DNA Sequence-]. DNA Sequencing and Mapping, Vol. 3, pp. 245-246 Reprints available directly from the publisher Photocopying permitted by license only
0 1992 Harwood Academic Publishers GmbH Printed in Great Britain
SHORT COMMU N1CAT10N
Cloning and sequencing of human K=caseincDNA
Mitochondrial DNA Downloaded from informahealthcare.com by McMaster University on 12/16/14 For personal use only.
SVEN BERGSTROM, LENNART HANSSON*, OLLE HERNELLt, BO LONNERDAL+, ANNA KARIN NILSSON* and MATS STROMQVIST* Department of Microbiology, University of Umea, S-90187 Umed, Sweden, *Symbicom AB, P.O. Box 1457, S-90124 Umea, Sweden, tDepartment of Pediatrics, University of Umea, S-90787 Umea, Sweden, +Department of Nutrition, University of California, Davis, CA 956 7 6, USA
EMBL X66417
A cDNA encoding K-casein of human milk was cloned and sequenced. The K-casein cDNA was isolated from a Agtll library generated from mRNA prepared from a mammary gland biopsy obtained from a lactating woman. The library was screened with polyclonal rabbit antibodies raised against purified native rc-casein. The obtained nucleotide sequence contained an ORF sufficient to encode the entire amino acid sequence of a K-casein precursor protein consisting of 182 amino acids. This includes a tentative signal peptide of 20 amino acids and a processed protein of 162 amino acids.
3c:o
340
320
ATGCCCAAA'PPXTCAGCGGCAATACCTGCCAAI41AGC ti A Q I P Q n Q 'i L P N s>
380
Mitochondrial DNA Downloaded from informahealthcare.com by McMaster University on 12/16/14 For personal use only.
' f !I
400
420
I'ACCTCGCCCAAACCI GCATCCATCATTTATTGCCATCCCCCCAAAGAWATTC it ! i P N I I1 P S F I A I P P K K Ii 4 '1 3
460
480
TACI:ATTAATACCATTGCTAC?G~~AACCTACACCAGCT~CTGCCA~Ci;,~ r' 1 N T I A ? V E F T I' A P A T F', :c.
J
520
540
560
1 ' A i ; ' r 1 ' A C T C C A G A A G C T ~ r T T C A T C A T ~ , A C A 'J V 1 P E A t ' S F S I I 'T S T P E 'T>
620
600
p U C l 8 . The complete nucleotide sequence o i both strands of the cDNA region was determined using a T7 sequencing kit (Pharmacia), on double stranded tern p I at cis. Spec if ic o I igon uc Ieot i des corm I:,Ie in en tary to pUC18 or Ic-casein seyuenc-es were used. The 857 bp nucleotide sequence contain!; an ORF sufficient to encode the entire amino acid sequence of Ic-casein precursor protein consisting of 182 amino acids. This includes a tentatirve signal peptide of 20 amino acids and a processed protein of 162 amino acids. The deduced amino acid sequence is different at eight positions compared to the published sequence determined hy amino acid analysis (Brignon et a/., 198.5). Based on the proposed cleavage site we suggest that the peptide chain contain four additional amino acid1 residues than previously published (Brignon et 3/., 1985). I t so, the amino acid sequence of the N-termiiial i s identical to that of rat and very similar to those reported for other species, with the following amino acid sequence; E-V-Q-N.
CP,CC?ACGGC~?IRk4AnCACCAAG(~TATCAAAf;~CACMC~CA~GAC
660
100
680
ACKNOWLEDGEMENTS
1LAGCCATTTGTCTGCCTTCAGTCAnCAACAGWWr
140
760
TCTCCTTACATTTTACATTCATGCCACATTCAATATTTTGATTCTTG 820
840
This work is supported by grant4 from Svmbicom AB, and the Swedish Medical Research Council (1 9X-05708; 16X-079221.
REFERENCES
not contain the entire Ic-casein c D N A . This 440 bp fr,igment w'15 used '1s a nucleotide probe in an a r I d I t II ) n' 1 I p I a (1ue hy b r id i z a t i on 5c r ee n I ng of the ni,imrn,\rv gland ( D N A library One positive 857 bp EcoRl insert was isopl,iqucJ cont,iining Ic~ted,puritied and digested with EcoRI. The r c ~ u l t i t i gfragment b a s cloned into EcoRl cleaved
Brignon, G., Chtourou, A. ancl Kihadc,iu-Dunias, H . 1 1 Y 8 5 ) Preparation and aniino acid scqucticc oi humriti li-, iirdlc=, ,Ind ( < i \ i > i n subunits in human milk. In Atkinson S.A. ,in(! I c.)nncd,il B (eds.), Protein and Non-proit,in Nilrogrn . in \/tirn,iri Milk (CRC Press, Boca Raton, FI., USA), pp. 10-27. Kunz, C. and Lhnnerdal B. (1'3901. Cawin litid c a w i n siihuniti in preterm milk, colostrum arid maluro human milk. / P c d a i r . Castroenterol. Nutr. 10, 454-36 I Lonnerdal, B. (1 985). Biocheniistry a t i d ph\/\ioIogtc
0 1992 Harwood Academic Publishers GmbH Printed in Great Britain
SHORT COMMU N1CAT10N
Cloning and sequencing of human K=caseincDNA
Mitochondrial DNA Downloaded from informahealthcare.com by McMaster University on 12/16/14 For personal use only.
SVEN BERGSTROM, LENNART HANSSON*, OLLE HERNELLt, BO LONNERDAL+, ANNA KARIN NILSSON* and MATS STROMQVIST* Department of Microbiology, University of Umea, S-90187 Umed, Sweden, *Symbicom AB, P.O. Box 1457, S-90124 Umea, Sweden, tDepartment of Pediatrics, University of Umea, S-90787 Umea, Sweden, +Department of Nutrition, University of California, Davis, CA 956 7 6, USA
EMBL X66417
A cDNA encoding K-casein of human milk was cloned and sequenced. The K-casein cDNA was isolated from a Agtll library generated from mRNA prepared from a mammary gland biopsy obtained from a lactating woman. The library was screened with polyclonal rabbit antibodies raised against purified native rc-casein. The obtained nucleotide sequence contained an ORF sufficient to encode the entire amino acid sequence of a K-casein precursor protein consisting of 182 amino acids. This includes a tentative signal peptide of 20 amino acids and a processed protein of 162 amino acids.
3c:o
340
320
ATGCCCAAA'PPXTCAGCGGCAATACCTGCCAAI41AGC ti A Q I P Q n Q 'i L P N s>
380
Mitochondrial DNA Downloaded from informahealthcare.com by McMaster University on 12/16/14 For personal use only.
' f !I
400
420
I'ACCTCGCCCAAACCI GCATCCATCATTTATTGCCATCCCCCCAAAGAWATTC it ! i P N I I1 P S F I A I P P K K Ii 4 '1 3
460
480
TACI:ATTAATACCATTGCTAC?G~~AACCTACACCAGCT~CTGCCA~Ci;,~ r' 1 N T I A ? V E F T I' A P A T F', :c.
J
520
540
560
1 ' A i ; ' r 1 ' A C T C C A G A A G C T ~ r T T C A T C A T ~ , A C A 'J V 1 P E A t ' S F S I I 'T S T P E 'T>
620
600
p U C l 8 . The complete nucleotide sequence o i both strands of the cDNA region was determined using a T7 sequencing kit (Pharmacia), on double stranded tern p I at cis. Spec if ic o I igon uc Ieot i des corm I:,Ie in en tary to pUC18 or Ic-casein seyuenc-es were used. The 857 bp nucleotide sequence contain!; an ORF sufficient to encode the entire amino acid sequence of Ic-casein precursor protein consisting of 182 amino acids. This includes a tentatirve signal peptide of 20 amino acids and a processed protein of 162 amino acids. The deduced amino acid sequence is different at eight positions compared to the published sequence determined hy amino acid analysis (Brignon et a/., 198.5). Based on the proposed cleavage site we suggest that the peptide chain contain four additional amino acid1 residues than previously published (Brignon et 3/., 1985). I t so, the amino acid sequence of the N-termiiial i s identical to that of rat and very similar to those reported for other species, with the following amino acid sequence; E-V-Q-N.
CP,CC?ACGGC~?IRk4AnCACCAAG(~TATCAAAf;~CACMC~CA~GAC
660
100
680
ACKNOWLEDGEMENTS
1LAGCCATTTGTCTGCCTTCAGTCAnCAACAGWWr
140
760
TCTCCTTACATTTTACATTCATGCCACATTCAATATTTTGATTCTTG 820
840
This work is supported by grant4 from Svmbicom AB, and the Swedish Medical Research Council (1 9X-05708; 16X-079221.
REFERENCES
not contain the entire Ic-casein c D N A . This 440 bp fr,igment w'15 used '1s a nucleotide probe in an a r I d I t II ) n' 1 I p I a (1ue hy b r id i z a t i on 5c r ee n I ng of the ni,imrn,\rv gland ( D N A library One positive 857 bp EcoRl insert was isopl,iqucJ cont,iining Ic~ted,puritied and digested with EcoRI. The r c ~ u l t i t i gfragment b a s cloned into EcoRl cleaved
Brignon, G., Chtourou, A. ancl Kihadc,iu-Dunias, H . 1 1 Y 8 5 ) Preparation and aniino acid scqucticc oi humriti li-, iirdlc=, ,Ind ( < i \ i > i n subunits in human milk. In Atkinson S.A. ,in(! I c.)nncd,il B (eds.), Protein and Non-proit,in Nilrogrn . in \/tirn,iri Milk (CRC Press, Boca Raton, FI., USA), pp. 10-27. Kunz, C. and Lhnnerdal B. (1'3901. Cawin litid c a w i n siihuniti in preterm milk, colostrum arid maluro human milk. / P c d a i r . Castroenterol. Nutr. 10, 454-36 I Lonnerdal, B. (1 985). Biocheniistry a t i d ph\/\ioIogtc
