BACKGROUND
after a short gestation period, marsupials give birth to a relatively immature newborn that is totally dependant on milk for growth and development during a relatively long lactation period.
important changes occur during the lactation cycle of marsupials in terms of mammary gland development, milk production, milk composition as well as development or behaviour of the young  <cit> .
by contrast, eutherian mammals have evolved with a larger investment in the development of the young during gestation  <cit>  and, apart from the initial colostrum during the immediate postpartum period, produce milk of a relatively constant composition  <cit> .
marsupial milk provides essential nutrients and putative growth factors for the development of the young and crossfostering experiments have shown that milk controls post-natal development  <cit> .
endocrine and others factors, potentially intrinsic to the mammary gland, are likely to control milk secretion  <cit>  and marsupial milk contains autocrine/paracrine regulators of the mammary gland  <cit> .
in special circumstances macropod marsupials such as the tammar wallaby macropus eugenii and red kangaroo macropus rufus may present asynchronous concurrent lactation, feeding concurrently two young of different ages with milk of different compositions from adjacent mammary glands; a new born pouch young and an few months older animal  <cit> .
however, the molecular control mechanisms of marsupial milk composition are not fully known.
the tammar wallaby  is an annual breeder characterised by a short pregnancy lasting  <dig> days followed by an extended lactation period of about a  <dig> days.
the lactation cycle is divided into  <dig> phases of approximately  <dig> days each based on the sucking pattern of the young  and milk composition.
shortly after birth, the single young weighing only  <dig> mg crawls into the pouch and attaches to one of four teats, each associated with a separate mammary gland.
the chosen teat will provide all the milk during the entire period of lactation with massive growth of the associated glandular tissue while the other three glands do not generally participate in any milk production.
changes in expression levels of milk protein genes have been described for a number of milk proteins in several marsupial species.
in particular, lactation stage specific genes, such as early lactation protein , mid-late whey acidic protein  and late lactation proteins  have been characterised in the tammar and other species  <cit> .
interestingly, with the exception of wap which is also found in milk of many eutherians  <cit>  but not in humans, goat and ewe  <cit> , all of these marsupial phase specific milk proteins as well as another ubiquitous marsupial milk protein, trichosurin  <cit>  have not been found in eutherian milk.
we now report expression of these and other new potential milk protein genes quantified by sampling the mammary transcriptome at specific stages of the tammar lactation cycle.
RESULTS
library construction, est sequencing and annotation
a comprehensive set of cdna libraries was derived from mammary tissue collected throughout the lactation cycle of the tammar:  <dig> stage specific libraries from pregnant day  <dig>  or lactating animals at day  <dig>  and day  <dig> ,  <dig> libraries from day  <dig> of lactation subtracted for major milk genes ,  <dig> libraries from the nonlactating glands  of day  <dig> lactating animals and, a normalized library from a mix of mammary tissues .
a total of  <dig>  expressed sequence tags  were characterized by dna sequencing , including  <dig>  from the normalised  and  <dig>  from lactation phase specific libraries .
blast searches  <cit>   resulted in hit rates of 50% against the non-redundant protein database, 60% against the genome of the american marsupial opossum monodelphis domestica and, 85% against the sequence archive of the macropus eugenii genome trace archive at ncbi.
furthermore, poorly annotated 3'ests often matched 3'non-coding regions of annotated genes from the opossum ensembl genome server  <cit>  with lower e-values.
sequence assembly and gene clustering
est clustering and sequence assembly were used to construct a comprehensive catalogue of transcripts.
the cap <dig> sequence assembly program  <cit>  produced  <dig>  contigs and  <dig>  singletons.
furthermore, blast was used to detect groups of high similarity within contig sequences.
a score cut off value of  <dig> resulted in the grouping of  <dig> contigs into  <dig> super-contigs.
additional validation was performed by inspection of multiple alignments to separate gene family clusters , identify splicing variants  and check for artefacts such as chimeras, incorrect vector clipping error, simple sequences or sequence rearrangements.
the final edited gene index contains  <dig> clusters from  <dig> original cap <dig> contigs and  <dig> validated supercontigs.
during this analysis  <dig> putative splice variants were identified in alpha-casein resulting in the in frame deletion of  <dig>  and  <dig>  amino acid fragments corresponding to short exons in other species.
in addition the alpha-casein c terminal sequence has been improved and found to be much more similar to the possum  sequence than previously reported  <cit> .
automatic contig sequence annotation using blast searches  resulted in similar hit rate of about 60% against a number of databases; the non redundant protein database, the set of genscan protein predictions from the opossum genome, the complete opossum genome sequence and 49% with the ensembl cdna predictions from the human genome.
however, 94% of contig sequences could be retrieved from the partial genome sequence archive of the tammar wallaby.
multi species comparison of mammary transcript sequences
in order to investigate the level of similarity between the tammar wallaby and other vertebrate cdnas, blastn was used to identify best hits in unigene sequence data sets  or ensembl genome cdna predictions from a number of species .
sequence identity distributions of best score hit alignments are represented in figure  <dig>  it can be seen that the distribution of similarity between the tammar and the other marsupial, the opossum monodelpis domestica is much higher  than the similarity between the tammar and any other mammal including eutherian species , the monotreme platypus  and more distantly related vertebrates such as the chicken  and the zebrafish .
in addition, the proportion of tammar contigs mapped across species varies from 23% in zebrafish or 35% in chicken to 59% with the marsupial opossum, while 49% can be mapped to human mrna sequences and 38% in the platypus.
these results have to be considered carefully due to the heuristic nature of alignment selection by the blast algorithm.
however, since only relatively long alignments were retained, this provided an estimate of similarity distributions between tammar mammary transcripts and the genomes of other species and is compatible with other evolutionary evidences  <cit> .
it also suggests that up to 10% of the mammary transcriptome may be marsupial specific  and up to an additional 15% therian or mammalian specific .
in this analysis the platypus results are disregarded because the platypus gene set based on automatic gene prediction is not complete.
estimation of gene expression from lactation stage specific libraries
lactation stage specific mammary transcript frequencies were evaluated from the distribution of ests within each stage specific library.
this approach provides an estimation of the relative abundance of major gene transcripts in the mammary gland which may be sampled multiple times in any cdna library.
figure  <dig> shows the proportion of ests assigned to contigs in each library when ignoring or including an additional  <dig> singletons with high similarity to contig sequences .
inclusion of singletons does not have a significant effect because they mainly represent chimeras associated with highly expressed transcripts.
interestingly, in all libraries from a milk-producing gland, more than 80% of ests are assigned to contigs irrespective of lactation phase  or sampling depth .
remarkably, the high coverage obtained with day  <dig> subtracted libraries is close to 100%, suggesting that a large proportion of the singletons at day  <dig> in other day  <dig> libraries are derived from cloning and sequencing artefacts induced, for the most part, by the high abundance of otherwise subtracted casein and betalactoglobulin milk protein transcripts.
the coverage in glands from pregnant animals was also consistently lower at  <dig> to 78%, and even much lower at 40– <dig> % in unsucked glands as well as in the combined and normalised libraries.
these results show that the sequence data capture a large proportion of the transcript population in lactating glands and suggests a shift of transcriptional complexity during the lactation cycle.
the transcript composition of each cdna library is represented in figure  <dig>
generally, it can be seen that the composition is similar in libraries from related lactation stages or sample preparations despite differences in the sampling depth.
the second observation is that, as previously noted, the proportion of singletons is highest in samples from un-sucked and pregnant glands .
in contrast, all lactating samples contain less than 20% singletons.
conversely, any major transcript in pregnant and un-sucked gland samples does not represent more than a few percent while the major milk protein gene may represent more than 25% of all transcripts in un-substracted lactating samples.
more significantly, in lactating samples, the top  <dig> to  <dig> most abundant genes, corresponding to highly expressed milk protein genes, represent more than 50% of all ests in contrast to no more than 10% in non-lactating samples.
thus, a handful of milk protein genes account for a large proportion of transcripts in lactating samples while the expression of these genes is less dominant in pregnant or un-stimulated gland.
this confirms how the lactating mammary gland is indeed specialised in the production of an increasingly massive amount of milk protein during lactation..
differential gene expression during the lactation cycle estimated from stage specific library sequencing data
in order to identify differentially expressed genes the data were normalized by pooling libraries from identical stages and used to perform a number of statistical tests using library sequence frequencies for a total subset of  <dig> informative super-clusters with at least one est in any of the lactation phase specific libraries.
the subtracted libraries at day  <dig> were kept in a separate pool.
tests were conducted using the ideg <dig> online tool with a p-value >  <dig>  applying the bonferroni correction  <cit> .
the analysis of library sequencing data resulted in the identification of a relatively small subset of  <dig> transcripts with significant differential expression across the lactation cycle of the tammar .
this low number may reflect the limitation of shallow library sequencing and therefore nay not directly represent the number of genes that might be differentially expressed in the mammary gland during lactation.
indeed most of the transcripts identified represent high abundance transcripts that can be grouped in  <dig> sub-sets based on their expression profile: 1) ubiquitous milk protein transcript with higher expression in lactating than pregnant mammary glands, 2) genes with inverse expression profile, high during pregnancy, lower in lactation, 3) transcripts with high expression in mid lactation, only identified using the subtracted libraries at day  <dig> with improved coverage, 4) late lactation specific transcripts, 5) highly expressed transcript isolated from the unsucked gland.
the first set consists of milk proteins more highly expressed in lactating than pregnant mammary glands: beta-casein, beta-lactoglobulin, kappacasein, alpha-lactalbumin, trichosurin , ferritin heavy chain and two new potential tammar milk proteins ptmp- <dig> and ptmp-2-glycam.
surprisingly, ptmp- <dig> seems to be highly specific to the tammar since no similarities could be found in the opossum or other available genome sequences.
significantly, this new predicted  <dig> amino acid open reading frame carries a high scoring secretory signal but blast or psi-blast  <cit>  searches did not retrieve significant public database hit.
peptide analysis did not identify any known conserved motif beside the signal peptide.
however, the partial genome sequence of the tammar wallaby contains sequence reads with high similarity to this transcript confirming the true tammar origin of this new candidate milk protein.
in spite of this, a similar gene is not detected in the genome sequence of the american marsupial opossum and further experiments will be needed to assess the significance of this sequence for marsupial lactation and its distribution in the marsupial lineage.
the other new putative tammar protein ptmp-2-glycam presents no clear similarity to known proteins except for a predicted glycosylation-dependent cell adhesion motif .
interestingly, this glycosylation dependent cell adhesion domain is found in only two characterised proteins with clear relation to milk: lactophorin precursor proteose peptone component  <dig>   <cit>  and glycosylation-dependent cell adhesion molecule  <dig>   <cit> .
glycam- <dig> is expressed in the mammary gland of pregnant and lactating mice as well as human milk cells while pp <dig> has been identified in bovine milk  <cit> .
in addition, expression of glycam- <dig> in mice was positively regulated by prolactin  <cit>  and further analysis has confirmed that bovine pp <dig> and mouse glycam- <dig> are homologous  <cit> .
the second group contains only two genes with inverse expression profile, namely, high during pregnancy, lower in lactation: elp previously described as an early phase specific whey protein in the tammar  <cit> , and cytochrome oxidase subunit i. the third set, which is only identified using the subtracted library at day  <dig>  contains transcripts with high expression in mid-late lactation including the major wap, known to be specifically expressed between day  <dig> and  <dig> of lactation  <cit> , polymeric immunoglobulin receptor, cystatin c and transferrin.
the fourth group represents late lactation specific transcripts containing the extensively characterised marsupial specific late lactation proteins llp-a and llp-b  <cit> .
the fifth group contains highly expressed transcripts isolated from the unsucked gland: galactosidase-beta- <dig>  three non coding transcripts ptnc- <dig>  ptnc- <dig>  and ptnc- <dig> as well as, specific to one of the libraries  <dig>   <dig> s ribosomal rna and two repetitive sequences.
remarkably, three novel non-coding rna-like sequences were identified amongst the  <dig> most abundant transcripts.
ptnc- <dig> is a novel major transcript .
ptnc- <dig> corresponds to a region of the genome that is highly conserved in all mammals including humans and opossum and overlaps the non-coding metastasis-associated lung adenocarcinoma transcript  <dig> .
expression of malat- <dig> has been associated with endometrial stromal sarcoma  <cit> .
ptnc- <dig> is apparently of mitochondrial origin.
in all, these results contain all the presently know tammar milk proteins and are in agreement with published phase-specific expression measurements.
in addition they exhibit two new tammar milk protein candidates ptmp- <dig> and ptmp- <dig>  suggest putative high mid-late lactation expression of a number of immune genes and indicate high expression of non coding transcripts in the 4-days unsucked gland..
massively parallel sequencing of lactating mammary gland at day  <dig> and  <dig> of lactation
to compensate for the limitation of sequencing cdna libraries from lactating mammary tissue with a high proportion of milk protein transcripts, a more comprehensive massive sequencing approach was used.
massively parallel signature sequencing data  <cit>  were obtained from the commercial provider lynx bioscience using polya+ mrna samples from the lactating mammary gland of individual tammar wallabies at day  <dig> and  <dig> of lactation.
this represented  <dig> , <dig> and  <dig> , <dig> 17-base pair sequence signature occurrences respectively from a total of  <dig>  distinct signature sequences including  <dig> signatures common to both samples.
simple clustering of signature sequences was used to compensate for sequencing errors, especially in high abundance transcripts leading to  <dig>  independent signature clusters.
searching for the sequence signatures in the complete est sequence database identified a total of  <dig> independent signature clusters  representing 72%  and 80%  of all mpss counts showing that although only about a quarter of all signature clusters can be identified in the sequence assembly, they represent a large proportion of the transcript population.
this process resulted in a comprehensive annotation of the tammar mammary gene catalogue with expression estimates from mpss data.
the resulting proportional compositions of transcript levels at day  <dig> and  <dig> of lactation are presented in figure  <dig>  showing a profile globally similar to lactating profiles obtained from est sequencing in figure  <dig>  the integrated normalized quantification from est and mpss sequencing data for the most abundant genes identified are shown in additional file  <dig>  a contrast analysis between day  <dig> and  <dig> lactation mpss data was performed using  <dig> est clusters with at least one signature occurrence in any sample.
this resulted in  <dig> transcripts with significant  differences in all tests available and  <dig> additional transcripts passing at least one of the proposed statistical tests.
the use of a much more stringent cut off  still identified  <dig> to  <dig> transcripts.
in total this represents about 60% of the clusters with mpss signature.
these results suggest that significant changes in transcript levels occur in the lactating tissue between day  <dig> and  <dig> of lactation corresponding to the phase at which milk protein production is strongly increased.
this contrast between shallow and deep sequencing results illustrates the effect of sampling on statistical resolution and is mainly influenced by the very small day- <dig> library sequencing sample.
there are also limitations in the mpss approach.
the use of a specific restriction site to target signature sequencing  excludes transcripts missing the restriction sequence.
for example llp-a, a major late lactation milk protein, is not detected in this experiment.
this might have a significant effect on expression level estimations when a high abundance gene is not detectable and missing data for such genes alone may bias the results of statistical tests.
prediction of secretory proteins and expression of the secretome
open reading frames were predicted using estscan <dig> and submitted to the identification of signal peptides by ipsort software  <cit>  or signalp web server  <cit> .
this includes  <dig> ipsort,  <dig> signalp neural network and  <dig> signalp hmm predictions.
supercontig clustering resulted in a total of  <dig> supercontigs predicted by either ipsort or signalp to encode proteins with a signal peptide.
this represented  <dig> ipsort,  <dig> signalp neural network and  <dig> signalp hmm predictions with  <dig> predictions common to all methods.
because different methods failed to identify all the previously described milk proteins, the larger union set of  <dig> predictions obtained by any method were retained for further analysis of the secretome in the mammary gland.
in order to address global expression levels of secreted proteins in the mammary gland during lactation, library sequencing and mpss experiments were combined to estimate the proportion of mammary transcripts encoding secreted proteins.
this is shown in figure  <dig>  in which a steady increase in expression of predicted secreted protein transcripts during lactation can be seen.
this increase in secreted protein expression correlates gene expression levels with growth of the mammary gland, growth of the young, increased milk production and milk protein synthesis, which all steadily increase during the lactation cycle  <cit> .
transcription profile in late pregnancy
in the pregnant mammary gland, milk protein gene expression  estimated from the pool of day  <dig> pregnant samples represent only 6% of transcripts compared to an increasingly massive 45–75% in lactating samples.
in addition, the most abundant transcript identified in this stage corresponds to elp.
however, this transcript is estimated at  <dig> %, also a sharp contrast with much higher 15–20% levels for alpha-casein or beta-lactoglobulin during lactation.
cathelicidin  <dig>  an immune related protein recently identified in the milk of mice and humans  <cit> , is found at levels of 1%.
beside milk protein genes, a number of cytochrome oxidase subunits are consistently detected at relatively high levels , siii 1%).
below 1% abundance, translational machinery , energy metabolism  and immune system components dominate.
these results paint a profile of the late pregnant mammary gland with a dominance of transcriptional machinery, energy metabolism and immune related components, except for a much lower level of milk protein transcripts reflecting how the late pregnant gland is prepared and primed for the rapid synthesis of milk after parturition.
transcription profile in mid lactation
milk protein genes largely predominate in the day- <dig> expression profile: alpha, beta and kappa-caseins, beta-lactoglobulin and trichosurin accounting together for 45% of total transcripts.
other highly expressed transcript represent the new potential milk protein identified above ptmp- <dig> , cytochrome oxidase , cathelicidin  <dig> , a novel putative non coding rna ptnc- <dig> , early lactation protein .
at lower frequencies , the profile is mainly dominated by: ribomosomal proteins, immunological proteins  and, components of energy metabolism .
in addition, two milk proteins typically expressed at high level in late lactation, wap and llp-a, are detected at low levels .
mpss data from a day  <dig> lactating gland shows a similar trend with the major milk protein represented by up to 63% of mpss signatures.
however this also includes higher levels of wap  consistent with the increased expression of wap between day  <dig> to  <dig>  <cit> .
expression of cytochrome c oxidase subunit  <dig>  and a number of hypothetical proteins are also identified with abundance above  <dig> %: a putative protein ptmp- <dig> with high similarity to a hypothetical protein  with homologues of unknown function in many species , the hypothetical new putative tammar specific milk proteins ptmp- <dig> and ptmp-2-glycam discussed above and, a short unassigned 3' est .
ptmp- <dig> is found only in the day  <dig> mpss sample and the normalized library.
tpmp-2-glycam is found in the day  <dig> mpss as well as day  <dig> and pregnant libraries.
ptmp-2-glycam may be a new milk protein expressed specifically during early-mid lactation in the tammar and ptmp- <dig> may play a role in mid lactation.
the remaining mpss profile is again largely dominated by transcription machinery and energy metabolism.
transcription profile in late lactation
interestingly, despite the relatively small number of sequence samples obtained, the day- <dig> library reveals some unique features of marsupial lactation.
out of a total of  <dig> sequences,  <dig>  are included in  <dig> distinct contigs.
three singletons contain chimeric dna with a region highly similar to contigs ,  <dig> are simple sequences probably resulting from poor sequencing while the remaining  <dig> unambiguously represent relatively short 3' cdna regions.
the most abundant transcript is late lactation protein a. expression of this marsupial specific protein with similarity to lipocalins has been shown to be highest in late lactation  <cit> .
llp-a represents 22% of sequences in the day  <dig> library.
the data also show that small but measurable amount of llp-a sequences can be identified in pregnant and day- <dig> lactation samples at low levels varying between  <dig>  to  <dig>  %.
in contrast, llp-b is also present a high expression level at day  <dig>  but, is not detected in other stages.
other milk proteins are beta-lactoglobulin , alpha and beta-caseins , alpha-lactalbumin , kappa-casein  and trichosurin .
expression of all these genes can be detected throughout the lactation cycle.
however, expression levels are consistently much higher in lactating than pregnant samples.
the other two genes consist of cytochrome c oxidase iii which represent  <dig> % at day  <dig> or  <dig> of lactation and  <dig>  % in pregnant samples and, the new putative tammar milk protein sequence  representing 8% of transcript and also expressed at seemingly increasing levels during lactation .
in parallel with sequencing the day- <dig> lactation cdna library, an mpss experiment was conducted on a slightly earlier sample from a day- <dig> lactating gland.
there are also limitations in this approach such as the failure to detect llp-a, a major late lactation milk protein due to absence of a dpnii restriction site.
llp-b is identified at about 1%.
this is consistent with the description of a sharp increase in llp-b expression after day  <dig>  <cit> .
other major milk proteins are found at high levels mostly correlated with the day- <dig> results.
except for alpha-casein which represent 8% of transcripts in the day- <dig> library and 18% in day- <dig> mpss and trichosurin , quantitative estimates for the major milk proteins including beta-lactoglobulin, beta and kappa-caseins, alpha-lactalbumin and transferrin show more comparable normalised levels  in day- <dig> and day- <dig> glands from deep mpss and shallow library sampling.
due to the limited cdna library sampling at day  <dig> it is difficult to extract further information from these data but, mpss data reveals two other genes that are expressed at high level including: the major whey acidic protein wap  known to be specifically expressed in phase 2b of lactation and, a 3' utr  with similarity to the solute carrier family  <dig> phosphate transporter scl20a <dig> , a protein that is likely expressed on the basolateral membranes of polarized epithelial cells, and is likely involved in cellular phosphate homeostasis  <cit> .
transcription profile in unsucked gland
in cdna libraries derived from un-sucked glands  <dig> days after parturition, milk protein transcripts represent  <dig> to  <dig> % of all transcripts, with predominance of betalactoglobulin , ptmp- <dig> , alpha- lactalbumin and alpha-casein , elp and cathelicidin .
important differences are also observed in the two independent library samples.
in general milk protein transcripts are less abundant in library  <dig> and this library is enriched in repetitive elements and ribosomal rna as well as immune response markers.
these observations may reflect stress, a pathological condition or a more advance stage of this sample toward regression of the gland.
high expression of beta-galactosidase  is specific to library  <dig>  this observation suggests carbohydrate degradation may play a role in un-sucked gland, possibly to reduce the contribution of free lactose in regulating milk volume by osmolarity.
this aspect is however controversial as tammar and more generally marsupial milk is poor in lactose, the canonical milk osmol in many mammals  <cit> .
it is noteworthy that the level of free lactose in wallaby milk is higher during the first  <dig> days of lactation when lactose is the main milk carbohydrate.
subsequently lactose declines to low levels when complex oligosaccharides first appear and increase gradually thereafter  <cit> .
this critical 4-day period may account in part for the difference between the two tissue samples taken around day  <dig>  currently, the data do not include equivalent lactating mammary gland at  <dig> days to confirm a specific role of beta-galactosidase in un-stimulated tissue.
nevertheless high expression of beta-galactosidase points to a possible role of sugar degradation in un-sucked glands.
remarkably,  <dig> sequences related to non-coding rna, including ptnc- <dig> , ptnc- <dig> and ptnc- <dig> , are identified at high level in both libraries.
transcripts for ribosomal proteins, immune related and energetic metabolism components were below 1% but dominated this background with differences between the two libraries as discussed above.
these results show slightly higher or comparable levels of milk protein gene transcripts in un-sucked compared to late pregnant glands, except for elp and cathelicidin  <dig> and suggest surprisingly high levels of three enigmatic non-coding rna.
in addition it is suggested beta-galactosidase may play a role in the inhibition of lactation in un-sucked glands.
CONCLUSIONS
cdna sequencing of the lactating tammar wallaby mammary gland mrna has permitted the identification of a large number of tammar genes expressed during lactation providing a catalogue representing about 25% of the tammar genome.
this currently is the largest cdna resource from a marsupial organism as library sequencing has been described in only one other species: the australian northern brown bandicoot  <cit> .
about 60% of cdnas possess significant similarity with genes from other mammalian species and a larger proportion  could be retrieved from the partial tammar wallaby genome sequence allowing for further characterisation of full-length sequences and associated genes.
sequence analysis suggested that about 10% of mammary transcripts might be marsupial specific and 15% mammal specific.
while many milk proteins are conserved between mammals, other major transcripts identified in this study provide extensive illustration for marsupial specific milk protein genes including elp, llp-a, llp-b, trichosurin and the new protein ptmp- <dig>  in addition, an ortholog of the new potential milk protein ptmp- <dig> cannot be recovered from the american marsupial opossum genome sequence representing, together with a number of specific repetitive elements, other examples of lineage specific transcripts.
at the other extreme, ptnc- <dig> a novel non-coding rna is derived from a region of the genome that is ultra-conserved in mammals suggesting an important functional role.
shallow sequencing of lactation phase specific cdna libraries has allowed the estimation of phase-specific gene expression of major mammary transcripts, the characterization of a number of new putative milk proteins including genes apparently unique to the tammar, the identification of phase specific markers and, suggested a role for non-coding rna and carbohydrate degradation during initialisation of lactation.
comparison of expression profiles estimated at different times of the lactation cycle from a combination of deep mpss and shallow cdna digital sequencing experiments revealed that, in general, actively lactating glands are expressing a limited number of common or phase specific milk protein genes at high and increasing levels accounting for up to 60% of all transcripts during late lactation.
in addition, a large proportion of genes represent translational machinery components, immune related product or genes involved in energy production.
not surprisingly, these observations show that the lactating mammary gland is an organ highly specialised in the synthesis of milk.
in addition, the recovery of all previously characterised phase-specific milk proteins and the identification of a new potential phase-specific milk protein genes demonstrate some of the unique aspects of marsupial lactation with significant changes in milk composition during an elongated lactation period.
our observations from the mammary tissue of late pregnant animals have shown how the late pregnant mammary gland is primed for the rapid commencement of milk production after parturition.
similarly, the large increase in protein content of tammar milk during mid to late lactation is accompanied by an increase in the mammary gland expression of genes encoding secretory proteins.
this increased gene expression correlates with growth of the mammary gland, growth of the young, milk production and milk protein synthesis, which all steadily increase during the lactation cycle  <cit> .
this global change of gene expression in the glandular tissue may reflect a combination of changes in cellular gene expression and cell type populations within the tissue.
as the mammary gland size steadily increases during the lactation cycle, the progressive replacement of the stroma by alveolar tissue during the course of pregnancy and lactation, and a marked increase of alveolar size during late lactation have been described  <cit> .
it is not possible to deduce from current data if the increase in relative abundance of milk protein transcripts corresponds to an increase of milk protein gene expression in mammary epithelial cells  only or, an increase in the number and proportion of secretory epithelial cells in the mammary gland during lactation .
our observations result most likely from a combination of these processes.
the combination of cdna and signature digital sequencing methodologies has highlighted some of the caveat and limitations of sequencing approaches for the study of gene expression in the highly specialised mammary gland.
in this tissue, a large dominance of milk protein transcripts makes cdna sequencing a less cost-effective method for gene discovery and produces a significant amount of noise in signature sequencing due to the accumulation of sequencing errors in dominant signatures.
these effects limit the depth and resolution of this approach for the analysis of the larger number of expressed genes.
one advantage of digital sequencing for the estimation of gene expression over differential gene expression estimation by microarray is that it provides a more precise quantitative estimation of relative mrna levels for the most highly expressed genes.
however, our data also include a larger number of ests obtained from a normalised cdna library using a combined set of samples covering the full duration of the lactation cycle.
the expression of many of these genes represented by a single or few est sequences cannot be discussed here but this unique est resource will allow the development of a tammar mammary cdna microarray to assess in more detail the changes in expression of a larger number of genes during lactation and also allow investigation of the molecular, hormonal and cellular mechanisms involved in the adaptation of lactation in the tammar wallaby and other marsupial species.

 after a short gestation period, marsupials give birth to a relatively immature newborn that is totally dependant on milk for growth and development during a relatively long lactation period. important changes occur during the lactation cycle of marsupials in terms of mammary gland development, milk production, milk composition as well as development or behaviour of the young. by contrast, eutherian mammals have evolved with a larger investment in the development of the young during gestation and, apart from the initial colostrum during the immediate postpartum period, produce milk of a relatively constant composition. marsupial milk provides essential nutrients and putative growth factors for the development of the young and crossfostering experiments have shown that milk controls post-natal development. endocrine and others factors, potentially intrinsic to the mammary gland, are likely to control milk secretion and marsupial milk contains autocrine/paracrine regulators of the mammary gland. in special circumstances macropod marsupials such as the tammar wallaby macropus eugenii and red kangaroo macropus rufus may present asynchronous concurrent lactation, feeding concurrently two young of different ages with milk of different compositions from adjacent mammary glands; a new born pouch young and an few months older animal. however, the molecular control mechanisms of marsupial milk composition are not fully known. the tammar wallaby is an annual breeder characterised by a short pregnancy lasting days followed by an extended lactation period of about a days. the lactation cycle is divided into phases of approximately days each based on the sucking pattern of the young and milk composition. shortly after birth, the single young weighing only mg crawls into the pouch and attaches to one of four teats, each associated with a separate mammary gland. the chosen teat will provide all the milk during the entire period of lactation with massive growth of the associated glandular tissue while the other three glands do not generally participate in any milk production. changes in expression levels of milk protein genes have been described for a number of milk proteins in several marsupial species. in particular, lactation stage specific genes, such as early lactation protein, mid-late whey acidic protein and late lactation proteins have been characterised in the tammar and other species. interestingly, with the exception of wap which is also found in milk of many eutherians but not in humans, goat and ewe, all of these marsupial phase specific milk proteins as well as another ubiquitous marsupial milk protein, trichosurin have not been found in eutherian milk. we now report expression of these and other new potential milk protein genes quantified by sampling the mammary transcriptome at specific stages of the tammar lactation cycle.  library construction, est sequencing and annotation a comprehensive set of cdna libraries was derived from mammary tissue collected throughout the lactation cycle of the tammar: stage specific libraries from pregnant day or lactating animals at day and day, libraries from day of lactation subtracted for major milk genes, libraries from the nonlactating glands of day lactating animals and, a normalized library from a mix of mammary tissues. a total of expressed sequence tags were characterized by dna sequencing, including from the normalised and from lactation phase specific libraries. blast searches resulted in hit rates of 50% against the non-redundant protein database, 60% against the genome of the american marsupial opossum monodelphis domestica and, 85% against the sequence archive of the macropus eugenii genome trace archive at ncbi. furthermore, poorly annotated 3'ests often matched 3'non-coding regions of annotated genes from the opossum ensembl genome server with lower e-values. sequence assembly and gene clustering est clustering and sequence assembly were used to construct a comprehensive catalogue of transcripts. the cap sequence assembly program produced contigs and singletons. furthermore, blast was used to detect groups of high similarity within contig sequences. a score cut off value of resulted in the grouping of contigs into super-contigs. additional validation was performed by inspection of multiple alignments to separate gene family clusters, identify splicing variants and check for artefacts such as chimeras, incorrect vector clipping error, simple sequences or sequence rearrangements. the final edited gene index contains clusters from original cap contigs and validated supercontigs. during this analysis putative splice variants were identified in alpha-casein resulting in the in frame deletion of and amino acid fragments corresponding to short exons in other species. in addition the alpha-casein c terminal sequence has been improved and found to be much more similar to the possum sequence than previously reported. automatic contig sequence annotation using blast searches resulted in similar hit rate of about 60% against a number of databases; the non redundant protein database, the set of genscan protein predictions from the opossum genome, the complete opossum genome sequence and 49% with the ensembl cdna predictions from the human genome. however, 94% of contig sequences could be retrieved from the partial genome sequence archive of the tammar wallaby. multi species comparison of mammary transcript sequences in order to investigate the level of similarity between the tammar wallaby and other vertebrate cdnas, blastn was used to identify best hits in unigene sequence data sets or ensembl genome cdna predictions from a number of species. sequence identity distributions of best score hit alignments are represented in figure it can be seen that the distribution of similarity between the tammar and the other marsupial, the opossum monodelpis domestica is much higher than the similarity between the tammar and any other mammal including eutherian species, the monotreme platypus and more distantly related vertebrates such as the chicken and the zebrafish. in addition, the proportion of tammar contigs mapped across species varies from 23% in zebrafish or 35% in chicken to 59% with the marsupial opossum, while 49% can be mapped to human mrna sequences and 38% in the platypus. these results have to be considered carefully due to the heuristic nature of alignment selection by the blast algorithm. however, since only relatively long alignments were retained, this provided an estimate of similarity distributions between tammar mammary transcripts and the genomes of other species and is compatible with other evolutionary evidences. it also suggests that up to 10% of the mammary transcriptome may be marsupial specific and up to an additional 15% therian or mammalian specific. in this analysis the platypus results are disregarded because the platypus gene set based on automatic gene prediction is not complete. estimation of gene expression from lactation stage specific libraries lactation stage specific mammary transcript frequencies were evaluated from the distribution of ests within each stage specific library. this approach provides an estimation of the relative abundance of major gene transcripts in the mammary gland which may be sampled multiple times in any cdna library. figure shows the proportion of ests assigned to contigs in each library when ignoring or including an additional singletons with high similarity to contig sequences. inclusion of singletons does not have a significant effect because they mainly represent chimeras associated with highly expressed transcripts. interestingly, in all libraries from a milk-producing gland, more than 80% of ests are assigned to contigs irrespective of lactation phase or sampling depth. remarkably, the high coverage obtained with day subtracted libraries is close to 100%, suggesting that a large proportion of the singletons at day in other day libraries are derived from cloning and sequencing artefacts induced, for the most part, by the high abundance of otherwise subtracted casein and betalactoglobulin milk protein transcripts. the coverage in glands from pregnant animals was also consistently lower at to 78%, and even much lower at 40– % in unsucked glands as well as in the combined and normalised libraries. these results show that the sequence data capture a large proportion of the transcript population in lactating glands and suggests a shift of transcriptional complexity during the lactation cycle. the transcript composition of each cdna library is represented in figure generally, it can be seen that the composition is similar in libraries from related lactation stages or sample preparations despite differences in the sampling depth. the second observation is that, as previously noted, the proportion of singletons is highest in samples from un-sucked and pregnant glands. in contrast, all lactating samples contain less than 20% singletons. conversely, any major transcript in pregnant and un-sucked gland samples does not represent more than a few percent while the major milk protein gene may represent more than 25% of all transcripts in un-substracted lactating samples. more significantly, in lactating samples, the top to most abundant genes, corresponding to highly expressed milk protein genes, represent more than 50% of all ests in contrast to no more than 10% in non-lactating samples. thus, a handful of milk protein genes account for a large proportion of transcripts in lactating samples while the expression of these genes is less dominant in pregnant or un-stimulated gland. this confirms how the lactating mammary gland is indeed specialised in the production of an increasingly massive amount of milk protein during lactation.. differential gene expression during the lactation cycle estimated from stage specific library sequencing data in order to identify differentially expressed genes the data were normalized by pooling libraries from identical stages and used to perform a number of statistical tests using library sequence frequencies for a total subset of informative super-clusters with at least one est in any of the lactation phase specific libraries. the subtracted libraries at day were kept in a separate pool. tests were conducted using the ideg online tool with a p-value > applying the bonferroni correction. the analysis of library sequencing data resulted in the identification of a relatively small subset of transcripts with significant differential expression across the lactation cycle of the tammar. this low number may reflect the limitation of shallow library sequencing and therefore nay not directly represent the number of genes that might be differentially expressed in the mammary gland during lactation. indeed most of the transcripts identified represent high abundance transcripts that can be grouped in sub-sets based on their expression profile: 1) ubiquitous milk protein transcript with higher expression in lactating than pregnant mammary glands, 2) genes with inverse expression profile, high during pregnancy, lower in lactation, 3) transcripts with high expression in mid lactation, only identified using the subtracted libraries at day with improved coverage, 4) late lactation specific transcripts, 5) highly expressed transcript isolated from the unsucked gland. the first set consists of milk proteins more highly expressed in lactating than pregnant mammary glands: beta-casein, beta-lactoglobulin, kappacasein, alpha-lactalbumin, trichosurin, ferritin heavy chain and two new potential tammar milk proteins ptmp- and ptmp-2-glycam. surprisingly, ptmp- seems to be highly specific to the tammar since no similarities could be found in the opossum or other available genome sequences. significantly, this new predicted amino acid open reading frame carries a high scoring secretory signal but blast or psi-blast searches did not retrieve significant public database hit. peptide analysis did not identify any known conserved motif beside the signal peptide. however, the partial genome sequence of the tammar wallaby contains sequence reads with high similarity to this transcript confirming the true tammar origin of this new candidate milk protein. in spite of this, a similar gene is not detected in the genome sequence of the american marsupial opossum and further experiments will be needed to assess the significance of this sequence for marsupial lactation and its distribution in the marsupial lineage. the other new putative tammar protein ptmp-2-glycam presents no clear similarity to known proteins except for a predicted glycosylation-dependent cell adhesion motif. interestingly, this glycosylation dependent cell adhesion domain is found in only two characterised proteins with clear relation to milk: lactophorin precursor proteose peptone component and glycosylation-dependent cell adhesion molecule. glycam- is expressed in the mammary gland of pregnant and lactating mice as well as human milk cells while pp has been identified in bovine milk. in addition, expression of glycam- in mice was positively regulated by prolactin and further analysis has confirmed that bovine pp and mouse glycam- are homologous. the second group contains only two genes with inverse expression profile, namely, high during pregnancy, lower in lactation: elp previously described as an early phase specific whey protein in the tammar, and cytochrome oxidase subunit i. the third set, which is only identified using the subtracted library at day contains transcripts with high expression in mid-late lactation including the major wap, known to be specifically expressed between day and of lactation, polymeric immunoglobulin receptor, cystatin c and transferrin. the fourth group represents late lactation specific transcripts containing the extensively characterised marsupial specific late lactation proteins llp-a and llp-b. the fifth group contains highly expressed transcripts isolated from the unsucked gland: galactosidase-beta- three non coding transcripts ptnc- ptnc- and ptnc- as well as, specific to one of the libraries s ribosomal rna and two repetitive sequences. remarkably, three novel non-coding rna-like sequences were identified amongst the most abundant transcripts. ptnc- is a novel major transcript. ptnc- corresponds to a region of the genome that is highly conserved in all mammals including humans and opossum and overlaps the non-coding metastasis-associated lung adenocarcinoma transcript. expression of malat- has been associated with endometrial stromal sarcoma. ptnc- is apparently of mitochondrial origin. in all, these results contain all the presently know tammar milk proteins and are in agreement with published phase-specific expression measurements. in addition they exhibit two new tammar milk protein candidates ptmp- and ptmp- suggest putative high mid-late lactation expression of a number of immune genes and indicate high expression of non coding transcripts in the 4-days unsucked gland.. massively parallel sequencing of lactating mammary gland at day and of lactation to compensate for the limitation of sequencing cdna libraries from lactating mammary tissue with a high proportion of milk protein transcripts, a more comprehensive massive sequencing approach was used. massively parallel signature sequencing data were obtained from the commercial provider lynx bioscience using polya+ mrna samples from the lactating mammary gland of individual tammar wallabies at day and of lactation. this represented, and, 17-base pair sequence signature occurrences respectively from a total of distinct signature sequences including signatures common to both samples. simple clustering of signature sequences was used to compensate for sequencing errors, especially in high abundance transcripts leading to independent signature clusters. searching for the sequence signatures in the complete est sequence database identified a total of independent signature clusters representing 72% and 80% of all mpss counts showing that although only about a quarter of all signature clusters can be identified in the sequence assembly, they represent a large proportion of the transcript population. this process resulted in a comprehensive annotation of the tammar mammary gene catalogue with expression estimates from mpss data. the resulting proportional compositions of transcript levels at day and of lactation are presented in figure showing a profile globally similar to lactating profiles obtained from est sequencing in figure the integrated normalized quantification from est and mpss sequencing data for the most abundant genes identified are shown in additional file a contrast analysis between day and lactation mpss data was performed using est clusters with at least one signature occurrence in any sample. this resulted in transcripts with significant differences in all tests available and additional transcripts passing at least one of the proposed statistical tests. the use of a much more stringent cut off still identified to transcripts. in total this represents about 60% of the clusters with mpss signature. these results suggest that significant changes in transcript levels occur in the lactating tissue between day and of lactation corresponding to the phase at which milk protein production is strongly increased. this contrast between shallow and deep sequencing results illustrates the effect of sampling on statistical resolution and is mainly influenced by the very small day- library sequencing sample. there are also limitations in the mpss approach. the use of a specific restriction site to target signature sequencing excludes transcripts missing the restriction sequence. for example llp-a, a major late lactation milk protein, is not detected in this experiment. this might have a significant effect on expression level estimations when a high abundance gene is not detectable and missing data for such genes alone may bias the results of statistical tests. prediction of secretory proteins and expression of the secretome open reading frames were predicted using estscan and submitted to the identification of signal peptides by ipsort software or signalp web server. this includes ipsort, signalp neural network and signalp hmm predictions. supercontig clustering resulted in a total of supercontigs predicted by either ipsort or signalp to encode proteins with a signal peptide. this represented ipsort, signalp neural network and signalp hmm predictions with predictions common to all methods. because different methods failed to identify all the previously described milk proteins, the larger union set of predictions obtained by any method were retained for further analysis of the secretome in the mammary gland. in order to address global expression levels of secreted proteins in the mammary gland during lactation, library sequencing and mpss experiments were combined to estimate the proportion of mammary transcripts encoding secreted proteins. this is shown in figure in which a steady increase in expression of predicted secreted protein transcripts during lactation can be seen. this increase in secreted protein expression correlates gene expression levels with growth of the mammary gland, growth of the young, increased milk production and milk protein synthesis, which all steadily increase during the lactation cycle. transcription profile in late pregnancy in the pregnant mammary gland, milk protein gene expression estimated from the pool of day pregnant samples represent only 6% of transcripts compared to an increasingly massive 45–75% in lactating samples. in addition, the most abundant transcript identified in this stage corresponds to elp. however, this transcript is estimated at %, also a sharp contrast with much higher 15–20% levels for alpha-casein or beta-lactoglobulin during lactation. cathelicidin an immune related protein recently identified in the milk of mice and humans, is found at levels of 1%. beside milk protein genes, a number of cytochrome oxidase subunits are consistently detected at relatively high levels, siii 1%). below 1% abundance, translational machinery, energy metabolism and immune system components dominate. these results paint a profile of the late pregnant mammary gland with a dominance of transcriptional machinery, energy metabolism and immune related components, except for a much lower level of milk protein transcripts reflecting how the late pregnant gland is prepared and primed for the rapid synthesis of milk after parturition. transcription profile in mid lactation milk protein genes largely predominate in the day- expression profile: alpha, beta and kappa-caseins, beta-lactoglobulin and trichosurin accounting together for 45% of total transcripts. other highly expressed transcript represent the new potential milk protein identified above ptmp-, cytochrome oxidase, cathelicidin, a novel putative non coding rna ptnc-, early lactation protein. at lower frequencies, the profile is mainly dominated by: ribomosomal proteins, immunological proteins and, components of energy metabolism. in addition, two milk proteins typically expressed at high level in late lactation, wap and llp-a, are detected at low levels. mpss data from a day lactating gland shows a similar trend with the major milk protein represented by up to 63% of mpss signatures. however this also includes higher levels of wap consistent with the increased expression of wap between day to. expression of cytochrome c oxidase subunit and a number of hypothetical proteins are also identified with abundance above %: a putative protein ptmp- with high similarity to a hypothetical protein with homologues of unknown function in many species, the hypothetical new putative tammar specific milk proteins ptmp- and ptmp-2-glycam discussed above and, a short unassigned 3' est. ptmp- is found only in the day mpss sample and the normalized library. tpmp-2-glycam is found in the day mpss as well as day and pregnant libraries. ptmp-2-glycam may be a new milk protein expressed specifically during early-mid lactation in the tammar and ptmp- may play a role in mid lactation. the remaining mpss profile is again largely dominated by transcription machinery and energy metabolism. transcription profile in late lactation interestingly, despite the relatively small number of sequence samples obtained, the day- library reveals some unique features of marsupial lactation. out of a total of sequences, are included in distinct contigs. three singletons contain chimeric dna with a region highly similar to contigs, are simple sequences probably resulting from poor sequencing while the remaining unambiguously represent relatively short 3' cdna regions. the most abundant transcript is late lactation protein a. expression of this marsupial specific protein with similarity to lipocalins has been shown to be highest in late lactation. llp-a represents 22% of sequences in the day library. the data also show that small but measurable amount of llp-a sequences can be identified in pregnant and day- lactation samples at low levels varying between to %. in contrast, llp-b is also present a high expression level at day but, is not detected in other stages. other milk proteins are beta-lactoglobulin, alpha and beta-caseins, alpha-lactalbumin, kappa-casein and trichosurin. expression of all these genes can be detected throughout the lactation cycle. however, expression levels are consistently much higher in lactating than pregnant samples. the other two genes consist of cytochrome c oxidase iii which represent % at day or of lactation and % in pregnant samples and, the new putative tammar milk protein sequence representing 8% of transcript and also expressed at seemingly increasing levels during lactation. in parallel with sequencing the day- lactation cdna library, an mpss experiment was conducted on a slightly earlier sample from a day- lactating gland. there are also limitations in this approach such as the failure to detect llp-a, a major late lactation milk protein due to absence of a dpnii restriction site. llp-b is identified at about 1%. this is consistent with the description of a sharp increase in llp-b expression after day. other major milk proteins are found at high levels mostly correlated with the day- results. except for alpha-casein which represent 8% of transcripts in the day- library and 18% in day- mpss and trichosurin, quantitative estimates for the major milk proteins including beta-lactoglobulin, beta and kappa-caseins, alpha-lactalbumin and transferrin show more comparable normalised levels in day- and day- glands from deep mpss and shallow library sampling. due to the limited cdna library sampling at day it is difficult to extract further information from these data but, mpss data reveals two other genes that are expressed at high level including: the major whey acidic protein wap known to be specifically expressed in phase 2b of lactation and, a 3' utr with similarity to the solute carrier family phosphate transporter scl20a, a protein that is likely expressed on the basolateral membranes of polarized epithelial cells, and is likely involved in cellular phosphate homeostasis. transcription profile in unsucked gland in cdna libraries derived from un-sucked glands days after parturition, milk protein transcripts represent to % of all transcripts, with predominance of betalactoglobulin, ptmp-, alpha- lactalbumin and alpha-casein, elp and cathelicidin. important differences are also observed in the two independent library samples. in general milk protein transcripts are less abundant in library and this library is enriched in repetitive elements and ribosomal rna as well as immune response markers. these observations may reflect stress, a pathological condition or a more advance stage of this sample toward regression of the gland. high expression of beta-galactosidase is specific to library this observation suggests carbohydrate degradation may play a role in un-sucked gland, possibly to reduce the contribution of free lactose in regulating milk volume by osmolarity. this aspect is however controversial as tammar and more generally marsupial milk is poor in lactose, the canonical milk osmol in many mammals. it is noteworthy that the level of free lactose in wallaby milk is higher during the first days of lactation when lactose is the main milk carbohydrate. subsequently lactose declines to low levels when complex oligosaccharides first appear and increase gradually thereafter. this critical 4-day period may account in part for the difference between the two tissue samples taken around day currently, the data do not include equivalent lactating mammary gland at days to confirm a specific role of beta-galactosidase in un-stimulated tissue. nevertheless high expression of beta-galactosidase points to a possible role of sugar degradation in un-sucked glands. remarkably, sequences related to non-coding rna, including ptnc-, ptnc- and ptnc-, are identified at high level in both libraries. transcripts for ribosomal proteins, immune related and energetic metabolism components were below 1% but dominated this background with differences between the two libraries as discussed above. these results show slightly higher or comparable levels of milk protein gene transcripts in un-sucked compared to late pregnant glands, except for elp and cathelicidin and suggest surprisingly high levels of three enigmatic non-coding rna. in addition it is suggested beta-galactosidase may play a role in the inhibition of lactation in un-sucked glands.  cdna sequencing of the lactating tammar wallaby mammary gland mrna has permitted the identification of a large number of tammar genes expressed during lactation providing a catalogue representing about 25% of the tammar genome. this currently is the largest cdna resource from a marsupial organism as library sequencing has been described in only one other species: the australian northern brown bandicoot. about 60% of cdnas possess significant similarity with genes from other mammalian species and a larger proportion could be retrieved from the partial tammar wallaby genome sequence allowing for further characterisation of full-length sequences and associated genes. sequence analysis suggested that about 10% of mammary transcripts might be marsupial specific and 15% mammal specific. while many milk proteins are conserved between mammals, other major transcripts identified in this study provide extensive illustration for marsupial specific milk protein genes including elp, llp-a, llp-b, trichosurin and the new protein ptmp- in addition, an ortholog of the new potential milk protein ptmp- cannot be recovered from the american marsupial opossum genome sequence representing, together with a number of specific repetitive elements, other examples of lineage specific transcripts. at the other extreme, ptnc- a novel non-coding rna is derived from a region of the genome that is ultra-conserved in mammals suggesting an important functional role. shallow sequencing of lactation phase specific cdna libraries has allowed the estimation of phase-specific gene expression of major mammary transcripts, the characterization of a number of new putative milk proteins including genes apparently unique to the tammar, the identification of phase specific markers and, suggested a role for non-coding rna and carbohydrate degradation during initialisation of lactation. comparison of expression profiles estimated at different times of the lactation cycle from a combination of deep mpss and shallow cdna digital sequencing experiments revealed that, in general, actively lactating glands are expressing a limited number of common or phase specific milk protein genes at high and increasing levels accounting for up to 60% of all transcripts during late lactation. in addition, a large proportion of genes represent translational machinery components, immune related product or genes involved in energy production. not surprisingly, these observations show that the lactating mammary gland is an organ highly specialised in the synthesis of milk. in addition, the recovery of all previously characterised phase-specific milk proteins and the identification of a new potential phase-specific milk protein genes demonstrate some of the unique aspects of marsupial lactation with significant changes in milk composition during an elongated lactation period. our observations from the mammary tissue of late pregnant animals have shown how the late pregnant mammary gland is primed for the rapid commencement of milk production after parturition. similarly, the large increase in protein content of tammar milk during mid to late lactation is accompanied by an increase in the mammary gland expression of genes encoding secretory proteins. this increased gene expression correlates with growth of the mammary gland, growth of the young, milk production and milk protein synthesis, which all steadily increase during the lactation cycle. this global change of gene expression in the glandular tissue may reflect a combination of changes in cellular gene expression and cell type populations within the tissue. as the mammary gland size steadily increases during the lactation cycle, the progressive replacement of the stroma by alveolar tissue during the course of pregnancy and lactation, and a marked increase of alveolar size during late lactation have been described. it is not possible to deduce from current data if the increase in relative abundance of milk protein transcripts corresponds to an increase of milk protein gene expression in mammary epithelial cells only or, an increase in the number and proportion of secretory epithelial cells in the mammary gland during lactation. our observations result most likely from a combination of these processes. the combination of cdna and signature digital sequencing methodologies has highlighted some of the caveat and limitations of sequencing approaches for the study of gene expression in the highly specialised mammary gland. in this tissue, a large dominance of milk protein transcripts makes cdna sequencing a less cost-effective method for gene discovery and produces a significant amount of noise in signature sequencing due to the accumulation of sequencing errors in dominant signatures. these effects limit the depth and resolution of this approach for the analysis of the larger number of expressed genes. one advantage of digital sequencing for the estimation of gene expression over differential gene expression estimation by microarray is that it provides a more precise quantitative estimation of relative mrna levels for the most highly expressed genes. however, our data also include a larger number of ests obtained from a normalised cdna library using a combined set of samples covering the full duration of the lactation cycle. the expression of many of these genes represented by a single or few est sequences cannot be discussed here but this unique est resource will allow the development of a tammar mammary cdna microarray to assess in more detail the changes in expression of a larger number of genes during lactation and also allow investigation of the molecular, hormonal and cellular mechanisms involved in the adaptation of lactation in the tammar wallaby and other marsupial species. 
results show a steady increase in expression of genes coding for secreted protein during the lactation cycle that is associated with high proportion of transcripts coding for milk proteins. sequence data from pregnant and early or late lactating specific cdna libraries and, data from early or late lactation massively parallel sequencing strategies were combined to analyse the variation of milk protein gene expression during the lactation cycle. marsupials, such as the tammar wallaby give birth to a relatively immature newborn and progressive changes in milk composition and milk production regulate early stage development of the young. sequence analysis and sequence assembly were used to construct a comprehensive catalogue of mammary transcripts. in order to investigate gene expression in the marsupial mammary gland during lactation, a comprehensive set of cdna libraries was derived from lactating tissues throughout the lactation cycle of the tammar wallaby. a number of potential new milk proteins or mammary gland remodelling markers, including noncoding rnas have been identified. lactation is an important aspect of mammalian biology and, amongst mammals, marsupials show one of the most complex lactation cycles. in addition, genes involved in immune function, translation and energy or anabolic metabolism are expressed across the lactation cycle. a total of express sequence tags were produced by cdna sequencing.
 lactation is an important aspect of mammalian biology and, amongst mammals, marsupials show one of the most complex lactation cycles. marsupials, such as the tammar wallaby give birth to a relatively immature newborn and progressive changes in milk composition and milk production regulate early stage development of the young.  in order to investigate gene expression in the marsupial mammary gland during lactation, a comprehensive set of cdna libraries was derived from lactating tissues throughout the lactation cycle of the tammar wallaby. a total of express sequence tags were produced by cdna sequencing. sequence analysis and sequence assembly were used to construct a comprehensive catalogue of mammary transcripts. sequence data from pregnant and early or late lactating specific cdna libraries and, data from early or late lactation massively parallel sequencing strategies were combined to analyse the variation of milk protein gene expression during the lactation cycle.  results show a steady increase in expression of genes coding for secreted protein during the lactation cycle that is associated with high proportion of transcripts coding for milk proteins. in addition, genes involved in immune function, translation and energy or anabolic metabolism are expressed across the lactation cycle. a number of potential new milk proteins or mammary gland remodelling markers, including noncoding rnas have been identified. 