High-level, stage- and mammary-tissue-specific expression of a caprine kappa-casein-encoding minigene driven by a beta-casein promoter in transgenic mice.
A 5' truncated caprine (ca) kappa-casein-encoding gene (kappa Cas) was fused to the 3' end of a 3' truncated ca beta Cas. The kappa Cas form comprised the 0.8-kb 3' end of intron 2, the remaining part of the transcription unit containing codons -2 to stop 172, and 0.43 kb of the 3' flanking region. The beta Cas form comprised a 3-kb 5' flanking region and the 5' end of the transcription unit terminating 69 bp downstream from exon 2 which encodes the 15-amino-acid (aa) signal peptide and the first 2 aa of mature beta Cas. The resulting hybrid gene driven by the beta Cas promoter was expressed in all eight lines of transgenic mice investigated, although at different levels. In two lines, the yield of recombinant (re-) kappa Cas was > or = 3 mg/ml of milk. The stage- and mammary tissue-specific expression was similar to that of endogenous beta Cas. The re-kappa Cas differed from its goat milk counterpart by the occurrence of four extra aa at the N-terminal end, indicating that the signal peptidase released the beta Cas signal peptide. According to sedimentation analyses of murine milk containing > or = 3 mg re-kappa Cas/ml, the latter essentially occurred in micelles. Preliminary comparative assays of the behavior of ca alpha s1Cas-kappa Cas and alpha s1Cas-re-kappa Cas mixtures upon incremental addition of Ca2+ showed that re-kappa Cas had the capacity to protect alpha s1Cas against Ca(2+)-induced precipitation in forming stable micelles.(ABSTRACT TRUNCATED AT 250 WORDS)
Persuy
M A
MA
Laboratoire de Génétique Biochimique et de Cytogénétique, INRA-CRJ, Jouy-en-Josas, France.
Legrain
S
S
Printz
C
C
Stinnakre
M G
MG
Lepourry
L
L
Brignon
G
G
Mercier
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000502:physiology; D002364:Caseins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D005260:Female; D018507:Gene Expression Regulation, Developmental; D006041:Goats; D007774:Lactation; D008321:Mammary Glands, Animal / Q000737:chemistry*; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008822:Mice, Transgenic; D008823:Micelles; D008892:Milk / Q000737:chemistry; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D021382:Protein Sorting Signals / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000096:biosynthesis; D017421:Sequence Analysis; D019076:Transgenes / Q000235:genetics*
D002364:Caseins; D008823:Micelles; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D002118:Calcium