BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1725-1731
Voi. 189, N6. 3, 1992 December 30, 1992
STRUCTURAL
ORGANIZATION
OF THE MOUSE LACTOFERRIN
GENE
GrainneA. Cunningham,Denis R. Headon*andOrla M . Conneelyl Departmentof Cell Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston,TX 77030 *Cell and Molecular Biology Group, Department of Biochemistry, University College, Galway, Ireland Received
November
17,
1992
The complete structure of the mouse lactoferrin gene is presented. Mouse lactoferrin (mLF) is encodedby a single copy geneof approximately30 kilobases(kb) in size. The ,oeneis organizedinto 17 exons separatedby 16 introns. The exons range in size from 48 base pairs (bp) to’190 bp whereasthe introns ransefrom 0.2 kb to 4.3 kb. Structuralanalysisof the moust: lactofemingene revealsthat this genesharesa similar intron-exondistribution pattern with both human transferrinand chicken ovotransfetrin. 0 1992Academic PES-, lnc.
Lactoferrin is a memberof the family of non-haem,iron-binding proteins known as the transferrins(1). This family includesserumtransferrin,the iron transportprotein in the blood of vertebratesand some invertebrates,ovotransferrinfrom egg-whiteand the membrane-associated melanotransferrinfrom melanocytes(2). The transferrinsare all monomericglycoproteinswith a molecularmassof approximately80 kDa, composedof two lobes,eachpossessingthe capacity to bind reversibly one ferric iron with the synergisticbinding of a bicarbonateor carbonic anion (1,3). It is thought that the transferrinsoriginate from a common primordial gene by a gene duplication event (4). This proposal is supportedby a two-fold internal repeat found in the primary structure of all the transferrinsstudied to date. For each of theseproteins, lactoferrin, serum transferrin, ovotransferrin and melanotransferrin,the N-terminal half was found to he approximately40% identical with the C-terminal half of the proteins(5). Lactoferrin is found in m ilk (6) and a variety of other exocrinesecretions(7) and also in the secondarygranules of neutrophils (8). The biological functions proposedfor lactoferrin include protection againstm icrobial infection (9). enhancedintestinal iron absorptionin infants (l(I), promotion of cell growth (II), regulation of myelopoiesis (12) and modulation of inflammatory responses(13). ’ To whom correspondence should be addressed. Abbreviations: kilobases= kb , basepairs = bp , mouselactoferrin = mLF.
1725
0006-291x/9? $4.00 Copyright 0 1992 by Academic Pres.v. Inc. All rights of reproduction in arty form rewnvd.
Vol.
189, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Partial structural organizationof the mouse lactofetrin gene was recently reported by both Liu et al. (14) and Shirsatet al. (15). In this paper,we extendtheseanalysesby presenting the complete structural organizationof the mouselactoferrin gene. We demonstratethat mouse lactoferrin is encoded by a single copy gene of approximately 30 kb in size that shares a structural relatedness,at the gene level, to both human transferrin and chicken ovotransferrin (16,17). The transcriptional unit is encoded by approximately 23 kb and is llanked by a previously characterizedpromoterregion of 7.5 kb (14).
Materials and Methods Genomic Southern Analysis: 10 ug of mouse genomic DNA was digested with the restriction enzymesindicatedin Figure 1, resolvedon a 0.8% agarosegel and then transferredto a nylon filter. Prehybridizationand hybridization of the nylon filter were performed in O S M Na2HP04, pH 7, 1 m M EDTA and 7%, SDS at 680C for 16 hours. The hybridization solution contained 1~10~cpm/ml (specific activity - 2x109 cpm/ug) of a [32P] labeled mLF genomic probe which containednucleotides997-1212 of the mLF cDNA (18), encoding part of exon 8 and all of exon 9, and the interveningintron (-800 bp). The filter was washedtwice in 0.04M Na2HP04/1%SDS at 680C for 30 m in. The filter was dried, exposedat -700C and developed by autoradiography. Amplification of mLF genomicDNA fragmentsusing the polymerasechain reaction(PCR): Amplification of individual regionsof the mouse lactoferrin gene was performed using 1 pg of mousegenomicDNA as templateand a variety of 30-meroligonucleotideprimers whose sequence was determined from the published mouse uterine LF cDNA sequence (18). Templateswere m ixed with 80 pmol of eachPCR primer in a total volume of 100 ul containing 6.7 m M MgC12, 16.6 m M (NH4)2 S04, 5 m M 2-mercaptoethanol, 6.8 P M EDTA, 67 m M TrisHCl pH 8.8, 1.5mM of each deoxyribonucleotidetriphosphateand 2.5 units of Taq DNA polymerase (Pharmacia, LKB, Biotechnology, M ilwaukee, Wisconsin). The reactions were heated at 940C for 3 m in prior to enzyme addition, overlayed with 100 ul m ineral oil and subjectedto 30 cycles of DNA polymerization(940C for 1 m in., denaturation;68OCfor 1 m in., annealing;680C for 2 m in., extension)in an automatedDNA thermocycler. All PCR primers were obtainedfrom National BiosciencesInc., Plymouth, MN. Cloning of mLF genomic DNA by hyridization screening: A genomic library preparedin h Dash II (Stratagene,La Jolla, CA) usin,@aOenomicDNA isolated from the 129/SvEv strain of mousewas screenedwith a PCR derived 1.0 kb mLF genomicprobe which included nucleotides 1902-2040of the mLF cDNA and encoded138 bp of exon 16 and -850 bp of the preceeding intron. Duplicate nitrocellulosetilters were prehybridizedovernight in 6XSSC, 10 m M EDTA, 2% nonfat dry m ilk. Filters were hybridizedfor 24 hours with the [32P] labeledprobe (specific activity - 2x109 cpmlyg, 1 x 107 cpm/mll underthe sameconditions.Filters were washedtwice in 1X SSC at 680C for 20 m in. prior to autoradiography.Four positive genomic clones were identified out of 0.5 x 106 plaquesscreened. A 13.3 kb mLF genomic clone and the PCR products representingthe mLF gene were subclonedinto pGEM-4 (Promega,Biotechnology, Madison, WI) and sequencedby the standarddideoxy chain terminationmethod (19) using the modified T7 DNA polymerase(Sequenase)in the presenceof [35S]dATP and in accordance with the manufacturer’sinstructions(USB, Cleveland,Ohio). 1726
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 189, No. 3, 1992
Results and Discussion It has previously
been shown that the gene for mouse lactoferrin
is located on
chromosome 9 (20). In order to confirm that mouse lactoferrin is encoded by a single copy gene we carried out Southern hybridization analysis of mouse penomic DNA using a [j2PJlabeled 1.0 kb mLF genomic DNA probe as described in the methods section. The results of the Southern analysis are shown in Figure 1. Dipestion of mouse genomic DNA with each restriction enzyme yielded a single hybridizing band as expected for a single copy gene with the exception of Hind111 which yielded two hybridizin g bands due to the presence of a Hind111 restriction site in the DNA probe used for Southern analysis. Taken together, these results confirm that mouse lactot’errin is encoded by a siqle copy gene in the haploid genome. The structural organization of the mouse lactoferrin gene and its alignment with the mLF cDNA are summarized in Figure 2. Usin,o a combination of the PCR (21) amplification of mouse genomic DNA and hybridization screening of mouse genomic DNA libraries we determined that :he mouse lactoferrin gene is approximately 30 kb in ler,gth and is :)rganized into 17 exons and 16 intmns. The exons are numbered in Figure 2 while the introns are marked alphabetically. The exons range in size from 48 bp to 190 bp and the introns vary from 0.2 kb to 4.3 kb.
The structural organization of the 5’ half of the gene was established by PCR
amplification of mouse genomic DNA using oligonucleotide primers generated from the mL.F cDNA sequence (18).
When the PCR primers were subjected to DNA polymerization using
mouse genomic DNA as a template, it was possible to amplify the intervening introns and to Nucleotide sequencing of the exonic
establish the sequence of the intron-exon boundaries.
Fiz. 1. Southern hybridization analysis of mouse coenomic DNA. 10 g of mouse penomic DNA was digested with the indicated enzymes, resolved on a 0.X% agarose gel, transferred to a nylon membrane and hybridized with a mLF genomic probe as described in the Methods section.
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Fip. 2. Structural organization oC the mLF gene and alignment with the mLF cDNA. Capital letters specify introns and numbers specify exons. The size of each exon, in base pairs. is indicated below the boxes.
regions and intron boundariesconfirmed that the genomicPCR productsrepresentedthe mouse lactoferrin gene. When the first three introns were mapped,we observedthat the exon-intron distribution was similar to that of human transferrin and chicken ovotransferrin (16,17). Therefore, to map the remaining introns, we assumedthat this similarity may extend to the rest of the gene and positioned the forward and reverse primers for amplification of mouse lactoferrin eenomic fragments accordingly. The sequenceof the 3’ half of the gene was completed by nucleotide sequenceanalysis of a 13.3 kb genomic DNA clone obtained by hybridization screening of a mouse genomic DNA library. Southern and sequenceanalysis showedthat this clone extendedfrom intron I to exon 16 of the mLF gene. Sequenceanalysis of the exon-intron boundariesestablisheda splicing pattern for the mouselactoferrin geneoutlined in Table 1. All the intron splice sites conform to the published consensussequencesof splice junctions (22). The splice patternshows a duplication of exons in the 5’and 3’halves of the gene that is identical in all the correspondingexon pairs (2/9, 3/10,
Table 1. Sequence of ExonAntron
lntron A B C D E F G Ii I J K
L M N 0 P
Splice Position 46/47 207/208 3X6/317 499/500 647/640 703/704 802/083 1057/105a 1212/1213 1303/1304 1351/1352 1507/15oa 1649/1650 1717/1710 1902/1903 2092/2093
Boundaries of Mouse Lactoferrin
Exon GCC CTT G ATT GTG AAAGAGC GAQWG TTG AC+ GTA TTT Q Q& TCA AATAAAA ATC AT'J AACCAGA GTDGAAE AAA TTT & =A &G ACT GAC G ---CPA CAG AGC TCC C
1728
Sequence Intron GTAAGT. . . . GTAAGC. . , . GTGAGT. . . . GTAAAA. . . . GTAAGC. . . . GTAAGG. . . . GTACAG. . . . GTAAGT. . . . GTAGGG. . . . GTAAGT. . . . GTAAGT. . . . GTAAGC. . . . GTAAGT. . . . GTATGT. . . . GTATGG. . . . GTGAGT. . . .
Gene
Exon .TCCCAG .TTTCAG .CTGCAG .TGACAG .TCACAG .CTCCAG .AAACAG .CCACAG .CTGCAG .CCTCAG .GACAAG .GTCTAG .TTTTAG .TACCAG .ACACAG .CCACAG
GA CTC ACA AAC AG CCC -m Q&i G TOT CTG s GAO GAG AA0 AG CAG AAG @ & a TCC @$ s AT GAG G TOT CTG s AA0 GTT CACj CA CTC
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189, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 2. Structural similarity between the Human Transterrin,Chicken Ovotransferrin and
Mouse Lactoferrin genes;H, human transferrin; Ov, chicken ovotransferrin;M, mouse lactoferrin. The line betweenexons 8 and 9 separatesthe exons of the N-terminal and Cterminal moieties of transferrins. Exon
Exon Size (bp)
Intron
Intron Size (bp)
H
ov
M
I
93
119
82
2
173
164
161
3
I Oh
109
109
4
177
192
183
5
133
136
14x
6
56
56
56
7
179
170
179
8
17x
187
175
9
15.5
155
155
10
94
94
91
11
33
36
48
I2
156
156
156
17
136
145
142
14
65
65
68
15
185
185
185
16
190
187
190
17
206
221
132
H
ov
M
-2100
1313
1600
A
-5000
317
1000
B
-750
986
200
c
-685
190
370
D
-810
396
700
E
-675
489
800
F
-765
124
550
G
-1070
573
800
H
-4900
757
1120
I
-900
215
922
.I
-1300
633
1120
K
-1400
749
1320
L
-2400
269
4300
M
-5300
448
3000
N
-1600
323
1360
0
-2600
418
1700
P
4/12, 5/13, 6/14, 7/15, g/16). This further strengthens the hypothesis that transferrins arose by a gene duplication event and that the primordial gene already contained introns (4,23). Using the above approaches we have mapped the mouse lactoferrin structural gene and show that the size of this region is -23 kb. This genomic region, together with the 7.5 kb ot promoter sequence including the transcriptional start site reported by Liu et al. (14) gives the mouse lactot’errin gene an overall size of -30 kb. The similarity between mouse lactoferrin, human transferrin and chicken ovotransferrin is summarized in Table 2. It is readily apparent that the three genes have a similar exon-intron distribution pattern. The three genes have four exons which are identical in length and the remaining thirteen exons are comparable in size. The 1729
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
varying size of the introns accountsfor the overall difference in size of the three genes. The human transferrin gene is -33.5 kb (16) while the chicken ovotransferringene is 10.5 kb (17). Johnstonet ul. have suggestedthat the humanlactoferrin geneis -35 kb (24) while the genefor mouse transferrin is -36 kb (25) and is similarly organized into 17 exons separatedby 16 introns. Our resultsconfirm that mouselactoferrin is encodedby a single copy genewith a size of approximately 30 kb. Previouspartial analysisof the mouselactofenin gene indicated that the genewas approximately 12 kb (15). The discrepanciesbetweenour analysisand that of Shirsat et ul. occur in the 3’half of the gene which was incompletely characterizedin the previous study. The characterizationof the mouse lactoferrin gene presentedin this study provides an important tool for subsequentgenetic disruption studies to establish the physiological role of mouselactoferrin in viva.
Acknowledgments: We thank Dr. P. Soriano(Baylor College of Medicine, Houston, TX) for kindly providing the mouse genomic DNA library, Deanne Gallup and Aileen Ward for excellent technical assistanceand Lisa Gambleand David Scarff for expert help in preparingthis manuscript. This work was supportedby NIH grant HD-27965(to OMC).
References 1. 2. 3.
4. 5. 6. 7. 8. 9.
10. 11.
Aisen, P. and Listowsky, I. (1980)Annu. Rev. Biochem.49, 357-393. Rose, T.M., Plowman, G.D., Teplow, D.B., Dreyer, W.J., Hellstrom, K.E. and Brown, J.P. (1986) Proc. Natl. Acad. Sci. USA 83,1261-1265. Montreuil, J., Mazurier, J., Legrand,D. and Spik, G. (1985) in, Proteins of iron storage and transport (Spik, G., Montreuil, J., Crichton, R.R. and Mazurier, J., Eds) p 25-38. Elsevier, Amsterdam. Williams, J. (1982) TrendsBiochem.Sci. 7, 394-397. Metz-Boutique, M .H., Jolles, J., Mazurier, J., Schoentgen,F., Legrand, D., Spik, G., Montreuil, J. and Jolles, P. (1984)Eur. J. Biochem. 145,659-676. Masson,P.L. and Heremans,J.F. (1971) Comp. Biochem.and Physiol. 39, 119-129. Masson,P.L., Heremans,J.F. and Dive, C. (1966)Clin. Chim. Acta. 14, 735-739. Masson,P.L., Heremans,J.F. and Schonne,E. (1969)J. Exp. Med. 130,643-658. Arnold, R.R., Cole, M .F. and McGhee,J.R. (1977)Science197,263-265. Nemet, K. and Simonovits,I. (19851Haematol.18, 3-12. Hashizume,S., Kuroda, K. and Murakami, M . (1983) Biochim. Biophys. Acta 763, 377382.
12. 13. 14. 15. 16. 17.
Broxemeyer, H.E., DeSousa,M ., Smithyman,A., Ralph, P., Hamilton, J., Kurland, J. and Bognacki, J. (1980) Blood 55, 324-333. Oseas,R., Yang, H.H., Baehner,R.L. and Boxer, L.A. (1981) Blood 57,939-945. Liu, Y. and Teng, CT. (19911J. Biol. Chem. 266,21880-21885. Shirsat,N.V., Bittenbender,S., Kreider, B.L. and Rovera,G. (1992) Gene 110,229-234. Schaeffer,E., Lucero, M .A., Jeltsch,J.-M., Py, M .C., Levin, M .J., Chambon,P., Cohen, G.N. and Zakin, M .M. (1987)Gene56, 109-l 16. Jeltsch, J.-M., Hen, R., Maroteaux,L., Garnier, J.M. and Chambon,P. (1987) Nucleic Acids Res. 15,7643-7645. 1730
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18. 19. 20. 21. 22. 23. 24. 25.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pentecost,B.T. and Teng, C.T. (1987)J. Biol. Chem. 262, 10134-10139. Sanger,F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 546% 5467. Teng, C.T., Pentecost,B.T., Marshall, A., Solomon,A., Bowman, B.H., Lalley, P.A. and Naylor, S.L. (1987) Somat.Cell Mol. Genet. 13,689-693. Mullis, K.B. and Faloona,F.A. (1987) MethodsEnzymol. 155, 335350. Mount, S.M. (1982) Nucleic Acids Res. 10,459-472. Park, I., Schaeffer,E., Sidoli, A., Baralle, F.E., Cohen, G.N. and Zakin, M .M. ( 1985) Proc. Natl. Acad. Sci. USA 82, 3149-3153. Johnston,J.J., Rintels, P., Chung, J., Sather,J., Benz, Jr., E.J. and Berliner, N. (1992) Blood 79, 2998-3006. Idzerda, R.L., Behringer, R.R., The&en, M ., Huggenvik, J.I., McKnight, G.S. and Brinster, R.L. (1989) Mol. Cell. Biol. 9, 5 154-5162.
1731
Voi. 189, N6. 3, 1992 December 30, 1992
STRUCTURAL
ORGANIZATION
OF THE MOUSE LACTOFERRIN
GENE
GrainneA. Cunningham,Denis R. Headon*andOrla M . Conneelyl Departmentof Cell Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston,TX 77030 *Cell and Molecular Biology Group, Department of Biochemistry, University College, Galway, Ireland Received
November
17,
1992
The complete structure of the mouse lactoferrin gene is presented. Mouse lactoferrin (mLF) is encodedby a single copy geneof approximately30 kilobases(kb) in size. The ,oeneis organizedinto 17 exons separatedby 16 introns. The exons range in size from 48 base pairs (bp) to’190 bp whereasthe introns ransefrom 0.2 kb to 4.3 kb. Structuralanalysisof the moust: lactofemingene revealsthat this genesharesa similar intron-exondistribution pattern with both human transferrinand chicken ovotransfetrin. 0 1992Academic PES-, lnc.
Lactoferrin is a memberof the family of non-haem,iron-binding proteins known as the transferrins(1). This family includesserumtransferrin,the iron transportprotein in the blood of vertebratesand some invertebrates,ovotransferrinfrom egg-whiteand the membrane-associated melanotransferrinfrom melanocytes(2). The transferrinsare all monomericglycoproteinswith a molecularmassof approximately80 kDa, composedof two lobes,eachpossessingthe capacity to bind reversibly one ferric iron with the synergisticbinding of a bicarbonateor carbonic anion (1,3). It is thought that the transferrinsoriginate from a common primordial gene by a gene duplication event (4). This proposal is supportedby a two-fold internal repeat found in the primary structure of all the transferrinsstudied to date. For each of theseproteins, lactoferrin, serum transferrin, ovotransferrin and melanotransferrin,the N-terminal half was found to he approximately40% identical with the C-terminal half of the proteins(5). Lactoferrin is found in m ilk (6) and a variety of other exocrinesecretions(7) and also in the secondarygranules of neutrophils (8). The biological functions proposedfor lactoferrin include protection againstm icrobial infection (9). enhancedintestinal iron absorptionin infants (l(I), promotion of cell growth (II), regulation of myelopoiesis (12) and modulation of inflammatory responses(13). ’ To whom correspondence should be addressed. Abbreviations: kilobases= kb , basepairs = bp , mouselactoferrin = mLF.
1725
0006-291x/9? $4.00 Copyright 0 1992 by Academic Pres.v. Inc. All rights of reproduction in arty form rewnvd.
Vol.
189, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Partial structural organizationof the mouse lactofetrin gene was recently reported by both Liu et al. (14) and Shirsatet al. (15). In this paper,we extendtheseanalysesby presenting the complete structural organizationof the mouselactoferrin gene. We demonstratethat mouse lactoferrin is encoded by a single copy gene of approximately 30 kb in size that shares a structural relatedness,at the gene level, to both human transferrin and chicken ovotransferrin (16,17). The transcriptional unit is encoded by approximately 23 kb and is llanked by a previously characterizedpromoterregion of 7.5 kb (14).
Materials and Methods Genomic Southern Analysis: 10 ug of mouse genomic DNA was digested with the restriction enzymesindicatedin Figure 1, resolvedon a 0.8% agarosegel and then transferredto a nylon filter. Prehybridizationand hybridization of the nylon filter were performed in O S M Na2HP04, pH 7, 1 m M EDTA and 7%, SDS at 680C for 16 hours. The hybridization solution contained 1~10~cpm/ml (specific activity - 2x109 cpm/ug) of a [32P] labeled mLF genomic probe which containednucleotides997-1212 of the mLF cDNA (18), encoding part of exon 8 and all of exon 9, and the interveningintron (-800 bp). The filter was washedtwice in 0.04M Na2HP04/1%SDS at 680C for 30 m in. The filter was dried, exposedat -700C and developed by autoradiography. Amplification of mLF genomicDNA fragmentsusing the polymerasechain reaction(PCR): Amplification of individual regionsof the mouse lactoferrin gene was performed using 1 pg of mousegenomicDNA as templateand a variety of 30-meroligonucleotideprimers whose sequence was determined from the published mouse uterine LF cDNA sequence (18). Templateswere m ixed with 80 pmol of eachPCR primer in a total volume of 100 ul containing 6.7 m M MgC12, 16.6 m M (NH4)2 S04, 5 m M 2-mercaptoethanol, 6.8 P M EDTA, 67 m M TrisHCl pH 8.8, 1.5mM of each deoxyribonucleotidetriphosphateand 2.5 units of Taq DNA polymerase (Pharmacia, LKB, Biotechnology, M ilwaukee, Wisconsin). The reactions were heated at 940C for 3 m in prior to enzyme addition, overlayed with 100 ul m ineral oil and subjectedto 30 cycles of DNA polymerization(940C for 1 m in., denaturation;68OCfor 1 m in., annealing;680C for 2 m in., extension)in an automatedDNA thermocycler. All PCR primers were obtainedfrom National BiosciencesInc., Plymouth, MN. Cloning of mLF genomic DNA by hyridization screening: A genomic library preparedin h Dash II (Stratagene,La Jolla, CA) usin,@aOenomicDNA isolated from the 129/SvEv strain of mousewas screenedwith a PCR derived 1.0 kb mLF genomicprobe which included nucleotides 1902-2040of the mLF cDNA and encoded138 bp of exon 16 and -850 bp of the preceeding intron. Duplicate nitrocellulosetilters were prehybridizedovernight in 6XSSC, 10 m M EDTA, 2% nonfat dry m ilk. Filters were hybridizedfor 24 hours with the [32P] labeledprobe (specific activity - 2x109 cpmlyg, 1 x 107 cpm/mll underthe sameconditions.Filters were washedtwice in 1X SSC at 680C for 20 m in. prior to autoradiography.Four positive genomic clones were identified out of 0.5 x 106 plaquesscreened. A 13.3 kb mLF genomic clone and the PCR products representingthe mLF gene were subclonedinto pGEM-4 (Promega,Biotechnology, Madison, WI) and sequencedby the standarddideoxy chain terminationmethod (19) using the modified T7 DNA polymerase(Sequenase)in the presenceof [35S]dATP and in accordance with the manufacturer’sinstructions(USB, Cleveland,Ohio). 1726
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 189, No. 3, 1992
Results and Discussion It has previously
been shown that the gene for mouse lactoferrin
is located on
chromosome 9 (20). In order to confirm that mouse lactoferrin is encoded by a single copy gene we carried out Southern hybridization analysis of mouse penomic DNA using a [j2PJlabeled 1.0 kb mLF genomic DNA probe as described in the methods section. The results of the Southern analysis are shown in Figure 1. Dipestion of mouse genomic DNA with each restriction enzyme yielded a single hybridizing band as expected for a single copy gene with the exception of Hind111 which yielded two hybridizin g bands due to the presence of a Hind111 restriction site in the DNA probe used for Southern analysis. Taken together, these results confirm that mouse lactot’errin is encoded by a siqle copy gene in the haploid genome. The structural organization of the mouse lactoferrin gene and its alignment with the mLF cDNA are summarized in Figure 2. Usin,o a combination of the PCR (21) amplification of mouse genomic DNA and hybridization screening of mouse genomic DNA libraries we determined that :he mouse lactoferrin gene is approximately 30 kb in ler,gth and is :)rganized into 17 exons and 16 intmns. The exons are numbered in Figure 2 while the introns are marked alphabetically. The exons range in size from 48 bp to 190 bp and the introns vary from 0.2 kb to 4.3 kb.
The structural organization of the 5’ half of the gene was established by PCR
amplification of mouse genomic DNA using oligonucleotide primers generated from the mL.F cDNA sequence (18).
When the PCR primers were subjected to DNA polymerization using
mouse genomic DNA as a template, it was possible to amplify the intervening introns and to Nucleotide sequencing of the exonic
establish the sequence of the intron-exon boundaries.
Fiz. 1. Southern hybridization analysis of mouse coenomic DNA. 10 g of mouse penomic DNA was digested with the indicated enzymes, resolved on a 0.X% agarose gel, transferred to a nylon membrane and hybridized with a mLF genomic probe as described in the Methods section.
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Fip. 2. Structural organization oC the mLF gene and alignment with the mLF cDNA. Capital letters specify introns and numbers specify exons. The size of each exon, in base pairs. is indicated below the boxes.
regions and intron boundariesconfirmed that the genomicPCR productsrepresentedthe mouse lactoferrin gene. When the first three introns were mapped,we observedthat the exon-intron distribution was similar to that of human transferrin and chicken ovotransferrin (16,17). Therefore, to map the remaining introns, we assumedthat this similarity may extend to the rest of the gene and positioned the forward and reverse primers for amplification of mouse lactoferrin eenomic fragments accordingly. The sequenceof the 3’ half of the gene was completed by nucleotide sequenceanalysis of a 13.3 kb genomic DNA clone obtained by hybridization screening of a mouse genomic DNA library. Southern and sequenceanalysis showedthat this clone extendedfrom intron I to exon 16 of the mLF gene. Sequenceanalysis of the exon-intron boundariesestablisheda splicing pattern for the mouselactoferrin geneoutlined in Table 1. All the intron splice sites conform to the published consensussequencesof splice junctions (22). The splice patternshows a duplication of exons in the 5’and 3’halves of the gene that is identical in all the correspondingexon pairs (2/9, 3/10,
Table 1. Sequence of ExonAntron
lntron A B C D E F G Ii I J K
L M N 0 P
Splice Position 46/47 207/208 3X6/317 499/500 647/640 703/704 802/083 1057/105a 1212/1213 1303/1304 1351/1352 1507/15oa 1649/1650 1717/1710 1902/1903 2092/2093
Boundaries of Mouse Lactoferrin
Exon GCC CTT G ATT GTG AAAGAGC GAQWG TTG AC+ GTA TTT Q Q& TCA AATAAAA ATC AT'J AACCAGA GTDGAAE AAA TTT & =A &G ACT GAC G ---CPA CAG AGC TCC C
1728
Sequence Intron GTAAGT. . . . GTAAGC. . , . GTGAGT. . . . GTAAAA. . . . GTAAGC. . . . GTAAGG. . . . GTACAG. . . . GTAAGT. . . . GTAGGG. . . . GTAAGT. . . . GTAAGT. . . . GTAAGC. . . . GTAAGT. . . . GTATGT. . . . GTATGG. . . . GTGAGT. . . .
Gene
Exon .TCCCAG .TTTCAG .CTGCAG .TGACAG .TCACAG .CTCCAG .AAACAG .CCACAG .CTGCAG .CCTCAG .GACAAG .GTCTAG .TTTTAG .TACCAG .ACACAG .CCACAG
GA CTC ACA AAC AG CCC -m Q&i G TOT CTG s GAO GAG AA0 AG CAG AAG @ & a TCC @$ s AT GAG G TOT CTG s AA0 GTT CACj CA CTC
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Table 2. Structural similarity between the Human Transterrin,Chicken Ovotransferrin and
Mouse Lactoferrin genes;H, human transferrin; Ov, chicken ovotransferrin;M, mouse lactoferrin. The line betweenexons 8 and 9 separatesthe exons of the N-terminal and Cterminal moieties of transferrins. Exon
Exon Size (bp)
Intron
Intron Size (bp)
H
ov
M
I
93
119
82
2
173
164
161
3
I Oh
109
109
4
177
192
183
5
133
136
14x
6
56
56
56
7
179
170
179
8
17x
187
175
9
15.5
155
155
10
94
94
91
11
33
36
48
I2
156
156
156
17
136
145
142
14
65
65
68
15
185
185
185
16
190
187
190
17
206
221
132
H
ov
M
-2100
1313
1600
A
-5000
317
1000
B
-750
986
200
c
-685
190
370
D
-810
396
700
E
-675
489
800
F
-765
124
550
G
-1070
573
800
H
-4900
757
1120
I
-900
215
922
.I
-1300
633
1120
K
-1400
749
1320
L
-2400
269
4300
M
-5300
448
3000
N
-1600
323
1360
0
-2600
418
1700
P
4/12, 5/13, 6/14, 7/15, g/16). This further strengthens the hypothesis that transferrins arose by a gene duplication event and that the primordial gene already contained introns (4,23). Using the above approaches we have mapped the mouse lactoferrin structural gene and show that the size of this region is -23 kb. This genomic region, together with the 7.5 kb ot promoter sequence including the transcriptional start site reported by Liu et al. (14) gives the mouse lactot’errin gene an overall size of -30 kb. The similarity between mouse lactoferrin, human transferrin and chicken ovotransferrin is summarized in Table 2. It is readily apparent that the three genes have a similar exon-intron distribution pattern. The three genes have four exons which are identical in length and the remaining thirteen exons are comparable in size. The 1729
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varying size of the introns accountsfor the overall difference in size of the three genes. The human transferrin gene is -33.5 kb (16) while the chicken ovotransferringene is 10.5 kb (17). Johnstonet ul. have suggestedthat the humanlactoferrin geneis -35 kb (24) while the genefor mouse transferrin is -36 kb (25) and is similarly organized into 17 exons separatedby 16 introns. Our resultsconfirm that mouselactoferrin is encodedby a single copy genewith a size of approximately 30 kb. Previouspartial analysisof the mouselactofenin gene indicated that the genewas approximately 12 kb (15). The discrepanciesbetweenour analysisand that of Shirsat et ul. occur in the 3’half of the gene which was incompletely characterizedin the previous study. The characterizationof the mouse lactoferrin gene presentedin this study provides an important tool for subsequentgenetic disruption studies to establish the physiological role of mouselactoferrin in viva.
Acknowledgments: We thank Dr. P. Soriano(Baylor College of Medicine, Houston, TX) for kindly providing the mouse genomic DNA library, Deanne Gallup and Aileen Ward for excellent technical assistanceand Lisa Gambleand David Scarff for expert help in preparingthis manuscript. This work was supportedby NIH grant HD-27965(to OMC).
References 1. 2. 3.
4. 5. 6. 7. 8. 9.
10. 11.
Aisen, P. and Listowsky, I. (1980)Annu. Rev. Biochem.49, 357-393. Rose, T.M., Plowman, G.D., Teplow, D.B., Dreyer, W.J., Hellstrom, K.E. and Brown, J.P. (1986) Proc. Natl. Acad. Sci. USA 83,1261-1265. Montreuil, J., Mazurier, J., Legrand,D. and Spik, G. (1985) in, Proteins of iron storage and transport (Spik, G., Montreuil, J., Crichton, R.R. and Mazurier, J., Eds) p 25-38. Elsevier, Amsterdam. Williams, J. (1982) TrendsBiochem.Sci. 7, 394-397. Metz-Boutique, M .H., Jolles, J., Mazurier, J., Schoentgen,F., Legrand, D., Spik, G., Montreuil, J. and Jolles, P. (1984)Eur. J. Biochem. 145,659-676. Masson,P.L. and Heremans,J.F. (1971) Comp. Biochem.and Physiol. 39, 119-129. Masson,P.L., Heremans,J.F. and Dive, C. (1966)Clin. Chim. Acta. 14, 735-739. Masson,P.L., Heremans,J.F. and Schonne,E. (1969)J. Exp. Med. 130,643-658. Arnold, R.R., Cole, M .F. and McGhee,J.R. (1977)Science197,263-265. Nemet, K. and Simonovits,I. (19851Haematol.18, 3-12. Hashizume,S., Kuroda, K. and Murakami, M . (1983) Biochim. Biophys. Acta 763, 377382.
12. 13. 14. 15. 16. 17.
Broxemeyer, H.E., DeSousa,M ., Smithyman,A., Ralph, P., Hamilton, J., Kurland, J. and Bognacki, J. (1980) Blood 55, 324-333. Oseas,R., Yang, H.H., Baehner,R.L. and Boxer, L.A. (1981) Blood 57,939-945. Liu, Y. and Teng, CT. (19911J. Biol. Chem. 266,21880-21885. Shirsat,N.V., Bittenbender,S., Kreider, B.L. and Rovera,G. (1992) Gene 110,229-234. Schaeffer,E., Lucero, M .A., Jeltsch,J.-M., Py, M .C., Levin, M .J., Chambon,P., Cohen, G.N. and Zakin, M .M. (1987)Gene56, 109-l 16. Jeltsch, J.-M., Hen, R., Maroteaux,L., Garnier, J.M. and Chambon,P. (1987) Nucleic Acids Res. 15,7643-7645. 1730
Vol. 189, No. 3, 1992
18. 19. 20. 21. 22. 23. 24. 25.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pentecost,B.T. and Teng, C.T. (1987)J. Biol. Chem. 262, 10134-10139. Sanger,F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 546% 5467. Teng, C.T., Pentecost,B.T., Marshall, A., Solomon,A., Bowman, B.H., Lalley, P.A. and Naylor, S.L. (1987) Somat.Cell Mol. Genet. 13,689-693. Mullis, K.B. and Faloona,F.A. (1987) MethodsEnzymol. 155, 335350. Mount, S.M. (1982) Nucleic Acids Res. 10,459-472. Park, I., Schaeffer,E., Sidoli, A., Baralle, F.E., Cohen, G.N. and Zakin, M .M. ( 1985) Proc. Natl. Acad. Sci. USA 82, 3149-3153. Johnston,J.J., Rintels, P., Chung, J., Sather,J., Benz, Jr., E.J. and Berliner, N. (1992) Blood 79, 2998-3006. Idzerda, R.L., Behringer, R.R., The&en, M ., Huggenvik, J.I., McKnight, G.S. and Brinster, R.L. (1989) Mol. Cell. Biol. 9, 5 154-5162.
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