanidium thiocyanate procedure (10) . Poly(A) RNA was isolated by oligo(dT)-cellulose type III (Collaborative Research, Inc., Bedford, MA) chromatography (11) .

RNA Analysis by Northern Blotting

Fifteen µg of total RNA from mouse kidney, fetal liver, spleen, and mouse proximal tubular cells were electrophoresed on 1% agarose, 2.2 M formaldehyde gels, transferred onto a Zeta-Probe nylon membrane (Bio-Rad) and hybridized to a P-labeled GT cDNA probe corresponding to bases -57 to +492 of rat GT cDNA (12) .

Construction of Mouse Fetal Liver GT cDNA Clones and DNA Sequence Analysis

Mouse fetal liver poly(A) RNA (2.5 µg) was used to generate 5` ends of GT cDNAs using 5`-Amplifinder RACE kit (Clontech Laboratories, Inc., Palo Alto, CA). The first strand of GT-specific cDNA was synthesized by reverse transcription of poly(A) RNA by avian myeloblastosis virus reverse transcriptase and primer p1 (5`-AACCACAGCCACCAGGCCCAG-3`) complementary to the coding sequence of mouse GT mRNA. The Amplifinder anchor was then ligated to the purified cDNA according to the supplier's instructions. PCR amplification was then performed on the anchor-ligated cDNA using the anchor primer and a GT cDNA-specific complementary primer p2 (5`-CACCAGAAACCGATTCTTCAT-3`) immediately upstream of primer p1 for 35 cycles at 94 °C for 20 s, 55 °C for 20 s, 72 °C for 40 s, with a final extension at 72 °C for 5 min. The amplified products were size-selected (200-900 bp) on 1% agarose/TAE gels using a Geneclean II Kit (Bio 101, Inc., Vista, CA) and cloned into a pT7 Blue® T-Vector (Novagen, Madison, WI). The plasmids with GT cDNA inserts were then purified using a QIA Prep Spin plasmid kit (QIAGEN, Inc., Chatsworth, CA). DNA sequencing was performed using a Prism Ready Reaction Dyedeoxy Terminator cycle sequencing kit and sequenced in an Applied Biosystems model 373A DNA sequencing system using T7 promoter primer and pUC/M13 forward primer.

Ribonuclease Protection Assay

In order to estimate the abundance of type III GT mRNA in the mouse fetal liver, antisense RNA probe was generated from a BamHI-linearized type III plasmid template and transcribed with T7 RNA polymerase in the presence of [-P]UTP using an in vitro transcription kit (Stratagene, La Jolla, CA). The type III GT probe used contains 28 bp of the 5` end of the coding region encompassing the translation initiation site, 138 bp of common 5`-untranslated region, and 140 bp of the unique type IIIb sequence (see Fig. 2A). Five µg of kidney or spleen poly(A) RNA and 20 µg of fetal liver poly(A) RNA were used for the assay. After 18 h of hybridization of about 1.5 10 cpm of probe to the mRNA, the hybrids were digested according to the previously described protocol (7) . The protected fragments were then resolved on a 7 M urea, 6% polyacrylamide gel and quantitated by the AMBIS radioanalytic imaging system and the AMBIS Quantiprobe software version 4.01 (AMBIS, Inc., San Diego, CA).


Figure 2: Organization and mapping of the GT type IIIA exon. A, diagrammatic representation of the longest type III cDNA obtained from mouse fetal liver. DNA sequences representing the IIIa and IIIb untranslated regions are represented by an openbox. The hatchedbox represents the first common untranslated exon and the solidbox the first coding exon shared by all GT RNAs. B, organization of the 5`-flanking region of mouse GT gene. Exons are drawn to scale. Solidboxes indicate the exons, and lines connecting them denote introns. Known splicing patterns are designated as illustrated by descending and ascendinglines. The openbox in exon I represents the common noncoding exon, and the openbox with ATG codon is the first coding exon.



The riboprobes used in the mapping of 5` transcription site(s) of types IIIA and IIIB exons were obtained as follows. (a) Probe I was obtained by PCR amplification of almost all type IIIa unique sequences and part of 5`-flanking region from the GT phage B with oligonucleotide p3 (5`-AGGTGCATGGGAGATCAGAGA-3`]) and p4 (5`-AGTCTGTTCCCCTGAGTGATT-3`), yielding a 513-bp fragment, which was subsequently cloned into pT7 Blue Vector; (b) Probe II was obtained by PCR amplification of the region containing most of the unique type IIIb sequences and the small intron between type IIIb and unique type IV exon with primers p5 (5`-GTAGGTGGGGGCAAATAGACT3`) and p6 (5`-CCGCCATCCTGGAGTGTGGTG-3`), yielding a 161-bp fragment, which was then cloned into pT7 Blue® T-vector. The plasmids were then linearized with BamHI and transcribed with T7 RNA polymerase in the presence of [-P]UTP to obtain uniformly labeled probes.

Mapping of Transcription of Start Site(s) by Primer Extension Analysis

The oligonucleotide primer p7 (5`-ATGTGTGAACCAGGGGGACCAGGGCACAGGAGC-3`) was end-labeled with [-P]ATP and T4 polynucleotide kinase. Primer extension reaction was carried out essentially as described before (7) , except that an end-labeled primer (1.2 10 cpm) was hybridized to 5 µg of poly(A) RNA from kidney or spleen and 20 µg of poly(A) RNA from fetal liver. The primer extended products were then analyzed on a 6% polyacrylamide gel containing 7 M urea. The sequencing reactions were performed using Sequenase 2.0 according to manufacturer's recommendations (U.S. Biochemical Corp.).

GT Type III CAT Plasmid Constructs

The following strategies were employed to subclone the 5`-flanking regions of GT type III into promoterless plasmid pJFCAT1 (13) . The genomic sequences encompassing the region downstream of type V unique exon and upstream of unique type IV exon have previously been reported (6) . Using primers p8 (5`-TACAGGTTTCTTCCTACCTCA-3`) and p9 (5`-AAGATCCAAGGAGTCTGTTCC-3`), a 1984-bp fragment was PCR-amplified from GT phage B and subcloned into pT7 Blue® T Vector. The fragment was then released by SphI and BamHI and then subcloned into the SphI and BglII sites of pJFCAT1 upstream of CAT to obtain PIIIa-1984. To obtain PIIIa-1025, oligonucleotide primers p10 (5`-CTCGAGCAGAGATGTCTCAGTGCGTACTGTT-3`) and p11 (5`-CTCGAGAAGATCCAAGGAGTCTGTTCC-3`) were used to amplify GT phage B, cut with XhoI to release the fragment, and then inserted into the XhoI site of pJFCAT1. To construct PIII-524, primer p12 (5`-CTCGAGAGGTGCATGGGAGATCAGAGA-3`) and primer p11 were used to amplify the phage, cut with XhoI, and then ligated into the XhoI site of pJFCAT1. Plasmid PIIIb-46 was obtained by PCR amplification of the phage with primers p5 and p7. This PCR fragment was subcloned with pT7 Blue (R) T Vector, released by SphI and BamHI, and then subcloned into the SphI and BglII sites of pJFCAT1.

Cell Culture and Transfections

Mouse kidney proximal tubular (MPT) cells were established from kidneys of transgenic mice previously reported (14) . These cell cultures and NIH-3T3 fibroblasts were used for transfections. The MPT cells were grown in the same medium as described for Cl.1 cell line (15) but in the presence of 7% fetal calf serum. The cells were transfected essentially as described in Ref. 7, except that 0.75 µg of pCMV (Clontech, Palo Alto, CA) was used for cotransfection. As a positive control, pRSVCAT (16) was used in all the transfection assays.

Sequence Analysis

Analysis of the genomic regions upstream of the transcription start sites of GT type III mRNA was done by the Quest program from IntelliGenetics, using release 6.0 of the Transcription Factors data base (17) .


RESULTS

Analysis and Quantitation of GT Expression in Mouse Fetal Liver by Northern Analysis and Ribonuclease Protection Assay

We have previously analyzed the expression of the six known GT RNAs in different tissues by reverse transcription PCR and found that fetal liver expresses only type III RNA (6) . We have recently identified a new GT type known as type VII, which is expressed in the mouse small intestine (8) . To determine if this GT RNA is made in fetal liver, a type VII-specific primer and a common primer in the first coding exon (8) were used to amplify any type VII RNA from mouse fetal liver using reverse transcription PCR. We were unable to detect a type VII-specific band in fetal liver RNA; however, the positive control (small intestine RNA) was strongly positive (data not shown). We also examined the relative abundance of GT RNA in mouse fetal liver, kidney, MPT cells, and spleen and found that, in fetal liver, GT RNA levels are about 10% of that found in kidney (Fig. 1A). These findings are in agreement with studies in rat (18) .


Figure 1: Analysis of mouse fetal liver GT type III expression. A, Northern analysis of total RNA. Total RNA (15 µg) from mouse kidney, MPT cells, fetal liver and spleen were blotted onto a Zeta Probe nylon membrane and probed with a rat GT probe (12) representing the common coding region of all transcripts. The migration distances of 18 and 28 S ribosomal RNAs have been marked. Inspection of ethidium bromide-stained gels revealed approximately equal loading of RNA in all lanes. Kidney lane was autoradiographed for 12 h, whereas the other three lanes were exposed for 2 days. B, quantitation of GT type III mRNA by ribonuclease protection. Poly(A) RNA from kidney (5 µg), spleen (5 µg), and fetal liver (20 µg) was hybridized to a type III-specific cDNA riboprobe and digested with RNase. Ribonuclease-protected fragments were separated on 7 M urea, 6% polyacrylamide gels, dried, and autoradiographed. Asterisk represents undigested probe. Type III-specific regions are indicated by Y and Y` and common region by X and X`, respectively (see ``Results'' for details). M, molecular size marker (bases) of X174 DNA-HinfI digest.



We used a ribonuclease protection assay to quantitate the relative fraction of type III mRNA in fetal liver and kidney (7, 8) and found that about 80% of the total GT RNA in fetal liver and 6% of the total GT RNA in the kidney is type III RNA (Fig. 1B). As expected (7) , the antisense probe that we used for the quantitation of type III mRNA protected a 306-bp band (Y) specific for type III RNA and a 166-bp (X) band for the common region of GT RNA (see ``Experimental Procedures''). We also found slightly smaller bands than those predicted for the common region (X`) and the type III band (Y`). These bands could possibly result from alternative splicing between the first coding exon and two acceptors 3 bp apart in the common 5`-untranslated region as a result of the mismatch between the probe and the GT mRNA and the resultant ribonuclease cleavage (7) .

Cloning of the 5` Termini of Mouse Type III GT by Anchored PCR

In order to elucidate the structure of the type III GT RNA in detail, we obtained cDNA clones representing the 5` termini of type III RNA by anchored PCR from mouse fetal liver RNA following the strategy described in detail under ``Experimental Procedures.'' Twelve clones containing cDNA inserts specific for type III were obtained from this library. After sequencing, we determined that two extended further 5` than the clone we had previously obtained (6) . The 5` end of the longer clone shows sequence identical to genomic sequences 5` of the transcription start of type IV RNA (Fig. 2, A and B; see also Ref. 7). These two clones contained all of the exon previously designated exon III (and now called exon IIIb) and a new exon (IIIa) that maps further 5` as well as all of the common 5` region and part of the first coding exon (Fig. 2B). The longest cDNA insert has 159 bases of type IIIb and 73 bases of the newly found type IIIa (Fig. 2A). Thus, the 5` region of type III mRNA consists of two unique exons that are interrupted by a small intron: the type IV exon and another intron of about 350 bp (Fig. 2B). Exons IIIa and IIIb are unique to type III RNA and are spliced to a common 5`-untranslated exon.

Determination of the Transcription Start Sites

We determined the transcription start sites of type III mRNA by a combination of primer extension and ribonuclease protection analyses (Fig. 3). Primer extension analysis using primer p7, which is located immediately upstream of the splice donor site of type IIIb (Fig. 3A), resulted in a major band of 245 bp and a minor species at 238 bp with both fetal liver and kidney poly(A) RNA (Fig. 3B). This finding indicates that transcription for type III starts about 245 bases upstream of the type IIIb splice donor site and about 86 bases upstream of the type IIIa splice donor site. This start site is 13 bases longer than the 5`-most end of the longest cDNA insert obtained from the anchored PCR cDNA library (see Fig. 2A). The ribonuclease protection experiments using Probe I (Fig. 3C) gave a cluster of protected fragments, the major one being 75 bp; this length maps the transcription start site to the same position as primer extension experiments. The ribonuclease protection experiments using riboprobe II protected a major 115-bp fragment, which maps the 5` end of the splice acceptor site for type IIIb and is 159 bases upstream of the splice donor site for IIIb. This finding suggests that no transcripts are initiated from type IIIb region (Fig. 3C). Control experiments using spleen poly(A) RNA did not show any protected fragments.


Figure 3: Determination of transcription start sites of type III GT RNA by primer extension and ribonuclease protection. A, schematic representation of the 5`-flanking region of GT type III and IV RNA. Openbox represents exons IIIa, IV, and IIIb; the hatchedbox denotes the common untranslated exon. The solidbox represents the first coding exon. A map of the region around exons IIIa and IIIb and the location of the oligonucleotide used in primer extension are shown. Solidlines indicate the location of the two probes used in nuclease protection, and the lengths of the protected are denoted by solidboxes. B, autoradiogram of primer extended products from kidney, fetal liver and spleen. Poly(A) RNA from kidney (5 µg), spleen (5 µg), and fetal liver (20 µg) was used in the reaction. An unrelated sequencing ladder is shown as a size marker (see ``Experimental Procedures'' for details). C, autoradiogram of ribonuclease protection assay using Probe I and Probe II (see Fig. 3A). Poly(A) from kidney (5 µg), spleen (5 µg), and fetal liver (20 µg) was hybridized to Probe I or Probe II (see ``Experimental Procedures'' for details of the reaction). Brackets indicate RNase-protected fragments. Labeled X174 DNA-HinfI fragments were used as size markers.



Analysis of sequences 5` of exon IIIa reveals putative AP1 and SP1 binding sites, a CCAAT box and a liver-specific factor binding site (LFA1) within -250 bp from the major transcription site (Fig. 4).


Figure 4: Nucleotide sequence of type III and IV promoters in the 5`-flanking region of GT (see Fig. 2B). The major transcription start site for type III is indicated as +1, and a minor start site as [daio]. Other putative regulatory sites such as LFA1, SP1, AP1 and CAT box are indicated. The exons are underlined. The putative initiator element is indicated in boldletters.



Analysis of Promoter Activity

To assess the ability of the 5`-flanking sequences of the type IIIa region to support transcription, we made CAT constructs and transfected them into MPT cells and NIH-3T3 fibroblasts. We made four different constructs of varying lengths and found that a construct containing a 1-kilobase fragment (pIII-1025) gave the highest CAT activity in MPT cells (Fig. 5, A and B). The same construct in the reverse orientation (PIII-1025-R) did not support CAT activity (Fig. 5, B and C). Addition of further upstream sequences (PIII-1984) or deletion (PIII-524) decreased CAT expression to about 60% of that of PIII-1025 (Fig. 5C). Only base-line activity was seen in the 3T3 cells with GT constructs; however, pRSVCAT showed the same level of activity in both cell lines (Fig. 5B). Construct PIII-46 that contains only sequences 5` of exon IIIb and 3` of the splice donor site of exon IV and most of exon IIIb (Fig. 2B) showed no CAT activity, indicating that transcription of type III RNA is not initiated in this region.


Figure 5: Analysis of promoter activity by transient expression. A, schematic representation of 5` deletion constructs of type IIIa and IIIb flanking sequences driving the CAT reporter gene. Solid box indicates exons V, IIIa, or IIIb; openbox indicates CAT sequences; lines represent the 5`-flanking region. Oligonucleotide primer p8 is located immediately downstream of the type V splice donor site. B1 and B2, autoradiograms of CAT assays of the cells transfected with the constructs depicted in A. B1, MPT cells; B2, NIH 3T3 fibroblasts. PIII-1025-R contains the 1025-bp fragment from PIII-1025 but in reverse orientation. FCAT is the promoterless CAT vector. See panelA for the nomenclature of other constructs. C, quantitative analysis of the expression of GT type III CAT constructs in MPT cells and NIH 3T3 fibroblasts. The construct pIIIb, which showed the highest CAT activity, is indicated as 100%. The activities of the rest of the constructs are shown as a percentage of PIII-1025. The solidboxes represent the activities of constructs in MPT cells, and openboxes, NIH-3T3 fibroblasts.




DISCUSSION

Our findings demonstrate that about 80% of GT RNA in mouse fetal liver is type III RNA. Both our previous studies (6, 8) and our present findings indicate that type III is the only one of the seven known types of GT RNA to be expressed in fetal liver. Thus, our findings suggest that there may be other GT RNAs in fetal liver that have not yet been identified.

GT type III RNA expression is interesting for two reasons. 1) The RNA is developmentally regulated in mouse liver and its expression is markedly reduced or shut off during maturation from fetus to adult; 2) it is a marker for the emergence of preneoplastic and/or neoplastic cells during liver carcinogenesis (19) . Ideally, one would like to study the mechanism for the silencing of GT during development and its activation during neoplasia. Recently it has been suggested that thyroid hormone may play a role in the modulation of GT expression during development in rat liver (20) . Our identification of type III as the major GT type in fetal liver and our mapping of the 5` end of this RNA and the transcription start should make these studies possible.

Because type III RNA is the major RNA in fetal liver, we made a 5` extended cDNA library from that tissue and obtained 12 cDNA clones, all containing sequences specific for type III. Two of the 12 clones extended further 5` than our previously obtained clone (6) ; the 5` end of this newly identified type III exon and its transcription start are located about 350 bp upstream of the unique type IV exon (Fig. 2B). Thus, to date we have identified 9 exons upstream of the exon containing the initiation codon; these are spliced in patterns that result in the generation of the seven known GT RNAs (Fig. 2B).

The phenomenon of genes coding for multiple mRNAs with tissue-specific 5` termini and promoters has also been observed for other eukaryotic genes. These include developmentally regulated genes such as aldolase A, fetoprotein, insulin-like growth factor II, aminopeptidase N, asparagine synthetase, and choline acetyltransferase (21, 22, 23, 24, 25, 26) . The gene encoding aromatase cytochrome P450 (CYP19) also seems to be regulated through the use of tissue-specific promoters. To date, at least five promoters have been identified for the CYP19 gene, which direct the transcription of at least five mRNAs all coding for the same protein (27) .

Mapping studies of the GT type III region by both primer extension and ribonuclease protection point to a major transcription initiation site and a few minor ones within 10 bases of it (Fig. 3, B and C). We have assigned the transcription start site to a deoxyadenosine in the sequence TTGCAGTCC (Fig. 4). The GT type III promoter appears to belong to a class of promoters which have no apparent TATA element, are regulated during development or differentiation, and initiate transcription at only one or a few highly clustered start sites (28, 29) . Comparable sequences (consensus YYCAYYYY) have been found in a family of TATA-less promoters, including the gene for terminal deoxynucleotide transferase (30). In this respect, the sequence in the vicinity of type III transcription start site closely resembles that of the type IV GT RNA start site (7) . The type III transcription initiator site shares sequence similarity with the class of promoters that have an initiator element within the transcription start site (31, 32) . This initiator element can act alone or in concert with another upstream element to initiate accurate transcription. At present, we do not yet know if these sequences actually function in the mouse GT gene.

The 5`-flanking region of type III GT is not unusually GC-rich but has a CCAAT box at -59 from the start site. There are also potential SP1 and AP1 factor binding sites located at -37 and -28 respectively. It is worth noting that SP1 may also direct transcription for the TFIID element in TATA-less promoters transcribed by RNA polymerase II (33) . A liver-specific factor (LFA1) binding site is located at -174 from the major start site. It remains to be seen if these regulatory elements are silent or play an active role in the transcriptional activation of the GT type III promoter.

Constructs from the 5`-flanking region of the GT type III promoter are able to drive the expression of the CAT reporter gene in a cell-specific and orientation-dependent manner (Fig. 5). Further studies of the type III promoter function should enable us to delineate the factors involved in the stage-dependent inactivation of this liver-specific promoter.

It is of interest to compare our findings with those in a recent report on the expression of a type III GT promoter in the rat (34) . Rat type III is initiated from a region where mouse type IV is located, whereas mouse type III start site is located upstream of type IV exon in mouse. Mouse type IIIb shares 79% sequence identity with rat type III at the 3` end. These results demonstrate that even highly conserved genes in related species may show important differences in their regulation.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA 39392. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Telephone: 713-798-6501; Fax: 713-798-6001.

The abbreviations used are: GT, -glutamyl transpeptidase; CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction; bp, base pair(s).


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