text:
The bovine lactation genome: insights into the evolution of mammalian milk
- Danielle G Lemay1Email author,
- David J Lynn2,
- William F Martin1,
- Margaret C Neville3,
- Theresa M Casey4,
- Gonzalo Rincon5,
- Evgenia V Kriventseva6,
- Wesley C Barris7,
- Angie S Hinrichs8,
- Adrian J Molenaar9,
- Katherine S Pollard10,
- Nauman J Maqbool11,
- Kuljeet Singh9,
- Regan Murney9,
- Evgeny M Zdobnov12, 13, 14,
- Ross L Tellam7,
- Juan F Medrano5,
- J Bruce German1, 15 and
- Monique Rijnkels16
DOI: 10.1186/gb-2009-10-4-r43
© Lemay et al.; licensee BioMed Central Ltd. 2009
Received: 12 September 2008
Accepted: 24 April 2009
Published: 24 April 2009
The newly assembled Bos taurus genome sequence enables the linkage of bovine milk and lactation data with other mammalian genomes.
Using publicly available milk proteome data and mammary expressed sequence tags, 197 milk protein genes and over 6,000 mammary genes were identified in the bovine genome. Intersection of these genes with 238 milk production quantitative trait loci curated from the literature decreased the search space for milk trait effectors by more than an order of magnitude. Genome location analysis revealed a tendency for milk protein genes to be clustered with other mammary genes. Using the genomes of a monotreme (platypus), a marsupial (opossum), and five placental mammals (bovine, human, dog, mice, rat), gene loss and duplication, phylogeny, sequence conservation, and evolution were examined. Compared with other genes in the bovine genome, milk and mammary genes are: more likely to be present in all mammals; more likely to be duplicated in therians; more highly conserved across Mammalia; and evolving more slowly along the bovine lineage. The most divergent proteins in milk were associated with nutritional and immunological components of milk, whereas highly conserved proteins were associated with secretory processes.
Although both copy number and sequence variation contribute to the diversity of milk protein composition across species, our results suggest that this diversity is primarily due to other mechanisms. Our findings support the essentiality of milk to the survival of mammalian neonates and the establishment of milk secretory mechanisms more than 160 million years ago.
Because bovine milk is a major human food and agro-economical product, comparison of bovine milk with the milk of other species in the context of the bovine genome sequence is important not only to improve our understanding of mammary evolution but also of bovine milk production and human nutrition. The importance of bovine milk consumption to humans is underscored by the domestication of cattle and the convergent evolution of lactase persistency in diverse human populations [7]. The availability of the bovine genome sequence provides unique opportunities to investigate milk and lactation. Lactation has been studied more extensively in Bos taurus than in other species, resulting in extensive milk proteome data, milk production quantitative trait loci (QTL), and over 100,000 mammary-related bovine expressed sequence tags (ESTs).
In the present study, we identified the bovine lactation genome in silico and examined its content and organization. Utilizing the genomes of the seven mammals listed above and in Table 1, we investigated gene loss and duplication, phylogeny, sequence conservation, and evolution of milk and mammary genes. Given the conspicuous absence of some known abundant proteins, such as beta-lactoglobulin and whey acidic protein, in the milk of some species [8], we hypothesized that variation in milk composition resides in part in variation in the milk protein genome. We show that gene duplication and genomic rearrangement contribute to changes in the milk protein gene complement of Bos taurus and other species. Although the casein proteins are highly divergent across mammalian milks [9, 10], we report that milk and mammary genes are more highly conserved, on average, than other genes in the bovine genome. Our findings illustrate the importance of lactation for the survival of mammalian species and suggest that we must look more deeply, perhaps into the non-coding regions of the genome that regulate milk protein gene expression, to understand the species-specificity of milk composition. Among mammals, we find milk proteins that are most divergent have nutritional and immunological functions, whereas the least divergent milk protein genes have functions that are important for the formation and secretion of mammalian milk. High conservation of milk fat globule membrane protein genes among the mammalian genomes suggests that the secretory process for milk production was firmly established more than 160 million years ago.
Results and discussion
Milk and mammary gene sets
The milk protein gene set is the most extensive curation to date of genes that give rise to milk proteins, the functions of which have not yet been comprehensively studied. To gain insight into the possible molecular functions of milk proteins, the milk protein gene set was analyzed for enriched molecular function Gene Ontology (GO) terms (see Materials and methods). Four significant, minimally redundant molecular function GO terms were identified: 'GTPase activity,' 'GTP binding,' 'pattern recognition receptor activity,' and 'calcium ion binding.' More than 30 milk proteins that were previously isolated in the milk fat globule membrane [11, 12] were associated with 'GTPase activity' or 'GTP binding'. GTPases are known to be involved in numerous secretory processes, and for this reason, it seems likely that these proteins have a role in assembly and secretion of the milk fat globule and possibly other milk components. The 'pattern recognition receptor activity' GO term was enriched due to the presence in milk of the cell surface and immune recognition components CD14 [GenBank:NM_174008], TLR2 [GenBank:NM_174197], TLR4 [GenBank:NM_174198], and DMBT1 [GenBank:S78981]. These proteins are involved in the activation of the innate immune system when they associate with cells. Further, the soluble forms of CD14 and TLR2, which can act as decoy receptors for microbial pathogens, could potentially modulate local inflammation following bacterial colonization in the neonate gut [13, 14]. Enrichment of the GO term 'calcium ion binding' was expected as many milk proteins are known to bind calcium, a mineral required in abundance by the growing neonate.
Milk is traditionally thought of as a food that provides the neonate with nutrients and some immune protection, such as that provided by immunoglobulins. Prior research also suggests that various milk proteins are resistant to digestion by gastric proteases at physiological pH [15] and that intact or partially intact milk proteins may either express their functions in the neonatal intestinal tract or may be absorbed and act on other organs [16]. To understand what signaling might be possible if milk proteins remain partially or wholly undigested, the milk protein gene set was interrogated for enriched pathway annotations (see 'Pathway analysis' in Materials and methods). The milk protein gene set contains elements of two marginally significant pathways that lead to activation of PPARalpha and LXR, two nuclear receptors involved in sensing nutrients and modifying metabolic responses at the level of gene transcription. Milk proteins that are associated with the LXR/RXR activation pathway include the cell surface or secreted molecules CD14 [GenBank: NM_174008], CD36 [GenBank:NM_174010], TLR4 [GenBank:NM_174198], and MSR1 [GenBank:NM_001113240], the apolipoproteins APOA1 [GenBank:NM_174242] and APOE [GenBank:NM_173991] and the lipid synthesis enzymes ACACA [GenBank:NM_174224] and FASN [GenBank:NM_001012669]. Those associated with the PPARalpha/RXRalpha activation pathway include the cell surface molecule CD36 [GenBank:NM_174010], the endoplasmic reticulum protein disulphide isomerase PDIA3 [GenBank:NM_174333], the apolipoprotein APOA1 [GenBank:NM_174242], the transcription factor STAT5B [GenBank:NM_174617], the heat shock protein HSP90AA1 [GenBank:NM_001012670], the regulator of adenylate cyclase GNAS [GenBank:NM_181021], and two enzymes involved in lipid synthesis, GPD2 [GenBank:NM_001100296] and FASN [GenBank:NM_001012669]. It is likely that the products of these genes, which are well known to be active at metabolic control points in many organs, are active in the mammary gland and then enter the milk via cytoplasmic crescents in the milk fat globules. Keenan and Patton [17] noted that cytoplasmic sampling, as part of milk fat globule formation, is present in all species examined to date, including humans, and that such evolutionary persistence suggests possible benefits for mother or offspring. Further research will be needed to determine whether these proteins are present in milk at sufficient quantities to have a physiological effect in the neonate.
All mammary gene sets were interrogated for enrichment of GO terms or pathway annotations, but the results did not further our knowledge of mammary biology. Consistent with our previous study [18], current GO term annotations were incomplete or generally out of context when applied to the mammary gland. Although bovine EST data indicate that more than 3,000 genes are expressed in the lactating mammary gland, a mere 22 genes are currently annotated with the GO term 'lactation.'
Bovine milk production QTL
Milk trait QTL delineate gene regions that harbor genes or cis-acting elements that are responsible for the milk trait phenotype. The dairy industry has invested enormous resources into the identification of these QTL for milk production traits in bovine, particularly milk yield, protein yield, fat yields, protein percentage, and fat percentage. Reviewing the literature, 238 milk trait QTL were identified for these five traits in 59 references (Additional data files 8-9). Of the 238 QTL, 63 were reported with flanking markers having a median interval size of approximately 17 million base pairs. Following a previously established method [19], the 175 remaining QTL that were reported with only a single peak marker were assigned this median interval size. Some QTL were reported for more than one milk trait; thus, these QTL span only 168 unique genome locations. These milk trait QTL span all 29 autosomes (Figure 2), with the highest densities of QTL occurring on chromosomes 27, 6, 20, and 14 (Additional data file 10). Possible differences in genetic architecture are most obvious between fat and protein percentage traits, where fat percentage QTL are present on fewer chromosomes with lower QTL density and protein percentage QTL are present on all but two chromosomes, most with higher QTL density (Additional data file 10). Fat percentage may be controlled by relatively fewer genes each with larger effects, whereas protein percentage may be controlled by far more genes each with smaller effects.
The milk trait QTL provide a very coarse map of genomic areas of interest that cover nearly half of the bovine assembly. Milk yield QTL overlap with 19.5% of the genomic assembly, fat yield QTL with 15.4%, protein yield QTL with 21.1%, fat percentage QTL with 12.3%, and protein percentage QTL with 33.6% of the genome assembly. The densities of genes within these QTL are very similar for each milk trait, with between 9.1 and 10.1 genes per million base pairs. Meanwhile, there are 8.4 genes per million base pairs in regions that do not overlap with any milk trait QTL. Given the gene density and number of QTL associated with each trait, each individual QTL is expected to contain between 105 and 127 genes.
To identify candidate genes within milk trait QTL, the lactation mammary gene set was intersected with the milk trait QTL. Between 12.5% and 13.7% of the genes within milk trait QTL are expressed during lactation. In other words, within a single milk trait QTL, between 13.9 and 17.1 genes are expected to be expressed during lactation. Thus, although the set of milk trait QTL reduces the search space for milk trait effectors by less than one order of magnitude, the use of expression data can contribute considerably towards the identification of candidate genes. Genes within milk trait QTL that are expressed in the mammary gland during lactation are listed in Additional data files 11-16. Milk trait effectors are likely to be near these candidate genes.
Genome organization of milk and mammary genes
Studies of eukaryotic genomes have demonstrated that genes with coordinated expression or shared ancestry appear in clusters across the genome [20]. Given that the clustering of the casein milk protein genes is essential to their coordinated transcription in the lactating mammary gland [9, 10], the arrival of the bovine genome sequence provides the opportunity to discover other gene clusters relevant to milk, lactation, or mammary biology. A genome-wide search was conducted for genomic intervals of 500 kb and greater that are statistically enriched with genes from the milk protein and mammary gene sets (see 'Genomic localization analysis' in Materials and methods). Among these gene sets, 190 non-overlapping statistically significant clusters were identified: four unique clusters in the milk protein gene set and 54, 60, 30, and 19 unique clusters in the pregnancy, lactation, involution, and mastitis mammary gene sets, respectively. Spreadsheets of all significant gene clusters are available in Additional data files 17 and 18.
The four significant milk protein gene clusters comprised the immunoglobulin genes, casein genes, fibrinogen genes, and genes that encode milk fat globule proteins. Because it is known that immunoglobulins, casein genes, and fibrinogen genes are each clustered in mammalian genomes [9, 10, 21, 22], this is a good verification of methodology. The cluster of genes that encode milk fat globule proteins contains FASN [GenBank:NM_001012669], ARHGDIA [GenBank:NM_176650], and P4HB [GenBank:NM_174135]. However, P4HB has only been isolated in mastitic milk [11]. By manual inspection, we found that these genes also cluster in the human, mouse, and other mammalian genomes. Based on EST data, other genes in this genomic region are expressed at various times in the mammary gland. Aside from these four clusters, there does not appear to be a preponderance of putative regulatory modules among genes in the milk protein gene set. Whereas only 6.6% of the milk protein genes were within a milk protein-specific cluster, 27.9% were within one of the mammary gene set clusters. Therefore, it is likely that milk protein genes are regulated along with other mammary genes independent of the function or cellular localization of the proteins they encode.
Next, we examined whether genes were clustered according to developmental stage, but found there were no gross differences in gene clustering using this parameter. Between 24% and 30% of the genes from each mammary gene set - virgin, pregnancy, lactation, and involution - were within one of the other mammary set clusters. Likewise, 28% of the genes from the mastitis mammary gene set fell within a mammary cluster. Thus, mammary genes are not differentially clustered by developmental stage or condition.
Genes may be clustered due to shared evolution, as duplicated genes are often co-localized in the genome. In our study, a significant cluster required a minimum of three genes that were not paralogs. When the paralog requirement was removed, only seven additional unique clusters of triplets or greater were identified. Significant clusters with more than one paralog appear to be confined to the major histocompatibility complex region on bovine chromosome 23. These data suggest that recent duplication is not a common driver of clustered mammary genes in the bovine genome.
In summary, the milk protein genes generally do not form clusters with each other but do appear to form clusters with other mammary genes. Milk protein genes may be regulated along with other lactation genes without regards to the final destination of the gene product. As mammary genes are generally clustered neither by developmental stage nor due to recent duplication, it appears that the need for co-expression in the mammary gland is the denominator for co-localization rather than co-functionality or shared ancestry. This organization in clusters of co-expressed mammary genes might be constrained by unidentified distal cis-acting elements [20], chromatin conformation [23], or coordinately expressed micro-RNAs [24].
Milk and mammary gene copy number trends in mammals
The absence of a milk or mammary gene in platypus or duplication after platypus (Figure 3) may be due to the expansion of gene families in the common therian ancestor. However, some of these genes may not be truly missing in the platypus genome, but may be undetectable by our methods due to incomplete or incorrect assembly of the platypus genome, lower sequence identity, or the inherent bias created by defining milk and mammary genes in the bovine genome. The identification of platypus orthologs of other genes in the bovine genome would also be affected by these biases; therefore, we next compared milk and mammary gene copy number trends to those genome-wide.
For each major trend shown in Figure 3, rates of occurrence among the uncurated orthologs of the milk protein and lactation mammary gene sets were compared with the orthologs of all bovine consensus gene models using a hypergeometric distribution to determine statistical significance. More bovine milk protein orthologs were found in all six studied mammalian genomes than would be expected given the rate at which other bovine orthologs were found in these genomes (P < 0.0001). Genes expressed during bovine lactation were also more likely than other genes to have orthologs in all of the mammalian genomes (P < 0.0001). In other words, milk and mammary genes are more likely than other genes to be found in all mammals. This result might be explained in part by an increased power to detect more conserved genes (see 'Conservation of milk and mammary genes in mammals' below). There were also statistically fewer lactation genes missing in the platypus (P < 0.005) and opossum genomes (P < 2.2 × 10-20); however, the number of milk protein genes missing in these genomes did not differ from the genome-wide rate. Finally, more milk protein and lactation genes were duplicated after platypus compared with the whole genome (P < 0.001 and P < 0.03, respectively). Together, these data support the essentiality of milk and mammary genes in Mammalia as well as suggest the possibility for expanded functionality in marsupials and placental mammals.
Milk protein gene copy number variation may potentially contribute to the diversity of milk composition. Ortholog analysis indicated that the gene for beta-lactoglobulin (LGB), one of the most abundant proteins in milk, is duplicated in the dog and bovine genomes (Figure 3). In the bovine genome, this gene is located at the position of a previously predicted pseudogene [25]. It has similarity to LGB-II genes in the horse and cat [26–29]. The similarity of this second gene to LGB-II in the horse, cat, and dog suggests that the LGB duplication existed in the common ancestor of the laurasiathians (Figure 1). Using two different primer pairs, we were unable to identify the LGB-II transcript in bovine mammary tissue samples using RT-PCR (see Additional data file 22 for details). It is likely that the duplicated LGB gene is not expressed in the bovine mammary gland and that the presence of this duplication does not influence the concentration of LGB in bovine milk.
LGB is apparently not present in human or mouse milk [30], although LGB-like proteins have been isolated from the milk of other primates [31–33]. A human protein, progestagen-associated endometrial protein (PAEP), has significant homology to the bovine and equine LGB-II-like genes [29, 34–36]. Although PAEP expression has been detected in the epithelial cells of human breast tissue [37], neither its presence nor that of an apparent LGB-like pseudogene [GenBank:AH011480] that flanks the PAEP gene [GenBank:NM_001018049] has been verified in human milk. We found that the LGB-like and PAEP genes are flanked by GLT6D1 [GenBank:NM_182974] and OBP2A [GenBank:NM_014582] in both the human and bovine genomes. This observation, combined with the fact that the baboon has both a PAEP gene [38] and a LGB gene [33], suggests that the primate genes arose by duplication of an ancestral gene before the Laurasiatheria and Eurochontoglires diverged. We were unable to find this region in the rodent or rabbit genomes, and an evolutionary break point is present in mouse and rat in this region [39], suggesting that these genes may have been lost after the split between primates and glires. Although the presence of LGB in laurasiathian milk and its absence in rodent milks has an obvious genetic basis, we cannot yet explain the absence of LGB in human milk.
Some immune components of milk are uniquely duplicated in certain species or clades. For example, SAA3 [GenBank:NM_181016], which is duplicated in the bovine and dog genomes (Figure 3), is thought to be involved in mucin induction in the gut [40, 41] and a human analog, SAA1, functions as an opsonin for Gram-negative bacteria [42]. The Cathelicidin gene family is greatly expanded in the bovine, opossum, and platypus genomes, with 10, 8, and 12 copies, respectively [43–45], but some of the opossum and platypus orthologs were not found in our automated analysis due to their high heterogeneity. Expansions in this gene family may reflect increased exposure to bacteria at epithelial surfaces in these species. Our results show that the CD36 gene [GenBank:NM_174010], which encodes a scavenger receptor, has duplications in the B. taurus and rat genomes. Beta-2-microglobulin [GenBank:NM_173893] has a second copy in the bovine genome and may also have a duplicate in the platypus genome. This gene encodes one of two chains in the IgG transporter FcRn, which transfers IgG molecules across epithelial cells [46]. Other variations in milk protein gene copy number (Figure 3) potentially give rise to diversity in milk protein composition.
Milk protein gene loss does not appear to be a common occurrence. Of the bovine milk protein genes with an ortholog identified in the platypus genome (Figure 3), all but ten genes were found in all of the other studied mammalian genomes. However, because the bovine milk proteome is used as the reference, the loss of some milk protein genes in placental mammals relative to the monotreme and marsupial mammals may have been missed in our analysis. For example, whey acidic protein has been identified in the milk of many mammals such as mouse, rat, opossum, and platypus, but it is absent in bovine milk due to a frameshift mutation in the whey acidic protein gene [47]. A full proteomic analysis of the milk samples from extant monotremes and marsupials will be needed to identify gene loss in placental mammals.
Our analysis of milk protein gene copy numbers has several other limitations. First, the mammalian orthologs of bovine consensus gene models derived on a genome-wide basis (see 'Orthology delineation' in Materials and methods) may be inaccurate for genes in which the bovine gene model is incorrect or may be incomplete when orthologs are too divergent to be detected by this method. Although we attempted to overcome these limitations by manually curating milk protein gene orthologs, the analysis is only as good as the available genome sequences, and some duplications and deletions may have been missed due to errors and gaps in the genome assemblies. Directed sequencing will be needed to confirm specific results. However, we can generally conclude that there is considerable copy number variation of milk protein genes that may contribute to the taxonomic diversity of milk composition.
Taxonomic relationships of the milk protein genes
Conservation of milk and mammary genes in mammals
Of the average pairwise PID distributions of the mammary gene sets in Figure 6, all are significantly different from the genome-wide distribution. The means of their distributions also differ from the genome-wide mean. As a group, mammary genes of every developmental stage and condition appear to be more conserved across Mammalia, on average, than other genes in the genome.
Highly conserved milk proteins
Protein name
Gene symbol
Average PID
Rab 11A
GTP binding protein G I G S G T beta subunit 1
GTP binding regulatory protein beta 2 chain
Rho C
Rab 11B
Rap 1b
GTP binding protein Sara
Rab 3A
Histone 2, H2ab
SAR1B protein
14-3-3 protein beta alpha
Leucine-rich repeat containing protein 8
Rab 18
Rho GDP-dissociation
Rab 5C
Rab 3C
Eukaryotic translation initiation factor 4, gamma 2
ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit
ARP3 (actin-related protein 3, yeast) homolog
GTP binding protein alpha 14
Endoplasmin precursor (GRP94/GP96)
Lymphocyte cytosolic protein 1 (65 K macrophage protein/L-plastin)
Retinal short-chain dehydrogenase/reductase
Nearly all of the highly conserved milk proteins (Table 2) are found in the milk fat globule membrane proteome. GO analysis of these proteins yields four enriched terms: 'GTPase activity,' 'GTP binding,' 'small GTPase mediated signal transduction,' and 'intracellular protein transport.' Twelve of the proteins listed in Table 2 are annotated with one or more of these GO terms. GTPases are known to be involved in the exocytotic pathway by which proteins are trafficked from the Golgi compartment to the plasma membrane. Further, GBB1 [GenBank:NM_175777], RAB11B [GenBank:NM_001035391], RAP1B [GenBank:NM_175824], YWHAB [GenBank:NM_174794], and RAB18 [GenBank:NM_001075499] listed in Table 2 have previously been isolated in Golgi fractions from the mammary glands of pregnant and lactating rats [48]. An additional four milk proteins, SAR1A [GenBank:NM_001034521], SAR1B [GenBank:NM_001035315], RAB3A [GenBank:NM_174446], and RAB3C [GenBank:NM_001046606], are annotated with the GO term 'secretory pathway.' The finding that so many of these secretion-related proteins are associated with the milk fat globule membrane suggests they may also be involved in the highly specialized process by which the milk fat globule is secreted or that the exocytotic and lipid secretion pathways intersect at some point during the secretion process. Because the conserved proteins listed in Table 2 are related to the generic molecular function of secretion, it seems highly likely that they facilitate the secretion of milk lipid.
Conservation of mammary genes relative to other genes in the genome suggests hypotheses about the evolution of milk production. First, conservation of mammary genes involved in all developmental stages supports the hypothesis that, at the genetic level, the basic biological transformation of the virgin gland through pregnancy, lactation, and involution is conserved among all mammals, and occurred by co-opting existing structures and developmental pathways. Second, many of the most highly conserved proteins found in milk are constituents of the milk fat globule membrane and are known to be part of the secretory process. High conservation of these genes between platypus, opossum, and the placental mammals indicates that molecular mechanisms of secretion were already in place 160 million years ago.
Divergent milk protein genes in mammals
Highly divergent milk proteins
Protein name
Gene symbol
Average PID
G protein Xlalphas
Kappa casein
Mucin 1
Beta casein
Rab 10
Immunoglobulin IgM
Polymeric-immunoglobulin receptor precursor
Keratin 9
Rab 7
Peptidoglycan recognition protein
Fibrinogen alpha chain
Apolipoprotein B
Apolipoprotein E
Glycoprotein antigen MGP57/53 (lactadherin/bP47 protein)
Toll-like receptor 4
MUC15 protein
Macrophage scavenger receptor types I and II
Toll-like receptor 2
Albumin (precursor)
Chitinase-like protein 1 (CLP-1)
The greatest inter-species divergence among milk protein sequences occurs with those proteins that are most abundant in milk (caseins, alpha-lactalbumin (LALBA)), those most abundant in plasma (fetuin, albumin), and with those contributing to immunity. The casein proteins are the most divergent of the milk proteins, with an average pairwise PID of only 44-55% across placental mammals. Nutritionally, the caseins provide the suckling neonate with a source of amino acids and with highly bioavailable calcium. Additionally, peptides derived from partially digested caseins have potential anti-microbial, immune-modulating, and other bioactive properties. The fact that the caseins are the most divergent of the milk proteins suggests that the nutritional and immunological functions of these proteins do not particularly constrain their amino acid sequence and structure.
The sequence divergence of LALBA is surprising given its essentiality to the synthesis of lactose, the primary source of digestible carbohydrate. LALBA encodes a protein that forms the regulatory subunit of the lactose synthase heterodimer. However, additional functions of LALBA have emerged. When human LALBA is partially unfolded and bound to oleic acid, it functions as an apoptotic factor that kills tumor cells and immature cells, but not healthy differentiated cells [49]. Thus, it is possible that this variant of LALBA protects the gut of the human neonate. Furthermore, the apoptotic capabilities of LALBA appear to be utilized in the regulation of involution of the mammary gland. A recent study suggests that Cape fur seals escape apoptosis and involution of the mammary gland during long foraging trips because they lack the LALBA protein [50]. While lactose synthesis may be a common essential function, it appears that it does not overly constrain the sequence divergence of LALBA. The sequence divergence of LALBA may rather be related to the potential of this protein to modulate species-specific strategies related to immune function and the regulation of the mammary gland.
The most divergent immune-related proteins in milk are products of the following genes: mucin 1 (MUC1) [GenBank:NM_174115], immunoglobulin IgM [GenBank:BC114809], polymeric-immunoglobulin receptor (PIGR) [GenBank:NM_174143], peptidoglycan recognition protein (PGLYRP1) [GenBank:NM_174573], CD14 [GenBank:NM_174008], Toll-like receptor 2 (TLR2) [GenBank:NM_174197], Toll-like receptor 4 (TLR4) [GenBank:NM_174198], macrophage scavenger receptor types I and II (MSR1) [GenBank:NM_001113240], and chitinase-like protein 1 (CHI3L1) [GenBank:NM_001080219]. In milk, CD14 and TLR2 are present in soluble forms and may neutralize pathogens by binding to them as decoy receptors [13, 14]. MUC1 prevents the binding of pathogenic bacteria to epithelial cells in vitro (R.L. Tellam, personal communication). Our finding that the most divergent milk protein genes are those that confer immunity presumably reflects a flexibility to confront a wide variety of pathogen challenges.
Evolution of milk and mammary genes along the bovine lineage
Milk and mammary gene average dN/dS
Gene set
Number of genes with computed dN/dS
Average dN/dS
Milk protein
Virgin mammary
Pregnancy mammary
Lactation mammary
Involution mammary
Mastitis mammary
Whole genome
Given the taxonomic diversity of milk composition, we expected that the processes of lactation would be under stronger selective pressure than the genes that give rise to proteins in milk. However, the average dN/dS of the milk protein gene set was similar to that of the lactation mammary gene set (Table 4). This result suggests that species-specific variation in milk composition is primarily due to mechanisms other than milk and mammary protein sequence variation.
Next, milk and mammary genes were evaluated for positive selection. A gene is inferred to be subject to positive selection when dN/dS is significantly greater than 1. Of the 6,530 genes from the milk protein and mammary gene sets, only two bovine genes with dN/dS >1 were significant under the likelihood ratio test (see 'Evolution analysis under the bovine lineage' in Materials and methods): ADP-ribosyltransferase 4 (ART4) [GenBank:AJ291442] and prenylcysteine oxidase 1 (PCYOX1) [GenBank:NM_001105474]. The ART4 gene product, which has previously been reported to be subject to positive selection in cattle [51], is an erythrocyte protein that carries antigens to the Dombrock blood group. PCYOX1 produces a protein that degrades a variety of prenylcysteines. Using RT-PCR to determine PCYOX1 and ART4 mRNA levels in alveolar mammary tissue from virgin, prepartum, lactating, involuting and dried-off cows (Additional data file 22), we found that PCYOX1 and ART4 are not differentially expressed in these tissues. The accelerated evolution of these genes may be unrelated to mammary biology.
Two abundant milk protein genes, beta-casein (CSN2) [GenBank:NM_181008] and kappa-casein (CSN3) [GenBank:NM_174294], were among those with dN/dS >1, but they were not statistically significant under the likelihood ratio test (see 'Evolution analysis along the bovine lineage' in Materials and methods). The requirement that the entire gene shows statistical evidence of positive selection may be too stringent. Evidence of positive selection within the family Bovidae has been previously detected in a 34-codon region of CSN3 [52]. Further site-specific evolutionary analysis of the casein genes may be warranted.
Despite the domestication of cattle for milk production, breeding regimes have not caused the apparent accelerated evolution of even a single milk protein or member of the lactation mammary gene set. Furthermore, milk and mammary genes are undergoing stronger purifying selection than other genes in the bovine genome. It has previously been theorized that the evolution of the mammary gland has been subject to forces that maximize the survival of the mother-child pair [53]. Because all components in the milk are produced at the expense of the mother, it can be argued that few superfluous components would survive evolution. Our findings are consistent with this hypothesis. Genes encoding milk components and other genes expressed in the mammary gland were found to be under significant negative selection compared to the whole genome, highlighting the essentiality of milk in mammalian evolution.
The availability of the B. taurus genome sequence assembly marks the beginning of a new era for the study of milk and mammary biology. Using this assembly, we identified 197 unique milk protein genes and over 6,000 mammary-related genes distributed across all bovine chromosomes. Intersecting these genes with 238 curated milk-trait QTL, we reduced the search space for milk trait effectors by more than an order of magnitude.
Comparing genes of seven mammalian species, we found that more milk and mammary genes are present in all mammals and more were duplicated after the common ancestor with platypus than other genes in the bovine genome. We also found that, on average, milk and mammary genes are more conserved than other genes among mammals and are evolving more slowly than other genes in the bovine genome. The higher conservation of these genes, as well as the strong negative selection and absence of positive selection, supports the hypothesis that the evolution of milk has been constrained to maximize the survival of both mother and offspring.
Our findings also suggest that the species-specific variation in milk composition is primarily due to mechanisms other than protein sequence variation. Variation in copy number of the milk protein genes may contribute to the taxonomic diversity of milk composition, as exemplified by duplications of some immune-related milk proteins. Additional mechanisms, such as transcriptional and translational regulation of genes expressed in the mammary gland as well as other organs involved in energy partitioning may be larger contributors to milk composition variation. In future studies, non-coding regions of the genome, particularly those with putative regulatory function, will need to be explored as potential sources of species-specific variation in milk composition.
We found that mammary genes are co-localized in the bovine genome, implying co-regulation of expression in mammary epithelial cells. Our study of the most complete milk proteome to date demonstrates that milk proteins do not generally cluster with each other, but do cluster with mammary genes, and therefore are likely co-regulated. The casein proteins are therefore unique among the milk protein genes in both their divergence between species and in their genomic organization.
Across the seven mammalian genomes, we found that the most divergent milk proteins are known to have nutritional and immunological properties, whereas the most conserved are within the milk fat globule proteome. The high conservation of genes apparently involved in milk fat globule secretion suggests that the cellular anatomy of secretion may be conserved across species and likely shared among secretory organs. Likewise, the higher conservation of mammary genes, compared with other genes, suggests that the ontogeny of the mammary gland occurred by co-opting existing structures and developmental pathways. Lactation may be less than 200 million years old, but its biological roots are far more ancient.
Materials and methods
Collection of the milk protein gene set
Amino acid sequences corresponding to the protein identifiers reported in two proteomic studies [11, 12] were collected from SwissProt, UniRef, TREMBL, and NCBI databases. A custom track of the bovine consensus gene models [43], or 'GLEAN' models, was created and uploaded to the University of California Santa Cruz (UCSC) Genome Browser [54]. The milk protein amino acid sequences were aligned against the bovine genome draft 3.1, also known as 'Aug. 2006,' using BLAT [55]. The best scoring hits were manually reviewed against the custom GLEAN track in the UCSC Genome Browser. From the protein sequence identifiers in the Smolenski and Reinhardt studies [11, 12], 189 unique milk proteins were mapped to GLEAN IDs. The GLEAN IDs corresponding to the six most abundant milk proteins, alpha-S1-casein, alpha-S2-casein, beta-casein, kappa-casein, LGB, and LALBA, were also added to this gene set. Additionally, a script was written to identify genes in the bovine annotation database annotated by the lactation group that did not already exist in our milk protein gene set. Those genes were then manually screened for proteins known to be in milk. Two additional proteins were identified: lactotransferrin and secreted phosphoprotein 1 (osteopontin). In total, the milk protein gene set consists of 197 unique genes that encode proteins known to be in bovine milk (Additional data files 1 and 2).
Collection of the mammary gene sets
Bovine mammary gland EST libraries available through the NCBI EST database [56] were surveyed to define condition- or developmental stage-specific mammary gene sets. Descriptions of the EST libraries used as well as reasons for exclusion of other EST libraries are given in Additional data file 21. The EST libraries used include the BMGA, BMLB, BMPA, BMUA, and BMVB libraries developed by AgResearch Ltd and Genesis Ltd in New Zealand as well as the FNM and FLM libraries developed by ViaLactia Biosciences Ltd in New Zealand. Custom tracks for the UCSC Genome and Table Browsers [54, 57] were created for all GLEAN models from the bovine genome project [43] and for all of these EST libraries. The intersection filter of the UCSC Table Browser was used to identify the GLEAN models that overlapped with ESTs in these libraries. Mammary gene sets were defined as follows: virgin = (BMVB or FNM); pregnancy = BMPA; lactation = (BMGA or FLM); involution = BMLB; and mastitis = BMUA. In total, these mammary gene sets contain 6,469 unique genes derived from over 94,000 ESTs. Gene identifiers for the genes that comprise each mammary gene set are given in the spreadsheets of Additional data file 1. UCSC custom tracks of these genes sets are provided in Additional data files 3-7.
Gene Ontology analysis
Bovine genes from the milk protein and mammary gene sets were mapped to human orthologs using a three-way reciprocal best BLAST hit approach between human, mouse, and bovine as implemented in the 'Ortholuge' program [58]. Using GO-Elite version 1.17 beta [59], the human orthologs of milk and mammary genes were analyzed for over-represented GO terms compared with the full set of human orthologs. GO-Elite calculates a Fisher's exact test z-score for unique genes corresponding to a GO term from the user's input list compared with the reference list. To calculate a P-value for each GO term, GO-Elite randomly selects the same number of input genes or probe sets in the user's input gene list from the reference list 2,000 times to determine the likelihood of obtaining a z-score greater than or equal to the empirically derived z-score. To adjust for multiple hypothesis testing, the Benjamini-Hochberg correction is used to calculate an adjusted P-value. GO-Elite determines the set of minimally redundant significant GO terms by examining the scores and relative positions of all high-scoring terms within the hierarchy to select those terms that maximally describe the biology without removing valuable information. GO terms with an adjusted P-value ≤ 0.05 were taken to be significant.
Pathway analysis
Ingenuity Pathways Analysis [60] was used to identify metabolic and signaling pathways that are over-represented by the human orthologs of milk and lactation-related bovine genes compared with the human orthologs of all of the genes from the bovine consensus gene model (GLEAN) set. The Ingenuity Pathways Analysis library of canonical pathways includes 80 metabolic and 72 signaling pathways that have been incorporated from various resources and hand-curated. A Fischer's exact test was used to calculate a P-value to determine the probability that the enrichment of the canonical pathway with the gene set of interest is explained by chance alone. In this paper, this P-value is referred to as the unadjusted P-value. Enriched pathways with an unadjusted P-value ≤ 0.05 and associated with at least three genes of interest are referred to as marginally significant. To improve the stringency of the test, a Benjamini and Hochberg multiple testing correction was applied to the unadjusted P-values with a custom R script using the multtest library [61, 62]. Pathways reported to be statistically significant were those with a Benjamini and Hochberg adjusted P-value ≤ 0.05.
Genomic localization analysis
The bovine genome was explored for clusters of milk and lactation-related genes that are more proximal to each other in the genome than would be expected by chance. Using the method developed by Salomonis et al. [63], 500 kb windows on the genome that start in the same position as a gene were tested as candidate co-location clusters. For each such candidate cluster, a list of genes that overlap the window was assembled. Strand information was ignored, so that a gene was considered to start at its lowest coordinate. Genomic sequence not assembled to chromosomes was excluded.
For each of the milk protein and mammary gene sets, the statistically expected number of genes from the cluster in the gene set was calculated, given the number of genes on chromosomes both inside and outside of the gene set and using a hypergeometric distribution. Genes were considered to comprise a co-location cluster if there were at least three non-paralogous genes of interest in the region and the number of candidate cluster genes from the gene sets was significantly higher than chance (P ≤ 0.05). A multiple test correction against all the clusters was performed with a Benjamini and Hochberg false discovery rate [61, 62]. When multiple clusters contained the same genes or subsets of genes, the cluster with the lowest P-value was reported.
Orthology delineation
Orthologs of the milk and mammary genes were filtered from the whole genome ortholog set [43]. Briefly, groups of orthologous genes were automatically identified using a previously employed strategy [64] that is based on all-against-all protein sequence comparisons using the Smith-Waterman algorithm, followed by clustering of best reciprocal hits from highest scoring ones to 10-3 e-value cutoff for triangulating best reciprocal hits or 10-6 cutoff for unsupported best reciprocal hits, and requiring a sequence alignment overlap of at least 30 amino acids across all members of a group. Furthermore, the orthologous groups were expanded by genes that are more similar to each other within a proteome than to any gene in any of the other species, and by very similar copies that share over 97% sequence identity. The procedure was applied to the initial bovine set of consensus gene models [43] and to the longest transcript per gene of the Ensembl v.45 [65] gene sets of human, mouse, rat, dog, opossum, and platypus.
Curation of milk protein orthologs
Orthologs of milk protein genes (see 'Orthology delineation' above) were curated manually if they were uniquely duplicated in B. taurus or missing copies in one or more of the placental mammals. Fifteen genes uniquely duplicated in B. taurus were removed from the curated ortholog set for the following reasons: four were pseudogenes - gene duplicates without introns; four were not assembled on a chromosome and, therefore, likely to represent reading of the same sequence; and seven were a result of an assembly error or poor GLEAN prediction. The pseudogenes identified were those of PFN1 (GLEAN_02143), RAB18 (GLEAN_21462), RAP1B (GLEAN_10112), and YWHAZ (GLEAN_11922). A perfect duplicate of GAPDH (GLEAN_13969) that exists in both Btau 3.1 and 4.0 assemblies was retained, but further research is needed to determine if it is a true expansion. To find missing orthologs of milk protein genes in placental mammals, the Ensembl databases of those species with orthologs not found by automated detection were searched using BLAST and the bovine consensus gene and protein sequences. Additional known orthologs were collected from UniProt. In total, 15 erroneous duplicate genes and 37 missing orthologs were identified through manual curation. This curated ortholog set is available in Additional data file 19.
For conservation analyses, amino acid sequences of bovine genes annotated as erroneous in the bovine genome annotation database [66] were replaced with manually curated full-length sequences from UniProt [67]. Replaced sequences are indicated by the following accession format in Tables 2 and 3: GLEAN_ID_ACCESSION where ACCESSION is the UniProt accession for the replacement amino acid sequence and GLEAN_ID is the bovine gene model identifier for the original sequence.
Milk protein gene copy clustering and visualization
Perl and shell scripts were written to create input files for the clustering tool, Cluster version 2.20 [68]. A K-means clustering algorithm was applied in Cluster, and the resulting clusters were seriated using a multiple-fragment heuristic in PermutMatrix [69]. Whether or not clustering was applied, all heatmaps were seriated and visualized in PermutMatrix.
Consensus tree construction
Single copy ortholog identifiers of milk protein genes were extracted from the whole genome set of orthologous clusters (see 'Orthology delineation' in Materials and methods). Amino acid sequences for these identifiers were downloaded from the Ensembl database release 45 [70]. Multiple alignments of the milk proteins were constructed using MUSCLE [71]. The multiple alignments were then concatenated into a super-alignment that was used to create a maximum likelihood consensus tree with PhyML [72, 73]. The tree was constructed based on the JTT model of amino acid sequence evolution [74] with rates assumed to vary among sites according to a gamma distribution. Support for the tree's nodes is given by 100 bootstraps.
Statistical analysis of PID distributions
A Wilcoxon rank sum test with continuity correction (Mann-Whitney U) from the R programming language was used to determine if the mean of the average PIDs across the seven taxa of each milk and lactation gene set is statistically different from the whole genome. A two-sample Kolmogorov-Smirnov test was used to determine if the average PIDs of milk and mammary genes are drawn from the same distribution as the genome. Significance was determined by a P-value ≤ 0.05.
Evolution analysis along the bovine lineage
For this analysis only, putative orthology was assigned using a three-way reciprocal best BLAST hit approach as implemented in the 'Ortholuge' program [58]. Ortholog sequences for the caseins and alpha-lactalbumin were manually curated. For each gene, dN/dS ratios were estimated from coding sequence alignments of the human-mouse-bovine orthologs by maximum likelihood using the codeml program from PAMLv4 [75]. Two models were implemented to test the statistical significance of variable selective pressures on each lineage. Under the one-ratio model, which acts as the null model (NSsites = 0, model = 0), each lineage was modeled to have the same dN/dS ratio. The ratio is constrained between 0 and 1, and does not allow for the presence of positive selection. The second model is a model of bovine-specific evolution, where the bovine lineage was selected as the 'foreground' lineage and dN/dS was specifically allowed to vary unconstrained on this lineage (model = 2). The two models were compared by likelihood ratio test, calculated from the log likelihood (lnL) values of both models. Twice the difference between lnLmodel 2 and lnLone-ratio was compared with a chi-square distribution to obtain the P-values.
Additional data files
The following additional data are available with the online version of this paper. Additional data file 1 is an Excel file with one spreadsheet listing the genes in each of the milk and mammary gene sets and their annotations. Additional data file 2 is a custom track in BED format for the UCSC Genome Browser, bovine assembly 3.1, that lists the genome locations of the milk protein gene set. Additional data file 3 is a custom track in BED format for the UCSC Genome Browser, bovine assembly 3.1, that lists the genome locations of the virgin mammary gene set. Additional data file 4 is a custom track in BED format for the UCSC Genome Browser, bovine assembly 3.1, that lists the genome locations of the pregnancy mammary gene set. Additional data file 5 is a custom track in BED format for the UCSC Genome Browser, bovine assembly 3.1, that lists the genome locations of the lactation mammary gene set. Additional data file 6 is a custom track in BED format for the UCSC Genome Browser, bovine assembly 3.1, that lists the genome locations of the involution mammary gene set. Additional data file 7 is a custom track in BED format for the UCSC Genome Browser, bovine assembly 3.1, that lists the genome locations of the mastitis mammary gene set. Additional data file 8 is an Excel file that lists genomic locations of curated milk trait QTL. Additional data file 9 is a custom track in BED format for the UCSC Genome Browser, bovine assembly 3.1, that lists genomic locations of curated milk trait QTL. Additional data file 10 is a Word document that provides additional analysis and discussion of milk trait QTL density. Additional data file 11 is an Excel file that lists candidate genes that occur within QTL with one spreadsheet per milk trait. Additional data file 12 is a custom track in BED format for the UCSC Genome Browser, bovine assembly 3.1, that lists genomic locations of candidate genes associated with the 'fat percentage' trait. Additional data file 13 is a custom track in BED format for the UCSC Genome Browser, bovine assembly 3.1, that lists genomic locations of candidate genes associated with the 'fat yield' trait. Additional data file 14 is a custom track in BED format for the UCSC Genome Browser, bovine assembly 3.1, that lists genomic locations of candidate genes associated with the 'milk yield' trait. Additional data file 15 is a custom track in BED format for the UCSC Genome Browser, bovine assembly 3.1, that lists genomic locations of candidate genes associated with the 'protein percentage' trait. Additional data file 16 is a custom track in BED format for the UCSC Genome Browser, bovine assembly 3.1, that lists genomic locations of candidate genes associated with the 'protein yield' trait. Additional data file 17 is an Excel file that lists the significant genomic clusters within each milk and mammary gene set. Additional data file 18 is a custom track in BED format for the UCSC Genome Browser, bovine assembly 3.1, that lists the genome locations of the significant genomic clusters. Additional data file 19 is an Excel file that lists accession numbers of mammalian orthologs of bovine milk protein genes. Additional data file 20 is a Word document that provides more detail on the conservation of milk protein genes in mammals. Additional data file 21 is an Excel spreadsheet that lists the EST libraries that were surveyed for this study, the number of ESTs in each library, a description of the physiological state of the animal and tissue used to derive the library, and, if the library was excluded from this study, the reason for exclusion. Additional data file 22 is a Word document that details the methods used to probe relative mRNA levels of LGB-II, PCYOX1, and ART4 in bovine mammary tissue at different stages of development and the RT-PCR results.
- dN:
rate of non-synonymous substitutions per non-synonymous site
- dS:
rate of synonymous substitutions per synonymous site
expressed sequence tag
Gene Ontology
progestagen-associated endometrial protein
percent identity
quantitative trait loci
University of California at Santa Cruz.
The authors acknowledge Cora J Dillard for editorial assistance with the manuscript, David Adelson for providing milk trait QTL from the Bovine QTL viewer, and Chris Elsik for providing the bovine consensus gene model (GLEAN) sequences. The authors would also like to acknowledge Tom Wheeler, Timothy Reinhardt, and W James Kent for helpful comments. DGL, WFM, and JBG are supported by the International Milk Genomics Consortium, NIEHS Superfund grant P42 ES04699, and by the CHARGE study grant P01 ES11269. MR is funded by the USDA Agricultural Research Service 6250-51000-048. MCN is supported by NIH PO1-HD38129 to MCN. AJM, NJM, KS, and RM are funded by the Foundation for Research Science and Technology from the New Zealand Government. JFM and GR are supported by the California Dairy Research Foundation Project No. 05 MEJ-01-NH to JFM. ASH is supported by the National Human Genome Research Institute 2 P41 HG002371-06. DJL is funded by Genome Canada and Genome BC through the Pathogenomics of Innate Immunity (PI2) project and holds a Postdoctoral Trainee Award from the Michael Smith Foundation for Health Research. EMZ and EVK are supported by the Swiss National Science Foundation 3100A0-112588. RLT is supported by Australia's Cooperative Research Centre for Innovative Dairy Products and the Gardiner Foundation.
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- The Universal Protein Resource (UniProt). Nucleic Acids Res. 2008, D190-D195. 36 Database
- Eisen MB, Spellman PT, Brown PO, Botstein D: Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA. 1998, 95: 14863-14868.PubMedPubMed CentralView ArticleGoogle Scholar
- Caraux G, Pinloche S: PermutMatrix: a graphical environment to arrange gene expression profiles in optimal linear order. Bioinformatics. 2005, 21: 1280-1281.PubMedView ArticleGoogle Scholar
- Flicek P, Aken BL, Beal K, Ballester B, Caccamo M, Chen Y, Clarke L, Coates G, Cunningham F, Cutts T, Down T, Dyer SC, Eyre T, Fitzgerald S, Fernandez-Banet J, Gräf S, Haider S, Hammond M, Holland R, Howe KL, Howe K, Johnson N, Jenkinson A, Kähäri A, Keefe D, Kokocinski F, Kulesha E, Lawson D, Longden I, Megy K, et al: Ensembl 2008. Nucleic Acids Res. 2008, D707-D714. 36 Database
- Edgar RC: MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004, 32: 1792-1797.PubMedPubMed CentralView ArticleGoogle Scholar
- Guindon S, Gascuel O: A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 2003, 52: 696-704.PubMedView ArticleGoogle Scholar
- Guindon S, Lethiec F, Duroux P, Gascuel O: PHYML Online - a web server for fast maximum likelihood-based phylogenetic inference. Nucleic Acids Res. 2005, W557-W559. 33 Web Server
- Jones DT, Taylor WR, Thornton JM: The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci. 1992, 8: 275-282.PubMedGoogle Scholar
- Yang Z: PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol. 2007, 24: 1586-1591.PubMedView ArticleGoogle Scholar
- Jensen RG: Miscellaneous factors affecting composition and volume of human and bovine milks. Handbook of Milk Composition. Edited by: Jensen RG. 1995, Academic Press, Inc, 237-267.View ArticleGoogle Scholar
- Oftedal OT, Iverson SJ: Phylogenetic variation in the gross composition of milks. Handbook of Milk Composition. Edited by: Jensen RG. 1995, Academic Press, Inc, 749-780.View ArticleGoogle Scholar
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This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Family: Icteridae, Blackbirds and Orioles view all from this family
Description ADULT MALE Has blackish plumage overall, but in good light note the bluish violet sheen on much of the body. ADULT FEMALE Has brown plumage overall, darkest on wings and tail, and warmest and palest on throat, chest, and supercilium. JUVENILE Similar to adult female, but with streaking on underparts, and dark eye.
Dimensions Length: Male, 16-17" (41-43 cm); female, 12-13" (30-33 cm)
Habitat Formerly primarily a Mexican species that is now very common in southern U.S., favoring open habitats from farmland to parks and gardens; year-round resident in most parts but range extends north in summer.
Observation Tips Noisy and conspicuous, hence hard to miss.
Voice Song is a strange mix of slurred whistles and electrical static-type sounds, usually ending in a staccato, mechanical rattle; call is a soft tchut.
Discussion Large and raucous, slim-bodied grackle with a long, daggerlike bill. Forms flocks outside breeding season. All birds have relatively long, narrow wings and all adults have a pale iris. Male is larger than female and has a bizarrely long, diamond-shaped tail (appreciably longer than female's tail). Sexes are separable on plumage differences too.

subset: commoncrawl


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This is a repeat of a thread I started on the old forum. Thought it might be worth bringing it up again.
How much do you think a hunter's level of physical fitness impacts their safety and success in the field? And secondly, what kinds of things do you do, if any, to physically prepare yourselves for hunting season.
Personally, I think it is a huge factor and widely overlooked. There is so little written or said about it. Maybe we can change that.


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The Special Education Advisory Committee (SEAC) works with the School Board, administrators, parents and teachers who are responsible for students receiving special education services.
What SEAC Does:
* Advise LCPS of the needs of students with disabilities;
* Assist LCPS in the development of long-range plans, which will provide needed services for children with disabilities;
* Submit reports and recommendations regarding the education of students with disabilities to the School Board;
* Review annually the LCPS updated special education plan and application for federal funding.
How We Do It:
* Informative presentations for parents and educators at the monthly meetings;
* Provide helpful and timely information via the SEAC website HERE and on Facebook
* Recognition for Excellence in Supporting Special Education Awards Program. This year's award ceremony will be held on May 15, 2014;
* Invites each school to appoint a SEAC representative.
* Host a bi-annual Town Hall Meeting to receive input from parents and caregivers, which assists SEAC to assess the needs of LCPS students with disabilities. The next Town Hall will be held in the Spring of 2015.
When Are Meetings?
SEAC meets monthly during the school year on the third Tuesday of each month from 7 to 9 p.m. Meetings are held in Room 102 at the Loudoun County Public Schools (LCPS) Administrative Offices, 2100 Education Court, Ashburn. There is a presentation at each meeting of interest to parents, staff, and educators.
This year's meeting dates and topics are:
September 17, 2013
Superintendent Dr. Edgar B. Hatrick Reflects On Special Education And Moving Forward As Parents, Citizens, and SEAC
October 15, 2013
What Happened At School Today? Effective Home/School Communications
November 19, 2013
Inclusive Practices: Creating A Whole School Culture
December 17, 2013
Behavior Supports From A to Z
January 21, 2014
Building Relationships: How To Work With Your School
February 18, 2014
Best Practices For Conflict Resolutions
March 18, 2014
Nuts & Bolts Of IEP Compliance: Monitoring, Timelines, Procedures, and more
April 22, 2014
Leveled & Postgraduate Transition Planning
May 15, 2014
Award Ceremony and Reception: Recognition for Excellence in Supporting Special Education
May 20, 2014
Social Skills: How To Build Social Skills Into The Curriculum
Presentation topic subject to change, please check back.
The SEAC Representatives for 2013-14 are Aundrea Foster and Julie Hauer.
Who May Attend Meetings?
All meetings are open to the public. SEAC welcomes anyone interested in special education. There is a designated time for public comment during the meeting. This is an opportunity to share successful educational experiences and express general concerns regarding educational issues for students with disabilities.
2013-2014 SEAC Board Members
Lisa Glasgow, Chair, email@example.com
Jodi Folta, Past Chair and Vice Chair, Planning, firstname.lastname@example.com
April Redmon, Vice Chair, Membership, email@example.com
Amy DiBari, Vice Chair, Communications, email@example.com
Megan Wagner, Secretary, firstname.lastname@example.com
Jill Turgeon, Vice Chair, LCPS School Board, firstname.lastname@example.com
Mary Kearney, LCPS Director of Special Education, firstname.lastname@example.com


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Ajith Kumar,Arya,Nayantara,Taapsee Pannu
Music director:
Yuvan Shankar Raja
AARAMBAM Movie Preview:
Aarambam is an upcoming Tamil Action film directed by Vishnuvardhan and starring by Ajith Kumar,Arya,Nayantara,Taapsee Pannu.
AARAMBAM Movie Casts:
Ajith Kumar,Arya,Nayantara,Taapsee Pannu
Aarambam Suggested Tags for Bookmarking:
Aarambam Online Booking, Aarambam movie Tickets, Aarambam Movie Online Booking, Aarambam Online Booking, Aarambam Movie tickets online, Aarambam Release Date , Internet Booking, Film tickets advance online booking, Book Cinema Tickets in Coimbatore, Coimbatore Release Date, Book Aarambam Tickets, Aarambam Theatre showtime, Aarambam Film, Book Aarambam Tickets online, Aarambam Releasing Theatres, Aarambam Releasing Date, Aarambam Online Movie Ticket Booking. Tickets Reservation Coimbatore, Aarambam Tickets Coimbatore Aarambam Release Date Coimbatore, Aarambam Release Date Film Releasing Theatres Coimbatore, Film Releasing Theatres Coimbatore, Aarambam Theatres Coimbatore


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Shuffle off to the patio to grill spicy kabobs that take chicken salad to new heights!
If you don't have the refrigerated potatoes, use fresh new red potatoes. Because the refrigerated potatoes are partially cooked, you'll need to slightly cook the fresh ones. Place 2 cups of new potato wedges in a glass casserole. Cover and microwave on High for 3 to 5 minutes, stirring once, until slightly cooked.
This is a super one-dish entrée salad, so all you need to add is warm garlic bread. Wrap it in foil, then grill for 5 to 10 minutes.
This recipe is based on the popular Buffalo Chicken Wings appetizer that originated at the Anchor Bar in Buffalo, New York.
© 2017 ®/TM General Mills All Rights Reserved


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Deeplinks Blog posts about Content Blocking
For many years, major U.S. entertainment companies have been trying to gain the power to make websites disappear from the Internet at their say-so. The Internet blacklist bills SOPA and PIPA were part of that strategy, along with the Department of Homeland Security's project of seizing websites that someone accused of copyright infringement. Hollywood's quest for more censorship power was on display again today at a House of Representatives committee hearing that was supposed to be discussing reforms at ICANN, the nonprofit organization that oversees the Internet's domain name system.
Two men are going to fight this weekend, and HBO and Showtime have already thrown the first punch in the legal fight over online streaming of the match. Taking advantage of an increasingly abused loophole in copyright law, they have just won a court order requiring a host of third parties to block access to sites that may stream the fight. In other words, if you run a Wi-Fi network (for example, you're a coffee shop) and someone may use your network to watch the Pacquiao/Mayweather fight via unauthorized sources, HBO and Showtime think they can force you to block your customers' access.
Brazil's Marco Civil law contains vigorous language intended to protect free expression, and a stable, secure and neutral network in Brazil. But as we have noted before, such laws must be interpreted and enforced appropriately to be effective. A good Internet law can quickly turn bad if incorrectly or improperly applied.
Every month, TorrentFreak reports on absolutely ridiculous takedown notices issued by copyright holders to Internet service providers related to allegedly infringing content, using the process created by the Digital Millennium Copyright Act (DMCA). This month, TorrentFreak tore apart a series of takedown notices sent to Google by the German-based Total Wipes Music Group targeting, among other things, an EFF webpage describing how to use PGP for Mac OS X—a webpage within our Surveillance Self-Defense guide.
Verizon told the New York Times on Friday that it plans to begin allowing its customers to opt out of its privacy-invasive header injection program. For customers that are aware of the Verizon program and visit the opt-out page, this means they will soon be able to protect themselves against privacy circumvention like Turn's zombie cookie.
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It is Day 6 of spring training, when, for the first time, hitters will face pitchers in live batting practice sessions. The exercise sounds a lot cooler on paper than in practice. Many of the hitters will not swing the bat, instead using the session to stand in the box and get acclimated to recognizing and timing pitches. The younger guys tend to be less patient.
Throughout the spring, we'll take a stab at projecting what the 25-man Opening Day roster will look like. What follows is our first attempt.
- Jimmy Rollins SS (ODDS OF MAKING ROSTER/DL: 100 percent)
- Michael Young 3B (100 percent)
- Chase Utley 2B (100 percent)
- Ryan Howard 1B (100 percent)
- Darin Ruf LF (60 percent)
- Domonic Brown RF (65 percent)
- Erik Kratz C (95 percent)
- Ben Revere CF (100 percent)
- Cole Hamels LHP (100 percent)
- Roy Halladay RHP (100 percent)
- Cliff Lee LHP (100 percent)
- Kyle Kendrick RHP (100 percent)
- John Lannan LHP (95 percent)
- Jonathan Papelbon RHP/CL (100 percent)
- Mike Adams RHP/SU (100 percent)
- Antonio Bastardo LHP/SU (98 percent)
- Chad Durbin RHP/MID (100 percent)
- Jeremy Horst LHP/MID (75 percent)
- Justin De Fratus RHP/MID (50 percent)
- Raul Valdes LHP/MID (50 percent)
- Laynce Nix OF-1B/LHB (95 percent)
- Kevin Frandsen 3B-2B/RHB (95 percent)
- Humberto Quintero C/RHB (50 percent)
- Freddy Galvis SS-2B-3B/SHB (70 percent)
- John Mayberry Jr. OF-1B/RHB (80 percent)
- Delmon Young - OF/RHB (100 percent)
BREAKDOWN: The Phillies sound as if they expect Delmon Young to begin the season on the disabled list, which takes some drama out of an intense outfield battle. . .Mayberry seems likely to make the team as he is out of options and is one of the few members of the organization who has major league experience in center field, although Laynce Nix has played the position and Rule 5 pick Ender Inciarte will look to make the Phillies make a difficult decision on Mayberry. . .Young starting the season on the DL would leave the last few spots in the bullpen as the tightest roster battle. Mike Stutes, Jake Diekman and Phillippe Aumont would be the last three players off the roster in this scenario.


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text:
25 Years of Global Exchange
In 1988, four friends co-founded Global Exchange to fight a worldview based on greed, domination, and unvarnished worship of power. Global Exchange was founded to change the hearts and minds of those lured to believe that corporate profit would trickle down to create wealth for all. We aimed to speak truth to power. We sought to create awareness of what was being called, "global warming." We chose to stand in solidarity with South Africans seeking democracy and with peasant farmers seeking a fair economy.


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2.8: Physical Change
These stunning rock arches in Utah were carved by wind-blown sand. Repeated scouring by the sand wore away the rock, bit by tiny bit, like sandpaper on wood. The bits of rock worn away by the sand still contain the same minerals as they did when they were part of the large rock. They have not changed chemically in any way. Only the size and shape of the rock have changed, from a single large rock to millions of tiny bits of rock. Changes in size and shape are physical changes in matter.
What Is a Physical Change?
A physical change is a change in one or more physical properties of matter without any change in chemical properties. In other words, matter doesn't change into a different substance in a physical change. Examples of physical change include changes in the size or shape of matter. Changes of state—for example, from solid to liquid or from liquid to gas—are also physical changes. Some of the processes that cause physical changes include cutting, bending, dissolving, freezing, boiling, and melting. Four examples of physical change are pictured below. You can learn more about physical changes and why they occur by watching the video at this URL:
http://www.youtube.com/watch?v=RQTcSNVrNUk (13:35)
Q: In the Figure above, what physical changes are occurring?
A: The paper is being cut into smaller pieces, which is changing its size and shape. The ice cubes are turning into a puddle of liquid water because they are melting. This is a change of state. The tablet is disappearing in the glass of water because it is dissolving into particles that are too small to see. The lighthouse is becoming coated with ice as ocean spray freezes on its surface. This is another change of state.
Reversing Physical Changes
When matter undergoes physical change, it doesn't become a different substance. Therefore, physical changes are often easy to reverse. For example, when liquid water freezes to form ice, it can be changed back to liquid water by heating and melting the ice.
Q: Salt dissolving in water is a physical change. How could this change be reversed?
A: The salt water could be boiled until the water evaporates, leaving behind the salt. Water vapor from the boiling water could be captured and cooled. The water vapor would condense and change back to liquid water.
- A physical change in matter is a change in one or more of matter's physical properties. In a physical change, matter may change its size, shape, or state, but its chemical properties do not change.
- Because the chemical properties of matter remain the same in a physical change, a physical change is often easy to reverse.
- physical change: Change in one or more of matter's physical properties.
Watch the video about physical changes at the following URL. Then answer the questions below.
http://www.youtube.com/watch?v=WIOlnXbRpuk (3:03)
- Describe an example of temperature causing a change in the size of matter.
- How is temperature related to changes in the state of matter?
- Define physical change.
- What are some examples of physical change?
- The wood in the Figure below is being cut with a chainsaw. Is this a physical change? Why or why not?
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Bob Long Paintball Guns
Bob Long has been making high end paintball markers for over 10 years that have led to amazing innovations and now standard technology in most high end markers. These markers are poppet markers which makes them highly reliable and accurate while being easy to maintain also to clean. These are all electronic controlled which allows for high rates of fire and allows the marker to have adjustable modes of fire. The current Intimidator the G6R gives players a high end performing marker for intermediate to expert players at a mid range price, these markers are compact, light and fast while giving you an accurate shot that is ball on ball! Pick these up as these are a big favourite to the Paintballgear.ca crew as we have all at least owned or still currently use them today!


text: Looks like memphis is rocking. I got an answer to a question that had been bugging me for a while. The feedback rocks. Having it out there has made me motivated to work on getting it in better shape. I've made some incredible improvements over the last few days. I sort of wish I hadn't released it yet, but I would never have worked on it without releasing it. The next version will be much cooler. It is neat to see all the downloads. I think a lot of isp's probably have already written something like this and Cisco has a gui. I've never seen it though. But I am sure it is megabucks. We'll see. It is nice to have something of mine out there though.


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Although I quit smoking a long time back, I intend to start back up ASAP to help support my President's plan to provide health care for children. I expect all of you to do the same. After all, it's your patriotic duty.
Don't do it for me, do it for the children............the children!..........Think about the children!


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Oh shit!! The bolt gun killer revealed!!!
Did you watch? I don't want to spoil it if you didn't
They say Lucy loved the Kid but did she know that was her/Sara's son?
Can someone describe this show? Repeats come on SyFy all the time, so I've seen a few episodes, never full length but good sized chunks here and there.
Last Friday's 10PM episode of Haven contained scenes of fictitious violence in a high school setting and in light of the recent tragedy, we have decided to reschedule it along with the series' season three finale, originally slated to air this Friday, December 21st at 10PM. The final two episodes of season three of Haven will now air back-to-back on Thursday, January 17th from 8-10 PM (ET/PT) to maintain the integrity of the series' serialized nature
i'm a big sci-fi guy and ok with a lot of out there shite but the episode with the drawings made me lose faith for a couple of days. that was just god awful i mean you touch the paper and shite happens to that person or building or whatever, c'mon now. i had to just shake my head when he touched the top of the church and it broke
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text:
NZ First MPs
Andrea Vance at Stuff reports:
NZ First MPs are privately seething at Richard Prosser's call for Muslims to be banned from Western airlines.
I'm not surprised.
There are some good people in NZ First. I'm no fan of Winston but some of his MPs are trying to be productive Representatives in Parliament. I'm sure they are cringing at the thought of having to explain to their friends and families how they are part of a party where an MP can refer to Muslims coming from Wogistan and calls on anyone who is "or looks like" a Muslim being banned from air travel if they are a young male.
The list MP went to ground yesterday after his remarks were widely publicised and provoked controversy. NZ First leader Winston Peters has refused to sack him and says he doesn't have to apologise.
They should be equally angry at Winston. He has made it impossible for them to distance themselves from the views expressed, as not even the bare minimum of an apology has been given. They have no defence against people saying they are part of a party that effectively allows MPs to label some New Zealanders as wogs and that wogs shouldn't be allowed on our planes.
A NZ First source said its MPs were furious and believed Mr Prosser "acted like a stupid idiot".
Did no one in NZ First think to vet the column before publication. It is common in pretty much every party for columns to be go through the Leader's Office in advance. You can't allow an MP to claim they are a journalist and an MP, and what they write as a journalist is exempt.
And if I was in NZ First, I'd also be annoyed that the Leader knew about the column for three weeks and did nothing.
In a later interview he stood by his views and went on to say that an Israeli airline that bans Muslims is one of the world's safest carriers.
Incidentally this is wrong. They do not ban Muslims. And having travelled to Israel let me tell you the security there is far more rigorous than merely losing a pocket knife as happened to Prosser. It took me over an hour to clear security – and you know I don't "look like a Muslim" so the references to Israel are quite wrong.
"We are a mature party, we don't go round telling people off. After a rational discussion he has changed his mind," Mr Peters said. ". . . We can't take it any further than that. He's had a chance to reflect . . . he admits it was unbalanced."
The comments have gained some attention overseas, being well covered by Australian news organisations and turning up further afield in such publications as the Singapore-based Straits Times and Channel NewsAsia, Dubai-based Gulf News, and the UK edition of the Huffington Post.
I doubt there is another country on Earth where a legislator has actually seriously called for a travel ban on anyone who "looks likes a Muslim", and is young and male.
UPDATE: The spin doctors have now got to Prosser and after saying he stands by what he wrote yesterday, he today said on Nine to Noon he regrets his choice of words (yet defends the use of the word wog!) and that he should have called for a debate on profiling, not a travel ban. There is a world of difference between what he wrote and what he now says he should have written.
He's also trying to distract by saying my release of the story was part of a National strategy to cover up some (unidentified) other issue. Maybe he means the Papal abdication?
As I tweeted this morning, no one in National knew of my blog post before I made it. The only person who knew would be my flatmate after I showed him the article last night. You see, I only purchased a copy of Investigate on Monday afternoon, read the column with disbelief, and wrote my post that night timing it for 10 am Tuesday. So the conspiracy theory is just a desperate attempt to divert attention.
UPDATE 2: A very personal blog post from a Kiwi Muslim on what Prosser said.
I am a Bengali New Zealander. I am also a feminist, a bookworm, a foodie and a cinephile. Some days I have good taste in music and some days I have bad taste in music. I love the beach but hate getting sand everywhere. I speak several languages. I still get upset at the thought that Dumbledore dies. When I lived in Auckland, I thought of West Auckland as home. Now that I live in Wellington, I think of Auckland as home, and of Wellington as "my city," whatever that means. I bake decent brownies (oh the irony).
Oh, yes, I am also a Muslim. …
Richard Prosser's comments hurt because he's saying I'm not a real New Zealander. I am an Other. I am suspect because I don't, undoubtedly, look like his daughters. My little brother, now 20, studying a Bachelor of Commerce, with his terribly cheesy sense of humour and his kind, kind heart, who loves his sports and protein shakes but who I will partly always think of as that bossy toddler with chubby cheeks, is for no other reason than because Mr Prosser, from his position of power and privilege says so, not enough of a New Zealander. Because of only one thing, out of the many things that he is, because he is Muslim. Not enough of a New Zealander to deserve the minimum modicum of respect one would expect from an MP when talking about one of the citizens of this country, simply based on his faith, and, let's be honest, the colour of his skin. Not enough of a New Zealander to get on his Western planes.
A very personal and moving post.
UPDATE3: The Radio NZ audio is embdedded below.