From the
We have previously identified five promoters in the 5`-flanking
region of the mouse
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
Our findings demonstrate that about 80% of
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
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,
Mapping studies of the
The 5`-flanking region
of type III
Constructs from the 5`-flanking region of the
It is of interest to compare our findings
with those in a recent report on the expression of a type III
-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.
-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) .
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.
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
Mouse fetal liver poly(A)GT cDNA Clones and
DNA Sequence Analysis
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.).
The
following strategies were employed to subclone the 5`-flanking regions
of GT Type III CAT Plasmid Constructs
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) .
Analysis and Quantitation of
We have previously analyzed the expression of the six
known GT Expression in
Mouse Fetal Liver by Northern Analysis and Ribonuclease Protection
Assay
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
In order to elucidate the structure of the type III
GT by
Anchored PCR
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.
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.
GT RNAs (Fig. 2B).
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) .
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.
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.
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.
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.
GT,
-glutamyl transpeptidase; CAT, chloramphenicol acetyltransferase;
PCR, polymerase chain reaction; bp, base pair(s).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.