- Email: [email protected]
heart-fatty acid-binding protein
Gene. 147 ( 1994) ‘37 -242 0 1994 ElsevierScience B.V. All rights reserved.
GENE
237
0378-l 1 i9~94~$~7.0~
08136
Cloning and characterization of the mouse gene encoding mammary-derived growth inhibitor/heart-fatty acid-binding protein (Recombinant DNA; gene cloning; localization; mammary gland)
Mike Treunera,
Christine
Received by J.K.C. Knowles:
reverse transcription-polymerase
chain reaction;
A. Kozakb, Daniel Gallahan”,
7 January
1994; Accepted:
Richard
19 April 1994: Received at publishers:
pseudogene;
chromosome
Grossed and Thomas
Miiller”
30 May 1994
SUMMARY
From a mouse genomic library we isolated and characterized a gene, Fubphl, encoding mammary-derived growth inhibitor (MDGI)/heart fatty-acid-binding protein (H-FABP). Exon sequences were identical with a MDGI-encoding cDNA isolated previously from the mammary gland of pregnant mice. The product of this gene has also been detected in heart, where it had been termed H-FABP. It has an intron/exon structure similar to other FABP-encoding genes. In addition to this expressed gene, we isolated a related intronless pseudogene, Fabph-ps, with an open reading frame which was highly conserved when compared with Fubphf. Fubphl was positioned on chromosome (Chr) 4 using interspecies crosses. Analysis of somatic cell hybrids was used to assign Fubph-ps to Chr 10, and another MDGI/H-FABPrelated sequence locus, F~b~h-rsl, to Chr 8. A h4us spretus-specific related sequence, F~~J~h-rs2, was identified on Chr 17 by analysis of interspecies crosses. The 5’-flanking region of F~bph~ contains putative transcription factor-binding elements which could account for its constitutive expression in muscle tissue, as well as for its developmental stagedependent expression in mammary epithelium.
bovine
INTRODUCTION
Starting with the assumption that locally acting factors should be involved in regulation of the development of mammary epithelium (Oka et al., 1991), we isolated the mammary-derived growth inhibitor (MDGI) from Correspondence to: Dr. T. Mtiller,
Laboratory
of Molecular
NINDS. National Jnstitutes of Health, Bethesda, USA. Tel. (l-301) 496-3412: Fax (l-301) 402-1340; e-mail: [email protected]
Maryland
Biology, 20892.
Abbreviations: aa, amino acid(s); bp, base pair(s); Chr, chromosome(s); FABP, fatty acid-binding protein; FobphI, gene encoding MDGI/ H-FABP; Fuhph-ps, Fclhpltl-related pseudogene; Fahph-rs, F&phirelated sequence locus; I-t-FABP, heart-FABP; M., Mus; Mm., M. muscuiz~ MDGI. mammary-derived growth inhibitor; nt, nucleotide(s): ohgo. oligodeoxyribonucleotide~ ORF, open reading frame; RT-PCR. reverse transcripti(~ll-polyme~~s~ chain reaction; tsp. transcription start point(s); UTR. untranslated region(s). SSDl 037X-I
114f94)00347-U
lactating
mammary
gland (Biihmer
et al., 1987).
MDGI belongs to the family of fatty-acid-binding proteins (FABP) (Veerkamp et al., 1991) with highest homoiogies to the FABPs from bovine synthesis
is induced
in mammary
heart and brain. epithelium
MDGI
only during
late pregnancy and lactation (Miller et al., 1989; Kurtz et al., 1990) and strictly depends on lactogenic hormones (Binas
et al., 1992). Functionally,
acterized entiation
1992). The question FABP
MDGI
has been char-
as an endogenous growth inhibitor and differfactor of mammary epithelium (Grosse et al.,
(H-FABP)
of whether the proteins
MDGI
are in fact identical
one gene has been considered
previously
1990). While the high degree of similarity
and heart-
and encoded (Spener between
by
et al., MDGI
and H-FABP sequences is consistent with the conclusion that MDGI and H-FABP may be derived from a single
238 MDGI~H-FABP-encoding
gene, efforts
to chromosom-
ally map the genes MDGl/H-FABP-related
responsible have uncovered sequences on three mouse chro-
mosomes
cell hybrids
1987).
using somatic In man,
a single
(Peeters et al., 1991). The aim of the present
genetic study
(Heuckeroth locus
was
was to clone
et al.,
identified and map
(b) Analysis of ~~Gl/~-FA~P-r~Iat~d The coding served
region
when
transcripts
of the pseudogene
compared
is highly
with the gene and
cDNA. Three nt substitutions result in two aa exchanges (S14+C, G’” -+R). Furthermore, sequence conservation extends
beyond
section
d). Since examples
the tsp and the putative of expressed
TATA-box
(see
retroposons
are
the MDGI/H-FABP-encoding genes. The sequences of the isolated gene and of its regulatory regions will be
documented
instrumenta
tr~~llscribed. To this end, RNA was isolated
in the investigation
type-specific regulatory mechanisms operating in such diverse tissues
of common
and cell-
of gene tr~~nscription as mammary gland
and muscle.
mouse
5’-primer
clones
(a) Isolation of two mouse MDGI/H-FABP-related genes Approx. 800~0 plaque-forming units of a mouse genomic library &GEM-l 1, Promega, Madison. WI, USA) were screened using the coding region of mouse MDGI cDNA as probe (Binas et al., 1992, GenBank S48643, revised UO2883). Six different recombinant clones were identified and plaque purified. According to their rcstriction patterns they represent two different genomic clones. By Southern analysis with oligos from the 5’- and the 3’-region of the MDGI cDNA, phage clones hB and hD representing these two genes were found to contain the full-length coding region and were selected for subcloning into pBluescript SK( if. Overlapping genotnic DNA inserts in plasmid subclones were sequenced in both directions ( Fig. 1). Clone hB was found to contain a gene with four exons and three introns. Intron/exon boundaries were established by comparison with the cDNA sequence. They were found to obey the GT/AG rule (Breathnach and Chambon, 1981). lntrons are identically positioned as in all other FABP genes characterized (Matarese et al., 1989). The sequence of the exon regions of the gene encoded by clone hB was found to be identical with the mouse MDGI cDNA. Phage clone hD was found to contain an ORF which was not interrupted by introns and which differed by only three nt substitutions from the coding region of the gene contained in clone hB (Fig. 1). The region of high similarity between clones hB and hD covers I18 bp of S-UTR and 236 bp of 3’-UTR until an oligo(dA)-tract begins in clone hD. This region is flanked by 14-bp direct repeats. Thus, the genomic sequence contained in clone hD most likely represents a pseudogene which arose by retroposition from a processed RNA intermediate (Rogers, 198.5 1.
the question
106 and to allow
whether
the 3’-primer fragment
hD.
an additional
hD, representing
a potential
and the mouse
The
mRNAs
a subclone
in phage be distin-
since the pseusite at position derived
pseudogene-derived cDNA,
from cDNA
corresponding
in the PCR analysis.
liver and pancreas
MDGI/H-FABP-related
could
pattern
MDGI
to clone hB, were included Spleen,
from
contained
restriction
(cf., Fig. 1). As controls,
sequence,
for RT-PCR.
fragments
by their Hinfl restriction
dogene contains
is
from various
003 (cf., Fig. 1) were
sequences
These
et al., 1993).
the pseudogene
amplification
from genomic
hB and
guished f592
et al., 1991; Linnenbach
tissues and used as templates
chosen
AND DISCUSSION
(Kuhn
we addressed
transcribed
EXPERIMENTAL
con-
the MDGI
tissue did not contain
transcripts
(not shown).
any From
total RNA samples of lung, kidney, testis, skeletal muscle, heart, brain and mammary
gland from pregnant
and from
lactating animals RT-PCR fragments of the expected size were obtained (Fig. 2A). Fragments amplified from tissue RNAs, as well as fragments amplified from the control DNAs were subsequently digested using the restriction enzyme Hi&. Restriction patterns of PCR fragments obtained from the various tissue RNAs were all identical to the pattern obtained from MDGT cDNA (Fig. 2B). Omitting the reverse transcription step, small amounts of PCR fragments HinfI digestion
were obtained
pattern
similar
which gave rise to a
to the one resulting
from
hD DNA. This indicates probable contamination of RNA preparations with minor amounts of genomic DNA. Furthermore, the sequences of the PCR fragments obtained from heart and mammary gland RNA were found to be identical with the exon sequences of the gene contained in clone hB. These findings indicated that the mammary gland derived MDGI and the heart-derived H-FABP are both transcribed from the MDGI/H-FABPencoding gene contained in clone hB. This conclusion is in accordance with peptide sequencing data (Bansal and Medina, 1993). No transcripts from the MDGI/H-FABPrelated pseudogene contained in clone hD could be detected. It cannot be ruled out completely that expression of the pseudogene takes place in certain cells or at certain developmental stages. The MDGI/H-FABP-encoding gene and the pseudogene are designated F~z~?~~land F~~~~~?-~s, respectively.
239 )cB ...tttgagagagtatcttgccttggctcagactggCCCcCaaCtCaggaatctcttgCctccaggtgca~t~CCgtattaaCtgttggCtCaagCCagagCagCggCaCa~~ta E-box ha
-1068
E-box
accatgcCCtgaagtaggctacaaccatcaatagtcgggtcttatttaataacgtactttaaggtgacaagcagtctag~~gaagtcaggggaaaaaactgacttcagcagagggtc -__ NFl
hB
-1240 E-box
atgctgacttggtaataattaaacagaaactgctgaactaaggaataggctccactgaggttcccttactcacctgtaaaaaggggatgataccacctaccaacgaaaaagttgagtgtg APl
hB
E-box
-948
API
gcggctttccgggagttaaggtggccgaggccggaagaaccctctgaatagacaaattgtcttcgcggagtgaagaacgaccctggcacaagctc~gaggtcagtaaataaagcctgaag NF1
-828
DR-1
hB cgctttcaggcagcggcgaCgggtgggaCtgCggagaaaggCgC~ggCggg~g~CattCCgCaggg~ggggCt~g~tggggCt~gC~tg~gg3~~gCa~ggtC~CgttCtCCgCC E-box hB
agc~gq~_gaggcgctgggcagctcagccatccgcgg~~~caaggcaactcttttcc~tctggtaggagcaagagggctcaaaggccactagaccatgctctctgtccaggctcca E-LXX
ha
-708
MAF
TIE
-588
E-box
attctttttta~ttacggcgaccgcgt~~~~~~ctccgagcctctgagcctcttctacaagaagaggac~taggaccgttgagatgggttt... M-CAT
210
bp ...tttctccagcgfqq
HRE
-272
NFl
hB ~CcagctcaagggcgagtttcctttC~gt~tggCC~gggg~tgCtCt~CttgggttgCg~~gCCCCgC~gCC~ggCC~ggg~tgggt~~g~~~CC~~C~gg~~~g~ggg~g~ MPBS
-152
NFl
hD
...ctgg~g~ga~t~~~~gctttCaacagcctgcttCttg~~tgttgta~acaca~cgtaa~~aaattuttcacratttcgggagcgaggggtgtgggccactttcatcatgtgatgcga
hB
cgctgacgtaggcgacgggagggctgtggggg~tggg=C~=~gCCCtttgCggg~gtgC~~g=CC~gg=ttCCtt~tttCgggagCgaggggtgtgggCCaCtttCatCatgtgatgCga
hD gggctattt.aagaggctgtccagccgggagCTGCGGTTCTCAGTGCCTGCCCGCCTCCTCACTCATCGCACCATGGCGGACGCCTTTGTCGGTACCTGGAAGCTAGTGGACTGCAAG~ hB gggctattt.aagaggctgtccagccgggagcTGCGGTTCTCAGTGCCTGCTCGCCTCCTCACTCATCGCACCATGGCGGACGCCTTTGTCGGTACCTGGAAGCTAGTGGACAGCAAGAA MADAFVGTWKL"D*SKN m primer761 TTTTGATGACTACATGAAGTCACTCG-------------------~-------------GTGTGGGCTTTGCCACCAGGCAGGTGGCTAGCATGACC~GCCTACTACCATCATCGAG~G hB TTTTGATGACTACATGAAGTCACTCGgtgag...intrOn 1. 3.4 kb...cttagGTGTGGGCTTTGCCACCAGGCAGGTGGCTAGCATGACC~GCCTACTACCATCATCGAGAAG F D DYMKSL GVGFATRQVASMTKPTTI I E K
hD
-primer
-33 -33 +88 t88 16 t176 t3617 45
1064
hD AACGGGGATACTATCACCATAAAGACACAAAGTACCTTCAGGTCAAG--------hB AACGGGGATACTATCACCATRAAGACACAAAGTACCTTCRAGAACACAGAGATC~CTTTCAGCTGGG~TAGAGTTCGACGAGGTGACAGCAGATGACCGG~GGTC~Ggtgag NGDTIT I K T Q STFKNTEINFQLGI EFDEVTADDRKVK
t287 t3733 82
hB intron 2, 1.5 kb...cacagTCACTGGTGACGCTGGACGGAGGCAAACTCATCCATGTGCAGAAGTGGAACGGGCAGGAGACAACACTAACTAGGGAGCTAGTTGACGGG~ACTC SLVTLDGGKLI Q ETTLTRELVD*GKL H V Q K W N*G
t383 +5314 114
hB ATCCTGgtaag...intron I L? Hinf I
+471 +6518 133
1 Hinf I hD ATCCTG------------------------------ACTCGGAGGCGTGACCTGGCTGCTCCGTCACTGACCGCCCGC
3. 1.1 kb...tctagACTCTCACTCATGGCAGTGTGGTGAGCACTCGGACTTATGAG~GGAGGCGTGACCTGGCTGCTCCGTCACTGACCGCCCGC TLTHGSVVSTRTYEKEA---
hD TCCTCTGCCAACTGGCCACCCCTCAGCTCAGCACCATGCTGCCTCATGGTTTTCCCCTCTGACATTTTGTAT~CATTCTTGGGTTGGGATTTTTCTGGAGATACGGGGCATCAGCCTG hB TCCTCTGCCAACTGGCCACCCCTCAGCTCAGCACCATGCTGCCTCATGGTTTTCCCCTCTGACATTTTGTAT~CATTCTTGGGTTGGGATTTTTCTGGAGATACGGGGCATCAGCCTG
+591 +6638
J Hinf I ho GACTCAGTTCCTACTATGTATGTGGTTTATTTTTTAAAACCAGAACCAAGGCClaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa hB GACCCAGTTCCTACTATGTATGTGGTTTATTTTTTAAAACTGTATCCAAAGGGTGCTCCAAGGTCMT~GCAGAACCAAGGCC~ACCCAgttgtctgtctttggt~=t~~tttC~tgt * * -primer OO%---
t710 16757
l
hD
aaaaaaaaaaaaaaaaq~~~~~~a~cacaaacaatagactcacaagatgagc...
1767 +6814
hB gtgtcaggttg~~~tg~aggcctataggtcacctggt~a~~tggg~~g~~g~~~tgt~~agg~g~...
Fig. 1. Sequences
of the mouse
MDGI/H-FABP-encoding
gene Fabphl (LB) and pseudogene
Fabph-ps (hD). The nt differences
are indicated
by *.
DNA fragments generated by Sac1 and KpnI digestions of phage DNA from clones hB and hD were subcloned into pBluescript SK(+) and sequences were determined by dideoxy-sequencing in both directions using Sequenase 2.0 (US Biochemical, Cleveland, OH, USA). Putative TATA-box and polyadenylation signal are bold-faced. Exon regions are printed in capital letters while portions of the introns, S- and 3’-CITR are shown in lower case letters. The deduced aa is placed under the second nt of each codon. The region of homology between the gene and the pseudogene is enclosed in brackets. Direct repeats flanking this region are double underlined. Primer 781 used for primer extension (cf., section d) and primers 106 (sense) and 003 (antisense) used for transcript analysis (cf., section b) are indicated by horizontal arrows. Hinfl sites within the PCR fragment (cf., section b) are marked with vertical arrows. Putative transcription factor binding sites are underlined and explained in section d. The GenBank accession Nos. are UO2884 (Fabphl) and U02885
(Fubph-ps).
(c) Genetic mapping Chinese hamster x mouse somatic cell hybrids have been previously described (Hoggan et al., 1988). 18 hybrids were selected from a larger panel of 76 hybrids for this study. They were typed for MDGI/H-FABP-related sequences using the MDGI cDNA probe (Binas et al., 1992) following digestion with EcoRI, BumHI and PstI. For each enzyme, the probe identified two major bands and several minor bands. The two major bands identified loci on two different chromosomes. The Fuhphl locus, identified by an EcoRI fragment of 8.6 kb, a BumHI fragment of 12.0 kb and a PstI fragment of 6.5 kb, could be assigned to Chr 4 based on the perfect correlation between rhe presence or absence of this chromosome with
the presence or absence of these fragments (data not shown). At least two discordant hybrids were identified for each of the 18 autosomes and the X chromosome. The second major fragment was identified by fragments of 6.0 kb (EcoRI), 13.5 kb (BarnHI) and 3.0 kb (PstI). This locus was mapped to Chr 10. A probe specific for Fubphps corresponding to its 5’-flanking region (nt -524 to -33, cf., Fig. 1) also identified this EcoRI fragment of 6.0 kb, indicating that this Chr 10 locus represents the cloned pseudogene, which we designated Fahph-ps. Finally, fragments characterized by a weaker hybridization signal were identified by a 7.0-kb PsfI fragment and a 5.0-kb EcoRI fragment which mapped to Chr 8. This locus is designated Fubphl -related sequence 1 (Fabph-rsl ).
240 LBhD +mgj y-p
+b _ +h _ +I _ +sm_ +t _ +k _
A’1
bp
B
270 230
4/X2
4 Y +/- 2.4
203 Fig. 2. RT-PCR
analysis
of MDGl!H-FABP-related
transcripts
in
mouse tissues. RNA was extracted from mouse tissues (mg-I, lactating mammary gland; mg-p, mammary gland from 16 days pregnant mouse; b. brain; h. heart; I. lung; sm, skeletal muscle; t, testis: k. kidney) according to (Chomczynski and Sacchi, 1987). 0.5 pg. of RNA was reverse transcribed using a ss-cDNA-synthesis kit and random hexamer primers (Pharmacia) (lanes marked with (+)) and used as template for PCR with primers 106 and 003 (cf., Fig. I ). In control samples. lanes marked with (-). the reverse transcription step was omitted. As further controls. the subcloned 5~1 fragment of clone >_D (P-D) and the mouse MDGI cDNA (p&B)were used as templates for PCR. (A) Reaction products were analysed by 1.2%agarose is indicated. (B) PCR products
electrophoresis. Expected fragment size were subsequently digested with Hirzfl
and analysed by electrophoresis. The 230-bp fragment is indicative of a transcript originating from the cloned MDGI/H-FABP-encoding gene (LB), the 203-bp fragment would indicate a transcript from the pseudogene OLD). Both have the 270-bp fragment in common, 27-bp fragment was not resolved in this electrophoresis.
The remaining
These data indicate that all MDGI/H-FABP-related sequences can be assigned to three chromosomes in the inbred mouse. Two genetic crosses were used to position MDGI/HFABP-related sequences on the mouse genetic map: (NFS/N or C58/J x M. 111.r?tusculus) x iz4. m. nzus&us (Kozak et al., 1990) and (NFS/N M. spretus) x (M. spretus or C58/J) (Adamson et al., 1991). The progeny of these crosses have now been typed for over 650 genetic markers including Lck (lymphocyte tyrosine kinase), Dsil (David Steffen integration-l), and Oprd (opioid receptor delta) on Chr 4 and rd2 (retinal degeneration slow, peripherin), C3 (complement component 3) and Hprt-psi (hypoxanthine phosphoribosyl transferase pseudogene- 1) on Chr 17. DNAs from the parental mice of both genetic crosses were typed for restriction enzyme polymorphisms using as probes the MDGI cDNA, as well as an intron sequence corresponding to nt + 60 to + 3 195 of Fcihphf (cf., Fig. 1). Each enzyme used identified multiple fragments, but for any given enzyme only one or two fragments were polymorphic and could be typed in the progeny. For the M. m. musculus cross, ApaI produced fragments of 6.5, 4.2 and 2.8 kb in NFS/N and 13.2, 6.5 and 2.8 kb in M. m. musculus. Analysis of the progeny showed that the 4.2-kb NFS/N fragment identified the Fuhphl locus on Chr 4 (Fig. 3). Use of the Fuhphl derived intron probe identified this same locus. Using the MDGI-encoding cDNA, analysis of the M. spretus cross identified two fragments in M. spretu.s which represent two loci. One locus, identified as a M. spretus
Fig. 3. Linkage
of F~hphl
on mouse
Chr 4 and of F~hph-rsZ
on M.
sprrru.s Chr 17. DNAs extracted from the progeny of the two genetic crosses (NFS/N or C58.iJ x M. tn. ~rrusc~u/us)x M. rri. musc~ulus (Kozak et al.. 1990) and (NFS/N x M. spreru.s) x(M. sprerus or C58.;J) (Adamson et al., 1991) were digested with Apd and Pstl to identify polymorphic fragments by Southern analysis using the MDGI cDNA probe and a Fuhphl-specific intron probe (nt +60 to +3195). Membranes
were
washed
in 0.2 x SSC;2%
SDS
at 60-65 C.
Both
probes positioned the gene Fuhphl on Chr 4. The MDGI cDNA probe also identified the locus FuhplwsZ011IV.spwru.sChr 17. For each cross a recombination fraction is given for adjacent loci, and percent recombination and standard error (SE) are calculated according to Green ( 19X1 ). No double
recombinants
were identified
on either chromosome.
PstI fragment also identified
of 8.8 kb mapped to the Chr 4 position in the M. m. musculus cross. The second
locus, identified as a M. spretus PstI fragment of 6.0 kb mapped to the distal region of Chr 17 (Fig. 3). The failure to identify F&p&related sequences on Chr 17 using somatic cell hybrids or using the M. m. musculus cross suggests that these sequences represent M. spretusspecific F&p&related sequences. This locus is designated Fubph-rs2. Previous studies had used a rat H-FABP-encoding cDNA probe to assign related fragments of HindIII-digested genomic DNA to mouse Chr 4, 8 and either 10 or 15 (Heuckeroth et al., 1987). Two Hind111 fragments were mapped to Chr 4, designated as loci Fuhphl and Fuhph4 (Lyon and Searle, 1989), the locus on Chr 8 was named Fuhph2, and the locus on either Chr 10 or 15 was referred to as Fubph3. While the FABP-encoding sequences on Chr 10 and Chr 8 have not been positioned, a previous study positioned the locus Fuhphl on Chr 4 just distal to Lck (Bahary et al., 1991). Using the mouse MDGI-encoding cDNA probe and the intron probe derived from clone hB, our results identify Fubphl as the expressed MDGI/H-FABP-encoding gene. Since this gene has Hind111 sites in its first and second introns, the formerly defined locus Fuhph4 is most likely contiguous with Fuhphl. Our data indicate that the cloned pseudogene Fubph-ps corresponds to the locus Fubph3 and that this locus can now be assigned unequivocally to Chr 10. In man, the human H-FABP-encoding gene was assigned to chromosome lpter-q31 (Peeters et al., 1991) in a region of linkage homology with mouse Chr 4.
241 (d) Analysis of the S-flanking FABP-encoding
The S-boundary primer
extension
(1989).
of the first exon was determined analysis
Comparison
poly(A)‘RNA the sequencing
region of the MDGIfH-
gene according
of the
to Sambrook
extension
product,
by
Fig. 1 the T residue
Synthesis in various
of MDGI/H-FABP tissues.
Postnatally,
duced in the heart in very high amounts extent
in skeletal
of nt in
muscle,
testis, brain
pro-
and to a lesser and other
tissues
(Heuckeroth et al., 1987; Veerkamp et al., 1991). In bovine and mouse mammary epithelium MDGI/H-FABP is only detectable during pregnancy and lactation and strictly depends on the lactogenic hormones prolactin and hydrocortisonc (Miiller et al., 1989; Kurtz et al., 1990; Binas et al., 1992). Sequence inspection of the upstream region of Fuhphf revealed a TATA-box at -21 with the sequence TTTAAA (Fig. 1) characteristic of a variety of milk protein genes (Yoshimura and Oka, 1989). With regard to musclespecific expression of Fahphl, two clusters of E-boxes
yeast heart RNA
RNA
involved related
3’5’ CG GC GC CG CG CG TA CG GC A T* CG G C* CG CG AT AT GC AT GC TA CG AT 5’3’
Fig. 4. Analysis of the rsp of Fahphl. The DNA sequencing ladder and the product of the primer extension reaction using primer 781 (cf. Fig. 1) were separated in a sequencing gel. Primer extension reaction was carried out as described (Sambrook et al., 1989) using 2p.g of heart poly(A)~RNA or in thecontrol 2 ~gofbaker’sy~~st tRNA as templates.
sites for the MyoD-family
factors in muscle
gene expression
Sequences
(MPBF) activating
binding
(Wright,
1992) and a
tissue
specificity
(Mar and Ordahl, to milk protein
several CTF/NFl
half palindromic
binding
elements
of
1990) were
(Watson et al., 1991) and mammary factor (MAF) (Mink et al., 1992) binding
factor cellsites,
(Jones et al.,
1987) and a hormone responsive element (HRE) (Bailly et al., 1986) are present in the S-flanking region of F~~~~zf and might be instrumental
in
regulated
it is constitutively
motif
found.
in ~~~bph-pscontained is differentially
transcription
M-CAT
using
was set to + 1 in ~~~~~~ contained
in clone hB and, by analogy, clone AD as well.
of myogenic troponin-T
from mouse heart tissue as template, with ladder both originating from the antisense
start site (Fig. 4). For numbering
potential
et al.,
oligo 78 1 (cf., Fig. 1) indicated that transcription initiates at the T and the C residues 41 and 39 bp upstream from the translatiol~
representing
in the hormone
and differen-
tiation-dependent expression in mammary epithelium. In addition, a direct repeat (DR-1) with a spacing of one base pair, similar
to binding
sites of retinoic
tors (RAR, RXR) and peroxisome receptor
(PPAR)
1992) could
(Durand
be detected
proliferator
acid recepactivated
et al., 1992; Kliewer at position
-84.5.
et al.,
Evidence
is
accumulating that fatty acids and arachidonic acid metabolites may regulate gene expression (Distel et al., 1992; Grimaldi et al., 1992) possibly by activating similar transcription factors (Eager et al., 1992; Keller et al., 1993). Interestingly, one of the ligands of MDGI/H-FABP is arachidonic acid (Veerkamp et al., 1991; Wallukat et al., 1991). Since we have found that in mammary gland organ culture MDGI/H-FABP promotes its own expression (R.G., unpublished), an involvement of MDGI/H-FABP in transport or metabolism of a ligand of a member of this transcription factor family could be postulated. TGFP was recently shown to inhibit functional differentiation of mammary epithelium during pregnancy (Jhappan et al., 1993). We have found that TGFP downregulates F~~~~zi expression in di~erentiated primary mouse mammary epithelial cells (T.M.. LInpublished). In this respect, a putative TGF@l inhibitory element (TIE) (Kerr et al., 1990) in the FdphI promoter might be functionally important (cf., Fig. 1). Suppression of MDGI/HFABP-encoding gene expression during early ductal morphogenesis may be essential in order to allow development of a functional mammary gland (Binas et al.. 1992). The functional importance of putative regulatory elements for MDGI/H-FABP-encoding gene expression in mammary epithelial and in muscle cells will be investigated in future studies.
ACKNOWLEDGEMENTS
We wish to thank Drs. Claus Scheidereit and Matthias Gaestel for critical reading the manuscript and Andrea Dell’Oro for skilfull technical assistance. The work was supported by grant SFB 344 of the Deutsche Forsch~lngsgemeinschaft to T.M.
242 REFERENCES Adamson, M.C.. Silver, J. and Kozak. C.A.: The mouse homolog of the Gibbon ape leukemia virus receptor: genetic mapping and a possible receptor function in rodents. Virology 183 (1991) 778-781. Bahary, N., Zorich, G., Pachter, J.E.. Leibel, R.L. and Friedman, J.M.: Molecular genetic linkage maps of mouse chromosomes 4 and 6. Genomics 11 (1991) 33 47. Bailly, A., Le Page. C.. Rauch, M. and Milgrom, E.: Sequence-specific DNA binding of the progesterone receptor to the uteroglobin gene: effects of hormone, antihormone and receptor phosphorylation. EMBO J. 5 (1986) 3235-3241. Bansal. M.P. and Medina, D.: Expression of fatty acid-binding proteins in the developing mouse mammary gland. Biochem. Biophys. Res. Commun. 191 (1993) 61-69. Binas, B., Spitzer, E., Zschiesche, W., Erdmann. B., Kurtz, A., Miiller. T., Niemann, C., Blenau, W. and Grosse, R.: Hormonal induction of functional differentiation and mammary-derived growth inhibitor expression in cultured mouse mammary gland explants. In Vitro Cell Dev. Biol. 28A ( 1992) 625-634. BBhmer, F.D., Kraft, R., Otto, A., Wernstedt, C., Hellman, U., Kurtz, A., Mtiller, T., Rohde, K., Etzold, G., Lehmann, W. et al.: Identification of a polypeptide growth inhibitor from bovine mammary gland. Sequence homology to fatty acid- and retinoid-binding proteins. J. Biol. Chem. 262 (1987) 15137-15143. Breathnach, R. and Chambon, P.: Organization and expression of eucaryotic split genes coding for proteins. Annu. Rev. Biochem. 50 (1981) 349-383. Chomczynski, P. and Sacchi, N.: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162 (1987) 156-159. Durand, B., Saunders, M., Leroy, P., Leid. M. and Chambon, P.: Alltrans and 9-cis retinoic acid induction of CRABPII transcription is mediated by RAR-RXR heterodimers bound to DRl and DR2 repeated motifs. Cell 71 (1992) 73-85. Eager, N.S., Brickell, P.M., Snell, C. and Wood, J.N.: Structural and functional evidence for activation of a chick retinoid X receptor by eicosanoids. Proc. R. Sot. Lond. Biol. 250 ( 1992) 63-69. Green, E.L.: Genetics and Probability in Animal Breeding Experiments. Oxford University Press, New York, NY. 1981. Grimaldi, P.A., Knobel, S.A., Whitesell, R.R. and Abumrad, N.A.: Induction of aP2 gene expression by nonmetabolized long-chain fatty acids. Proc. Natl. Acad. Sci. USA 89 (1992) 10 930-10 934. Grosse, R., BBhmer, F.D., Binas, B., Kurtz, A., Spitzer. E., Miiller, T. and Zschiesche, W.: Mammary-derived Cancer Treat. Res. 61 (1992) 69-96.
growth
inhibitor
(MDGI).
Heuckeroth, R.O., Birkenmeier, E.H., Levin, M.S. and Gordon, J.I.: Analysis of the tissue-specific expression, developmental regulation, and linkage relationships of a rodent gene encoding heart fatty acidbinding protein. J. Biol. Chem. 262 (1987) 9709-9717. Hoggan, M.D., Halden, N.F.. Buckler, C.E. and Kozak. C.A.: Genetic mapping of the mouse +/ins proto-oncogene to chromosome 18. J. Virol. 62 (1988) 1055-1056. Jhappan, C.. Geiser, A.G., Kordon, E.C., Bagheri. D., Hennighausen, L., Roberts, A.B., Smith. G.H. and Merlino, G.: Targeting expression of a transforming growth factor-beta-l transgene to the pregnant mammary gland inhibits alveolar development and lactation. EMBO J. 12 (1993) 1835-1845. Jones. K.A.. Kadonaga. J.T.. Rosenfeld. P.J., Kelly, T.J. and Tjian, R.: A cellular DNA-binding protein that activates eukaryotic transcription and DNA replication. Cell 48 (1987) 79 -89. Keller, H.. Dreyer, C., Medin, J., Mahfoudi, A., Ozato, K. and Wahli. W.: Fatty acids and retinoids control lipid metabolism through activation of peroxisome prohferator-activated receptor-retinoid X receptor heterodimers. Proc. Nat]. Acad. Sci. USA 90 (1993) 2160.2164.
Kerr, L.D., Miller, D.B. and Matrisian, L.M.: TGF-beta 1 inhibition of transin/stromelysin gene expression is mediated through a Fos binding sequence. Cell 61 (1990) 267-278. Kliewer, S.A., Umesono. K., Noonan, D.J., Heyman, R.A. and Evans, R.M.: Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors. Nature 358 (1992) 771-774. Kozak, C.A., Peyser. M., Krall, M., Mariano, T.M., Kumar, C.S.. Pestka, S. and Mock, B.A.: Molecular genetic markers spanning mouse chromosome 10. Genomics 8 (1990) 519-524. Kuhn, R., Monuki, ES. and Lemke, G.: The gene encoding the transcription factor SCIP has features of an expressed retroposon. Mol. Cell. Biol. 11 ( 1991) 4642-4650. Kurtz, A.. Vogel, F., Funa. K., Heldin, C.H. and Grosse. R.: Developmental regulation of mammary-derived growth inhibitor expression in bovine mammary tissue. J. Cell Biol. 110 ( 1990) 1779 -1789. Lyon, M.F. and
Searle,
A.G.:
Genetic
Variants
and
Strains
of the
Laboratory Mouse. Oxford University Press, Oxford. 1989. Mar, J.H. and Ordahl, C.P.: M-CAT binding factor, a novel transacting factor governing muscle-specific transcription. Mol. Cell. Biol. IO (1990) 4271-4283. Matarese, V., Stone, R.L., Waggoner, D.W. and Bernlohr, D.A.: Intracellular fatty acid trafficking and the role of cytosolic lipid binding proteins. Progr. Lipid Res. 28 (1989) 245-272. Mink. S., Hartig, E., Jennewein. P., Doppler, mammary cell-specific enhancer in mouse
W. and Cato, A.C.: A mammary tumor virus
DNA is composed of multiple regulatory elements including binding sites for CTFINFI and a novel transcription factor, mammary cellactivating factor. Mol. Cell. Biol. 12 ( 1992) 4906&4918. Miiller, T., Kurtz, A., Vogel, F.. Breter, H., Schneider, F.. Angstrom, U.. Mieth, M., Biihmer, F.D. and Grosse, R.: A mammary-derived growth inhibitor (MDGI) related 70 kDa antigen identified in nuclei of mammary epithelial cells. J. Cell Physiol. 138 (1989) 415-423. Oka. T., Yoshimura, M., Lavandero, S., Wada, K. and Ohba, Y.: Control of growth and differentiation of the mammary gland by growth factors. J. Dairy Sci. 74 (1991) 2788-2800. Peeters. R.A., Veerkamp. J.H., Geurts van Kessel, A., Kanda, T. and Ono, T.: Cloning of the cDNA encoding human skeletal-muscle fatty-acid-binding protein. its peptide sequence and chromosomal localization. Biochem. J. 276 (1991) 203-207. Rogers. J.H.: The origin and evolution of retroposons. Int. Rev. Cytol. 93 (1985) 187-279. Sambrook,
J., Fritsch.
E.F. and
Maniatis,
T.: Molecular
Laboratory Manual, 2nd ed. Cold Spring Harbor Cold Spring Harbor. NY, 1989. pp. 7.79-7.83.
Cloning.
Laboratory
A
Press,
Spener, F., Unterberg, C., Biirchers, T. and Grosse, R.: Characteristics of fatty-acid binding proteins and their relation to mammaryderived growth inhibitor. Mol. Cell. Biochem. 98 (1990) 57-68 Veerkamp, J.H.. Peeters. R.A. and Maatman, R.G.: Structural and functional features of different types of cytoplasmic fatty acid-binding proteins. Biochim. Biophys. Acta 1081 (1991) I-24. Wallukat. G., Boehmer, F.D., Engstroem. U., Langen, P., Hollenberg, M.. Behlke, J., Kuehn. H. and Grosse, R.: Modulation of the betaadrenergic-response in cultured rat heart cells, II. Mammary-derived growth inhibitor (MDGI) blocks induction of beta-adrenergic supersensitivity. Dissociation from lipid-binding activity of MDGI. Mol. Cell Biochem. 102 (1991) 49-60. Watson, C.J., Gordon, K.E., Robertson, M. and Clark, A.J.: Interaction of DNA-binding proteins with a milk protein gene promoter in vitro: identification of a mammary gland-specific factor. Nucleic Acids Res. 19 (1991) 6603-6610. Wright, W.E.: Muscle basic helix-loop-helix proteins and the regulation of myogenesis. Curr. Opin. Genet. Dev. 2 (1992) 243-248. Yoshimura, M. and Oka, T.: Isolation and structural analysis of the mouse p-casein gene. Gene 78 (1989) 267-275.
GENE
237
0378-l 1 i9~94~$~7.0~
08136
Cloning and characterization of the mouse gene encoding mammary-derived growth inhibitor/heart-fatty acid-binding protein (Recombinant DNA; gene cloning; localization; mammary gland)
Mike Treunera,
Christine
Received by J.K.C. Knowles:
reverse transcription-polymerase
chain reaction;
A. Kozakb, Daniel Gallahan”,
7 January
1994; Accepted:
Richard
19 April 1994: Received at publishers:
pseudogene;
chromosome
Grossed and Thomas
Miiller”
30 May 1994
SUMMARY
From a mouse genomic library we isolated and characterized a gene, Fubphl, encoding mammary-derived growth inhibitor (MDGI)/heart fatty-acid-binding protein (H-FABP). Exon sequences were identical with a MDGI-encoding cDNA isolated previously from the mammary gland of pregnant mice. The product of this gene has also been detected in heart, where it had been termed H-FABP. It has an intron/exon structure similar to other FABP-encoding genes. In addition to this expressed gene, we isolated a related intronless pseudogene, Fabph-ps, with an open reading frame which was highly conserved when compared with Fubphf. Fubphl was positioned on chromosome (Chr) 4 using interspecies crosses. Analysis of somatic cell hybrids was used to assign Fubph-ps to Chr 10, and another MDGI/H-FABPrelated sequence locus, F~b~h-rsl, to Chr 8. A h4us spretus-specific related sequence, F~~J~h-rs2, was identified on Chr 17 by analysis of interspecies crosses. The 5’-flanking region of F~bph~ contains putative transcription factor-binding elements which could account for its constitutive expression in muscle tissue, as well as for its developmental stagedependent expression in mammary epithelium.
bovine
INTRODUCTION
Starting with the assumption that locally acting factors should be involved in regulation of the development of mammary epithelium (Oka et al., 1991), we isolated the mammary-derived growth inhibitor (MDGI) from Correspondence to: Dr. T. Mtiller,
Laboratory
of Molecular
NINDS. National Jnstitutes of Health, Bethesda, USA. Tel. (l-301) 496-3412: Fax (l-301) 402-1340; e-mail: [email protected]
Maryland
Biology, 20892.
Abbreviations: aa, amino acid(s); bp, base pair(s); Chr, chromosome(s); FABP, fatty acid-binding protein; FobphI, gene encoding MDGI/ H-FABP; Fuhph-ps, Fclhpltl-related pseudogene; Fahph-rs, F&phirelated sequence locus; I-t-FABP, heart-FABP; M., Mus; Mm., M. muscuiz~ MDGI. mammary-derived growth inhibitor; nt, nucleotide(s): ohgo. oligodeoxyribonucleotide~ ORF, open reading frame; RT-PCR. reverse transcripti(~ll-polyme~~s~ chain reaction; tsp. transcription start point(s); UTR. untranslated region(s). SSDl 037X-I
114f94)00347-U
lactating
mammary
gland (Biihmer
et al., 1987).
MDGI belongs to the family of fatty-acid-binding proteins (FABP) (Veerkamp et al., 1991) with highest homoiogies to the FABPs from bovine synthesis
is induced
in mammary
heart and brain. epithelium
MDGI
only during
late pregnancy and lactation (Miller et al., 1989; Kurtz et al., 1990) and strictly depends on lactogenic hormones (Binas
et al., 1992). Functionally,
acterized entiation
1992). The question FABP
MDGI
has been char-
as an endogenous growth inhibitor and differfactor of mammary epithelium (Grosse et al.,
(H-FABP)
of whether the proteins
MDGI
are in fact identical
one gene has been considered
previously
1990). While the high degree of similarity
and heart-
and encoded (Spener between
by
et al., MDGI
and H-FABP sequences is consistent with the conclusion that MDGI and H-FABP may be derived from a single
238 MDGI~H-FABP-encoding
gene, efforts
to chromosom-
ally map the genes MDGl/H-FABP-related
responsible have uncovered sequences on three mouse chro-
mosomes
cell hybrids
1987).
using somatic In man,
a single
(Peeters et al., 1991). The aim of the present
genetic study
(Heuckeroth locus
was
was to clone
et al.,
identified and map
(b) Analysis of ~~Gl/~-FA~P-r~Iat~d The coding served
region
when
transcripts
of the pseudogene
compared
is highly
with the gene and
cDNA. Three nt substitutions result in two aa exchanges (S14+C, G’” -+R). Furthermore, sequence conservation extends
beyond
section
d). Since examples
the tsp and the putative of expressed
TATA-box
(see
retroposons
are
the MDGI/H-FABP-encoding genes. The sequences of the isolated gene and of its regulatory regions will be
documented
instrumenta
tr~~llscribed. To this end, RNA was isolated
in the investigation
type-specific regulatory mechanisms operating in such diverse tissues
of common
and cell-
of gene tr~~nscription as mammary gland
and muscle.
mouse
5’-primer
clones
(a) Isolation of two mouse MDGI/H-FABP-related genes Approx. 800~0 plaque-forming units of a mouse genomic library &GEM-l 1, Promega, Madison. WI, USA) were screened using the coding region of mouse MDGI cDNA as probe (Binas et al., 1992, GenBank S48643, revised UO2883). Six different recombinant clones were identified and plaque purified. According to their rcstriction patterns they represent two different genomic clones. By Southern analysis with oligos from the 5’- and the 3’-region of the MDGI cDNA, phage clones hB and hD representing these two genes were found to contain the full-length coding region and were selected for subcloning into pBluescript SK( if. Overlapping genotnic DNA inserts in plasmid subclones were sequenced in both directions ( Fig. 1). Clone hB was found to contain a gene with four exons and three introns. Intron/exon boundaries were established by comparison with the cDNA sequence. They were found to obey the GT/AG rule (Breathnach and Chambon, 1981). lntrons are identically positioned as in all other FABP genes characterized (Matarese et al., 1989). The sequence of the exon regions of the gene encoded by clone hB was found to be identical with the mouse MDGI cDNA. Phage clone hD was found to contain an ORF which was not interrupted by introns and which differed by only three nt substitutions from the coding region of the gene contained in clone hB (Fig. 1). The region of high similarity between clones hB and hD covers I18 bp of S-UTR and 236 bp of 3’-UTR until an oligo(dA)-tract begins in clone hD. This region is flanked by 14-bp direct repeats. Thus, the genomic sequence contained in clone hD most likely represents a pseudogene which arose by retroposition from a processed RNA intermediate (Rogers, 198.5 1.
the question
106 and to allow
whether
the 3’-primer fragment
hD.
an additional
hD, representing
a potential
and the mouse
The
mRNAs
a subclone
in phage be distin-
since the pseusite at position derived
pseudogene-derived cDNA,
from cDNA
corresponding
in the PCR analysis.
liver and pancreas
MDGI/H-FABP-related
could
pattern
MDGI
to clone hB, were included Spleen,
from
contained
restriction
(cf., Fig. 1). As controls,
sequence,
for RT-PCR.
fragments
by their Hinfl restriction
dogene contains
is
from various
003 (cf., Fig. 1) were
sequences
These
et al., 1993).
the pseudogene
amplification
from genomic
hB and
guished f592
et al., 1991; Linnenbach
tissues and used as templates
chosen
AND DISCUSSION
(Kuhn
we addressed
transcribed
EXPERIMENTAL
con-
the MDGI
tissue did not contain
transcripts
(not shown).
any From
total RNA samples of lung, kidney, testis, skeletal muscle, heart, brain and mammary
gland from pregnant
and from
lactating animals RT-PCR fragments of the expected size were obtained (Fig. 2A). Fragments amplified from tissue RNAs, as well as fragments amplified from the control DNAs were subsequently digested using the restriction enzyme Hi&. Restriction patterns of PCR fragments obtained from the various tissue RNAs were all identical to the pattern obtained from MDGT cDNA (Fig. 2B). Omitting the reverse transcription step, small amounts of PCR fragments HinfI digestion
were obtained
pattern
similar
which gave rise to a
to the one resulting
from
hD DNA. This indicates probable contamination of RNA preparations with minor amounts of genomic DNA. Furthermore, the sequences of the PCR fragments obtained from heart and mammary gland RNA were found to be identical with the exon sequences of the gene contained in clone hB. These findings indicated that the mammary gland derived MDGI and the heart-derived H-FABP are both transcribed from the MDGI/H-FABPencoding gene contained in clone hB. This conclusion is in accordance with peptide sequencing data (Bansal and Medina, 1993). No transcripts from the MDGI/H-FABPrelated pseudogene contained in clone hD could be detected. It cannot be ruled out completely that expression of the pseudogene takes place in certain cells or at certain developmental stages. The MDGI/H-FABP-encoding gene and the pseudogene are designated F~z~?~~land F~~~~~?-~s, respectively.
239 )cB ...tttgagagagtatcttgccttggctcagactggCCCcCaaCtCaggaatctcttgCctccaggtgca~t~CCgtattaaCtgttggCtCaagCCagagCagCggCaCa~~ta E-box ha
-1068
E-box
accatgcCCtgaagtaggctacaaccatcaatagtcgggtcttatttaataacgtactttaaggtgacaagcagtctag~~gaagtcaggggaaaaaactgacttcagcagagggtc -__ NFl
hB
-1240 E-box
atgctgacttggtaataattaaacagaaactgctgaactaaggaataggctccactgaggttcccttactcacctgtaaaaaggggatgataccacctaccaacgaaaaagttgagtgtg APl
hB
E-box
-948
API
gcggctttccgggagttaaggtggccgaggccggaagaaccctctgaatagacaaattgtcttcgcggagtgaagaacgaccctggcacaagctc~gaggtcagtaaataaagcctgaag NF1
-828
DR-1
hB cgctttcaggcagcggcgaCgggtgggaCtgCggagaaaggCgC~ggCggg~g~CattCCgCaggg~ggggCt~g~tggggCt~gC~tg~gg3~~gCa~ggtC~CgttCtCCgCC E-box hB
agc~gq~_gaggcgctgggcagctcagccatccgcgg~~~caaggcaactcttttcc~tctggtaggagcaagagggctcaaaggccactagaccatgctctctgtccaggctcca E-LXX
ha
-708
MAF
TIE
-588
E-box
attctttttta~ttacggcgaccgcgt~~~~~~ctccgagcctctgagcctcttctacaagaagaggac~taggaccgttgagatgggttt... M-CAT
210
bp ...tttctccagcgfqq
HRE
-272
NFl
hB ~CcagctcaagggcgagtttcctttC~gt~tggCC~gggg~tgCtCt~CttgggttgCg~~gCCCCgC~gCC~ggCC~ggg~tgggt~~g~~~CC~~C~gg~~~g~ggg~g~ MPBS
-152
NFl
hD
...ctgg~g~ga~t~~~~gctttCaacagcctgcttCttg~~tgttgta~acaca~cgtaa~~aaattuttcacratttcgggagcgaggggtgtgggccactttcatcatgtgatgcga
hB
cgctgacgtaggcgacgggagggctgtggggg~tggg=C~=~gCCCtttgCggg~gtgC~~g=CC~gg=ttCCtt~tttCgggagCgaggggtgtgggCCaCtttCatCatgtgatgCga
hD gggctattt.aagaggctgtccagccgggagCTGCGGTTCTCAGTGCCTGCCCGCCTCCTCACTCATCGCACCATGGCGGACGCCTTTGTCGGTACCTGGAAGCTAGTGGACTGCAAG~ hB gggctattt.aagaggctgtccagccgggagcTGCGGTTCTCAGTGCCTGCTCGCCTCCTCACTCATCGCACCATGGCGGACGCCTTTGTCGGTACCTGGAAGCTAGTGGACAGCAAGAA MADAFVGTWKL"D*SKN m primer761 TTTTGATGACTACATGAAGTCACTCG-------------------~-------------GTGTGGGCTTTGCCACCAGGCAGGTGGCTAGCATGACC~GCCTACTACCATCATCGAG~G hB TTTTGATGACTACATGAAGTCACTCGgtgag...intrOn 1. 3.4 kb...cttagGTGTGGGCTTTGCCACCAGGCAGGTGGCTAGCATGACC~GCCTACTACCATCATCGAGAAG F D DYMKSL GVGFATRQVASMTKPTTI I E K
hD
-primer
-33 -33 +88 t88 16 t176 t3617 45
1064
hD AACGGGGATACTATCACCATAAAGACACAAAGTACCTTCAGGTCAAG--------hB AACGGGGATACTATCACCATRAAGACACAAAGTACCTTCRAGAACACAGAGATC~CTTTCAGCTGGG~TAGAGTTCGACGAGGTGACAGCAGATGACCGG~GGTC~Ggtgag NGDTIT I K T Q STFKNTEINFQLGI EFDEVTADDRKVK
t287 t3733 82
hB intron 2, 1.5 kb...cacagTCACTGGTGACGCTGGACGGAGGCAAACTCATCCATGTGCAGAAGTGGAACGGGCAGGAGACAACACTAACTAGGGAGCTAGTTGACGGG~ACTC SLVTLDGGKLI Q ETTLTRELVD*GKL H V Q K W N*G
t383 +5314 114
hB ATCCTGgtaag...intron I L? Hinf I
+471 +6518 133
1 Hinf I hD ATCCTG------------------------------ACTCGGAGGCGTGACCTGGCTGCTCCGTCACTGACCGCCCGC
3. 1.1 kb...tctagACTCTCACTCATGGCAGTGTGGTGAGCACTCGGACTTATGAG~GGAGGCGTGACCTGGCTGCTCCGTCACTGACCGCCCGC TLTHGSVVSTRTYEKEA---
hD TCCTCTGCCAACTGGCCACCCCTCAGCTCAGCACCATGCTGCCTCATGGTTTTCCCCTCTGACATTTTGTAT~CATTCTTGGGTTGGGATTTTTCTGGAGATACGGGGCATCAGCCTG hB TCCTCTGCCAACTGGCCACCCCTCAGCTCAGCACCATGCTGCCTCATGGTTTTCCCCTCTGACATTTTGTAT~CATTCTTGGGTTGGGATTTTTCTGGAGATACGGGGCATCAGCCTG
+591 +6638
J Hinf I ho GACTCAGTTCCTACTATGTATGTGGTTTATTTTTTAAAACCAGAACCAAGGCClaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa hB GACCCAGTTCCTACTATGTATGTGGTTTATTTTTTAAAACTGTATCCAAAGGGTGCTCCAAGGTCMT~GCAGAACCAAGGCC~ACCCAgttgtctgtctttggt~=t~~tttC~tgt * * -primer OO%---
t710 16757
l
hD
aaaaaaaaaaaaaaaaq~~~~~~a~cacaaacaatagactcacaagatgagc...
1767 +6814
hB gtgtcaggttg~~~tg~aggcctataggtcacctggt~a~~tggg~~g~~g~~~tgt~~agg~g~...
Fig. 1. Sequences
of the mouse
MDGI/H-FABP-encoding
gene Fabphl (LB) and pseudogene
Fabph-ps (hD). The nt differences
are indicated
by *.
DNA fragments generated by Sac1 and KpnI digestions of phage DNA from clones hB and hD were subcloned into pBluescript SK(+) and sequences were determined by dideoxy-sequencing in both directions using Sequenase 2.0 (US Biochemical, Cleveland, OH, USA). Putative TATA-box and polyadenylation signal are bold-faced. Exon regions are printed in capital letters while portions of the introns, S- and 3’-CITR are shown in lower case letters. The deduced aa is placed under the second nt of each codon. The region of homology between the gene and the pseudogene is enclosed in brackets. Direct repeats flanking this region are double underlined. Primer 781 used for primer extension (cf., section d) and primers 106 (sense) and 003 (antisense) used for transcript analysis (cf., section b) are indicated by horizontal arrows. Hinfl sites within the PCR fragment (cf., section b) are marked with vertical arrows. Putative transcription factor binding sites are underlined and explained in section d. The GenBank accession Nos. are UO2884 (Fabphl) and U02885
(Fubph-ps).
(c) Genetic mapping Chinese hamster x mouse somatic cell hybrids have been previously described (Hoggan et al., 1988). 18 hybrids were selected from a larger panel of 76 hybrids for this study. They were typed for MDGI/H-FABP-related sequences using the MDGI cDNA probe (Binas et al., 1992) following digestion with EcoRI, BumHI and PstI. For each enzyme, the probe identified two major bands and several minor bands. The two major bands identified loci on two different chromosomes. The Fuhphl locus, identified by an EcoRI fragment of 8.6 kb, a BumHI fragment of 12.0 kb and a PstI fragment of 6.5 kb, could be assigned to Chr 4 based on the perfect correlation between rhe presence or absence of this chromosome with
the presence or absence of these fragments (data not shown). At least two discordant hybrids were identified for each of the 18 autosomes and the X chromosome. The second major fragment was identified by fragments of 6.0 kb (EcoRI), 13.5 kb (BarnHI) and 3.0 kb (PstI). This locus was mapped to Chr 10. A probe specific for Fubphps corresponding to its 5’-flanking region (nt -524 to -33, cf., Fig. 1) also identified this EcoRI fragment of 6.0 kb, indicating that this Chr 10 locus represents the cloned pseudogene, which we designated Fahph-ps. Finally, fragments characterized by a weaker hybridization signal were identified by a 7.0-kb PsfI fragment and a 5.0-kb EcoRI fragment which mapped to Chr 8. This locus is designated Fubphl -related sequence 1 (Fabph-rsl ).
240 LBhD +mgj y-p
+b _ +h _ +I _ +sm_ +t _ +k _
A’1
bp
B
270 230
4/X2
4 Y +/- 2.4
203 Fig. 2. RT-PCR
analysis
of MDGl!H-FABP-related
transcripts
in
mouse tissues. RNA was extracted from mouse tissues (mg-I, lactating mammary gland; mg-p, mammary gland from 16 days pregnant mouse; b. brain; h. heart; I. lung; sm, skeletal muscle; t, testis: k. kidney) according to (Chomczynski and Sacchi, 1987). 0.5 pg. of RNA was reverse transcribed using a ss-cDNA-synthesis kit and random hexamer primers (Pharmacia) (lanes marked with (+)) and used as template for PCR with primers 106 and 003 (cf., Fig. I ). In control samples. lanes marked with (-). the reverse transcription step was omitted. As further controls. the subcloned 5~1 fragment of clone >_D (P-D) and the mouse MDGI cDNA (p&B)were used as templates for PCR. (A) Reaction products were analysed by 1.2%agarose is indicated. (B) PCR products
electrophoresis. Expected fragment size were subsequently digested with Hirzfl
and analysed by electrophoresis. The 230-bp fragment is indicative of a transcript originating from the cloned MDGI/H-FABP-encoding gene (LB), the 203-bp fragment would indicate a transcript from the pseudogene OLD). Both have the 270-bp fragment in common, 27-bp fragment was not resolved in this electrophoresis.
The remaining
These data indicate that all MDGI/H-FABP-related sequences can be assigned to three chromosomes in the inbred mouse. Two genetic crosses were used to position MDGI/HFABP-related sequences on the mouse genetic map: (NFS/N or C58/J x M. 111.r?tusculus) x iz4. m. nzus&us (Kozak et al., 1990) and (NFS/N M. spretus) x (M. spretus or C58/J) (Adamson et al., 1991). The progeny of these crosses have now been typed for over 650 genetic markers including Lck (lymphocyte tyrosine kinase), Dsil (David Steffen integration-l), and Oprd (opioid receptor delta) on Chr 4 and rd2 (retinal degeneration slow, peripherin), C3 (complement component 3) and Hprt-psi (hypoxanthine phosphoribosyl transferase pseudogene- 1) on Chr 17. DNAs from the parental mice of both genetic crosses were typed for restriction enzyme polymorphisms using as probes the MDGI cDNA, as well as an intron sequence corresponding to nt + 60 to + 3 195 of Fcihphf (cf., Fig. 1). Each enzyme used identified multiple fragments, but for any given enzyme only one or two fragments were polymorphic and could be typed in the progeny. For the M. m. musculus cross, ApaI produced fragments of 6.5, 4.2 and 2.8 kb in NFS/N and 13.2, 6.5 and 2.8 kb in M. m. musculus. Analysis of the progeny showed that the 4.2-kb NFS/N fragment identified the Fuhphl locus on Chr 4 (Fig. 3). Use of the Fuhphl derived intron probe identified this same locus. Using the MDGI-encoding cDNA, analysis of the M. spretus cross identified two fragments in M. spretu.s which represent two loci. One locus, identified as a M. spretus
Fig. 3. Linkage
of F~hphl
on mouse
Chr 4 and of F~hph-rsZ
on M.
sprrru.s Chr 17. DNAs extracted from the progeny of the two genetic crosses (NFS/N or C58.iJ x M. tn. ~rrusc~u/us)x M. rri. musc~ulus (Kozak et al.. 1990) and (NFS/N x M. spreru.s) x(M. sprerus or C58.;J) (Adamson et al., 1991) were digested with Apd and Pstl to identify polymorphic fragments by Southern analysis using the MDGI cDNA probe and a Fuhphl-specific intron probe (nt +60 to +3195). Membranes
were
washed
in 0.2 x SSC;2%
SDS
at 60-65 C.
Both
probes positioned the gene Fuhphl on Chr 4. The MDGI cDNA probe also identified the locus FuhplwsZ011IV.spwru.sChr 17. For each cross a recombination fraction is given for adjacent loci, and percent recombination and standard error (SE) are calculated according to Green ( 19X1 ). No double
recombinants
were identified
on either chromosome.
PstI fragment also identified
of 8.8 kb mapped to the Chr 4 position in the M. m. musculus cross. The second
locus, identified as a M. spretus PstI fragment of 6.0 kb mapped to the distal region of Chr 17 (Fig. 3). The failure to identify F&p&related sequences on Chr 17 using somatic cell hybrids or using the M. m. musculus cross suggests that these sequences represent M. spretusspecific F&p&related sequences. This locus is designated Fubph-rs2. Previous studies had used a rat H-FABP-encoding cDNA probe to assign related fragments of HindIII-digested genomic DNA to mouse Chr 4, 8 and either 10 or 15 (Heuckeroth et al., 1987). Two Hind111 fragments were mapped to Chr 4, designated as loci Fuhphl and Fuhph4 (Lyon and Searle, 1989), the locus on Chr 8 was named Fuhph2, and the locus on either Chr 10 or 15 was referred to as Fubph3. While the FABP-encoding sequences on Chr 10 and Chr 8 have not been positioned, a previous study positioned the locus Fuhphl on Chr 4 just distal to Lck (Bahary et al., 1991). Using the mouse MDGI-encoding cDNA probe and the intron probe derived from clone hB, our results identify Fubphl as the expressed MDGI/H-FABP-encoding gene. Since this gene has Hind111 sites in its first and second introns, the formerly defined locus Fuhph4 is most likely contiguous with Fuhphl. Our data indicate that the cloned pseudogene Fubph-ps corresponds to the locus Fubph3 and that this locus can now be assigned unequivocally to Chr 10. In man, the human H-FABP-encoding gene was assigned to chromosome lpter-q31 (Peeters et al., 1991) in a region of linkage homology with mouse Chr 4.
241 (d) Analysis of the S-flanking FABP-encoding
The S-boundary primer
extension
(1989).
of the first exon was determined analysis
Comparison
poly(A)‘RNA the sequencing
region of the MDGIfH-
gene according
of the
to Sambrook
extension
product,
by
Fig. 1 the T residue
Synthesis in various
of MDGI/H-FABP tissues.
Postnatally,
duced in the heart in very high amounts extent
in skeletal
of nt in
muscle,
testis, brain
pro-
and to a lesser and other
tissues
(Heuckeroth et al., 1987; Veerkamp et al., 1991). In bovine and mouse mammary epithelium MDGI/H-FABP is only detectable during pregnancy and lactation and strictly depends on the lactogenic hormones prolactin and hydrocortisonc (Miiller et al., 1989; Kurtz et al., 1990; Binas et al., 1992). Sequence inspection of the upstream region of Fuhphf revealed a TATA-box at -21 with the sequence TTTAAA (Fig. 1) characteristic of a variety of milk protein genes (Yoshimura and Oka, 1989). With regard to musclespecific expression of Fahphl, two clusters of E-boxes
yeast heart RNA
RNA
involved related
3’5’ CG GC GC CG CG CG TA CG GC A T* CG G C* CG CG AT AT GC AT GC TA CG AT 5’3’
Fig. 4. Analysis of the rsp of Fahphl. The DNA sequencing ladder and the product of the primer extension reaction using primer 781 (cf. Fig. 1) were separated in a sequencing gel. Primer extension reaction was carried out as described (Sambrook et al., 1989) using 2p.g of heart poly(A)~RNA or in thecontrol 2 ~gofbaker’sy~~st tRNA as templates.
sites for the MyoD-family
factors in muscle
gene expression
Sequences
(MPBF) activating
binding
(Wright,
1992) and a
tissue
specificity
(Mar and Ordahl, to milk protein
several CTF/NFl
half palindromic
binding
elements
of
1990) were
(Watson et al., 1991) and mammary factor (MAF) (Mink et al., 1992) binding
factor cellsites,
(Jones et al.,
1987) and a hormone responsive element (HRE) (Bailly et al., 1986) are present in the S-flanking region of F~~~~zf and might be instrumental
in
regulated
it is constitutively
motif
found.
in ~~~bph-pscontained is differentially
transcription
M-CAT
using
was set to + 1 in ~~~~~~ contained
in clone hB and, by analogy, clone AD as well.
of myogenic troponin-T
from mouse heart tissue as template, with ladder both originating from the antisense
start site (Fig. 4). For numbering
potential
et al.,
oligo 78 1 (cf., Fig. 1) indicated that transcription initiates at the T and the C residues 41 and 39 bp upstream from the translatiol~
representing
in the hormone
and differen-
tiation-dependent expression in mammary epithelium. In addition, a direct repeat (DR-1) with a spacing of one base pair, similar
to binding
sites of retinoic
tors (RAR, RXR) and peroxisome receptor
(PPAR)
1992) could
(Durand
be detected
proliferator
acid recepactivated
et al., 1992; Kliewer at position
-84.5.
et al.,
Evidence
is
accumulating that fatty acids and arachidonic acid metabolites may regulate gene expression (Distel et al., 1992; Grimaldi et al., 1992) possibly by activating similar transcription factors (Eager et al., 1992; Keller et al., 1993). Interestingly, one of the ligands of MDGI/H-FABP is arachidonic acid (Veerkamp et al., 1991; Wallukat et al., 1991). Since we have found that in mammary gland organ culture MDGI/H-FABP promotes its own expression (R.G., unpublished), an involvement of MDGI/H-FABP in transport or metabolism of a ligand of a member of this transcription factor family could be postulated. TGFP was recently shown to inhibit functional differentiation of mammary epithelium during pregnancy (Jhappan et al., 1993). We have found that TGFP downregulates F~~~~zi expression in di~erentiated primary mouse mammary epithelial cells (T.M.. LInpublished). In this respect, a putative TGF@l inhibitory element (TIE) (Kerr et al., 1990) in the FdphI promoter might be functionally important (cf., Fig. 1). Suppression of MDGI/HFABP-encoding gene expression during early ductal morphogenesis may be essential in order to allow development of a functional mammary gland (Binas et al.. 1992). The functional importance of putative regulatory elements for MDGI/H-FABP-encoding gene expression in mammary epithelial and in muscle cells will be investigated in future studies.
ACKNOWLEDGEMENTS
We wish to thank Drs. Claus Scheidereit and Matthias Gaestel for critical reading the manuscript and Andrea Dell’Oro for skilfull technical assistance. The work was supported by grant SFB 344 of the Deutsche Forsch~lngsgemeinschaft to T.M.
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