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Molecular characterization and in-vitro hormonal requirements for expression of two casein genes from a marsupial

C. Collet, R. Joseph, and K. Nicholas

ABSTRACT

Two marsupial casein genes have been isolated from a tammar wallaby (Macropus eugenii) mammary gland cDNA library. Comparisons of the tammar α- and β-casein genes with their eutherian homologues reveal extensive divergence at the levels of nucleotide and amino acid sequences. Regions of similarity between the tammar and eutherian Ca2+-sensitive caseins are restricted to the major phosphorylation sites and the signal peptides. Quantification of casein mRNA levels in hormone-stimulated mammary gland explants from tammars in late pregnancy suggests that maximal induction of the β-casein gene is dependent upon prolactin and insulin, while maximal induction of the α-casein gene is dependent upon prolactin, insulin and cortisol. These results are in contrast to earlier studies which show that the maximal induction of a putative 19 kDa casein, α-lactalbumin and β-lactoglobulin in the tammar mammary gland is dependent upon prolactin alone. The expression of the latter three genes is not modulated by other hormones known to play a role in the in-vitro initiation of lactation in eutherians.

DOI:
https://doi.org/10.1677/jme.0.0080013
Volume 8: Issue 1,
Pages:
13–20 (8 total)
Restricted access

A marsupial β-lactoglobulin gene: characterization and prolactin-dependent expression

C. Collet, R. Joseph, and K. Nicholas

ABSTRACT

Analysis of the tammar wallaby β-lactoglobulin cDNA and inferred amino acid sequences reveal extensive sequence divergence from the eutherian β-lactoglobulins. Conserved residues include the cysteines and a number of individual amino acids involved in structure and ligand-binding. The only region of extended similarity is a heptapeptide sequence which may impart specificity to its interaction with a receptor protein. Northern analysis of total mammary RNA revealed two transcripts which result from differential utilization of polyadenylation signals. The concentration of β-lactoglobulin mRNA increased in late lactation and correlates with increases in milk production and levels of milk fat. Quantification of β-lactoglobulin mRNA levels in hormone-stimulated mammary gland explants from tammars in late pregnancy suggests that maximal induction of the gene is dependent on prolactin alone and that expression is not modulated by other hormones known to play a role in the initiation of lactation in eutherians.

DOI:
https://doi.org/10.1677/jme.0.0060009
Volume 6: Issue 1,
Pages:
9–16 (8 total)
Free access

Molecular characterisation and hormone-dependent expression of the porcine whey acidic protein gene

KJ Simpson, P Bird, D Shaw, and K Nicholas

A 17.5 kDa protein was isolated from porcine whey by reverse phase HPLC and identified as a putative whey acidic protein (WAP) homologue by sequencing 35 and 40 amino acid residues of the amino- and carboxy-terminus respectively. Degenerate oligonucleotides to both of these amino acid sequences were designed and used in reverse transcriptase PCR with RNA from lactating porcine mammary gland as a template. A 162 bp PCR fragment was detected and sequenced. Compilation of the deduced and determined amino acid sequence revealed a protein of 111 amino acids, which had approximately 75, 50, 40 and 35% similarity at amino acid level to camel, rabbit, rat and mouse WAP respectively. It also included the four-disulphide core characteristic of all WAP proteins and most Kunitz-type protease inhibitors. This provides the first unequivocal evidence for WAP secretion in the pig. SDS PAGE analysis of the whey fraction showed that WAP is secreted as a major protein in sow's milk from farrowing to weaning. The molecular mass of WAP in SDS PAGE was significantly greater than the 11.7 kDa determined from amino acid sequence, indicating that porcine WAP is possibly glycosylated. Northern analysis detected a single mRNA transcript of approximately 600 bp in porcine RNA from the mammary gland of lactating sows. To examine the hormone-regulated expression of the WAP gene the mammary glands of sows at day 90 of pregnancy were biopsied and explants cultured for 3 days in the presence of various combinations of porcine insulin (I), cortisol (F) and porcine prolactin (P). Northern analysis of RNA extracted from the tissue indicated that WAP gene expression was barely detectable in the mammary gland prior to culture and there was no increment in explants cultured in the presence of I and F. However, a significant increase in the accumulation of WAP mRNA was observed in explants cultured in I, F and P. A similar result was observed for beta-casein and alpha-lactalbumin gene expression.

DOI:
https://doi.org/10.1677/jme.0.0200027
Volume 20: Issue 1,
Pages:
27–35 (9 total)
Restricted access

Progressive changes in milk protein gene expression and prolactin binding during lactation in the tammar wallaby (Macropus eugenii)

P H Bird, K A K Hendry, D C Shaw, C J Wilde, and K R Nicholas

ABSTRACT

Changes in milk protein gene expression and specific prolactin binding were quantified in mammary tissue from the tammar wallaby (Macropus eugenii) at different stages of lactation. The transition from early (phase 2) lactation to late (phase 3) lactation was characterized by the induction of the gene for late lactation protein, a novel whey protein. During the same period, the levels of β-lactoglobulin and β-casein gene expression increased, whereas there was no change in the levels of expression of α-lactalbumin and α-casein genes. Prolactin binding in the mammary gland doubled during the latter half of phase 2 of lactation but declined significantly during the transition to phase 3 of lactation. These changes in prolactin binding resulted from changes in the number of receptors and not from a change in the affinity of the receptor for prolactin. Treatment of membranes with concanavalin A increased the number of prolactin-binding sites by 40% in membranes from phase 2 mammary tissue but decreased binding by 40% in membranes from phase 3 tissue, indicating that significant changes had occurred in the membranes of cells during this period. The tammar wallaby can secrete phase 2 and phase 3 milk from adjacent mammary glands (asynchronous concurrent lactation) and the developmental changes in milk protein gene expression and prolactin binding observed during lactation were reflected in these individual glands. Taken collectively, these findings suggest that mammary development and milk secretion in the tammar wallaby are regulated by both endocrine and local (intramammary) mechanisms.

DOI:
https://doi.org/10.1677/jme.0.0130117
Volume 13: Issue 2,
Pages:
117–125 (9 total)