We used 35S-labeled cRNA probes to localize the sites of a-lactalbumin, a-Sl-casein, and laaoferrin mRNA synthesis in sheep and forcibly weaned cattle mammary tissue. Expression of a-lactalbumin was absent in three of four "virgin" glands studied, present in some alveoli of "pregnant" glands but not in others, despite a similar histological appearance. In the early lactating gland, expression was high in those alveoli with few fat globules in their cells and lumen and was absent in alveoli with abundant fat globules. These observations suggest either that a-lactalbumin gene expression is linked to the long-term seaetory activity of cells and falls once cells are resting or regressing, or that there are cyclical variations in expression, or that in the lactating IntroductionThe mammary gland is a complex organ both in structure and function. After pubertal mammogenesis it undergoes three physiological transitions during a lactation cycle: from involution to colostrogenesis, to lactation, then back to involution. Marked changes occur in mammary gland size, structure, and secretion as the gland progresses to or from a state of active milk synthesis (33). An obvious question to ask is what happens to the expression of various milk protein genes during this time, and several investigators have indeed performed such studies (3,27,28,39). In general, the methodology has been based on the extraction ofmRNA from total mammary tissue so that the levels of gene expression being measured are actually the average for the whole tissue, and heterogeneities would not be detected. More recently in situ hybridization has been used, and although this is capable of detecting tissue-to-tissue and cell-to-cell variations in gene expression, the detection of such differential expression per se does not seem to have been the primary aim of most investigators. For example, a study of a-casein (21) neither set out to detect nor to comment on variations in mRNA levels throughout the mouse mammary gland.Correspondence t~: Adrian Molenaar, Molecular Biology, MAFTech, Ruakura Agricultural Centre, Hamilton, New Zealand. gland some groups of epithelial cells are synthesizing a-lactalbumin and some are synthesizing fat. Enpression patterns of adl-casein were similar to those of a-lactalbumin. Lactoferrin, in contrast, was expressed almost exclusively in the "fatty alveoli" of both species. Our results show that dramatic variations in milk gene expression can OCCUI throughout the mammary gland of sheep and cattle and that at no stage of pregnancy, lactation, or involution can the gland be considered metabolically homogeneous. (J I-iiktochem Cytochem 40:611418, 1992) KEY WORDS: Mammary; Milk proteins; a-lactalbumin; a-S1-casein; Lactoferrin; In situ hybridization; mRNA expression; Localization; Ovine; Bovine.The present study is aimed explicitly at addressing the possibility of heterogeneous expression of milk protein genes in the mammary glands of ruminants. We have chosen ruminants for three reasons: first, their economic importance; second, th...
Accumulation of lactoferrin mRNA in mammary tissue from virgin, pregnant, lactating, and involuting ewes and cows was localized using 35S-labeled cRNA probes. Expression of lactoferrin was low in the glands of virgin animals. In the glands of animals in early pregnancy, very high expression occurred in the ducts and immature alveoli, but expression tended to decrease as the alveoli matured. In the lactating and involuting gland, expression was generally low or absent in actively secreting alveoli and high in alveoli that had an accumulation of vesicles in the lumen and secretory epithelium, which was indicative of stasis. Occasionally, expression of lactoferrin was seen in cells that appeared to be secretory, particularly in involuting glands. Lactoferrin mRNA was expressed not only at different sites from other milk protein genes, such as alpha-lactalbumin and alpha s1-casein, but also during different stages of mammary development, supporting the view that the expression of lactoferrin is regulated differently from that of other milk proteins. For all ewes and cows, lactoferrin mRNA was detected in the epithelial ducts of the mammary parenchyma and the teat in a gradient that increased in ducts nearer the teats. The expression of lactoferrin in the ductal epithelium close to the teat was consistent with the antibacterial role of lactoferrin.
The STAT transcription factors form a family of signal transducers and activators of transcription. We sequenced the bovine STAT5B cDNA and both STAT5-encoding genes, STAT5A and STAT5B, representing the first complete description of any STAT5-encoding gene. DNA fiber FISH hybridization revealed that the genes reside only 40 kbp apart on BTA19. Both genes are segmented into 19 exons and all but two of the homologous exons are of equal size. The genes harbor a central block of nearly identical DNA sequence (97.5% sequence identity over 3373 bp), spanning from intron 5 to intron 9. Isolation and sequencing of the homologous segments from mouse revealed the same unusually high degree of intronic sequence conservation in these segments of the murine STAT5-encoding genes. However, the respective sequences are completely divergent between the two species. A comparison of the inter- and intragenic cDNA sequence preservation at nonsynonymous sites reveals that the DNA-binding domain is under the strongest selection pressure for both intergenic and factor-specific intragenic sequence preservation. The so-called "SH3" segment of the linker domain, in contrast, shows species-specific sequence identity in all but one amino acid residues in both factors, in cattle, human, and mouse. This indicates that the same species-specific selection pressure occurs on the linker domain from both factors, STAT5A and STAT5B. Thus, the comparison of evolutionary selection pressures resting on various domains suggests that the DNA-binding domain might contribute to differential DNA binding of STAT5A and STAT5B factors, while both might interact equally well with other cellular factors through a segment of the linker domain.
S U M M A R YThe activity of the enzyme acetyl-CoA-carboxylase ␣ (ACC-␣ ) is rate limiting for the de novo synthesis of fatty acids. The encoding gene is expressed from three promoters in ruminants (PI-PIII). Their individual contribution to the formation of milk fat is unknown. Promoter-specific molecular probes were hybridized in situ to serial sections of mammary glands from cows and sheep to determine their developmental and spatial expression profile in the udder. We show that all three promoters are active in mammary epithelial cells (MECs) of udders from both species. This implies that, in principle, none of these promoters can be singled out as the key element controlling the ACC-␣ -related contribution to establishment of milk fat content, although the activity of PIII only is known to be disproportionally stimulated by lactation in MECs. We propose that all three promoters may be relevant for milk fat synthesis in cattle, whereas PII and PIII are crucial for milk fat formation in sheep. We show also that ACC-␣ synthesis is not strictly coupled to casein synthesis, particularly during pregnancy and involution.
Comparative maps between ruminant species and humans are increasingly important tools for the discovery of genes underlying economically important traits. In this article we present a primary linkage map of the deer genome derived from an interspecies hybrid between red deer (Cervus elaphus) and Père David's deer (Elaphurus davidianus). The map is ~2500 cM long and contains >600 markers including both evolutionary conserved type I markers and highly polymorphic type II markers (microsatellites). Comparative mapping by annotation and sequence similarity (COMPASS) was demonstrated to be a useful tool for mapping bovine and ovine ESTs in deer. Using marker order as a phylogenetic character and comparative map information from human, mouse, deer, cattle, and sheep, we reconstructed the karyotype of the ancestral Pecoran mammal and identified the chromosome rearrangements that have occurred in the sheep, cattle, and deer lineages. The deer map and interspecies hybrid pedigrees described here are a valuable resource for (1) predicting the location of orthologs to human genes in ruminants, (2) mapping QTL in farmed and wild deer populations, and (3) ruminant phylogenetic studies.
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