Expression of novel lipocalin-like milk protein gene is developmentally- regulated during lactation in the tammar wallaby, Macropus eugenii Josephine F. Trott a, * , Michael J. Wilson a,1,2 , Russell C. Hovey b,1 , Denis C. Shaw c , Kevin R. Nicholas a a Division of Molecular Biology and Genetics, Victorian Institute of Animal Science, 475 Mickleham Road, Attwood, VIC 3049, Australia b Molecular and Cellular Endocrinology Section, Center for Cancer Research, NCI, NIH, Bethesda, MD 20892-1402, USA c Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, The Australian National University, Canberra, ACT 2600, Australia Received 24 August 2001; received in revised form 9 November 2001; accepted 11 December 2001 Received by A.J. van Wijnen Abstract We have identified a novel whey protein (late lactation protein B; LLPB) that is first secreted in the milk of the tammar wallaby around day 200 of lactation. The LLPB cDNA clone of 843 base pairs encodes a mature protein of 156 amino acids. LLPB shares 65 and 48% nucleotide and deduced amino acid identity, respectively, with a previously identified late lactation protein A (LLPA). Both these proteins share significant amino acid sequence homology with the lipocalin protein family. Expression of the LLPB gene is induced between days 200 and 240 of lactation, in contrast to expression of the LLPA gene, which is induced at around 145 days of lactation. Maximal expression of both genes in mammary explants from tammars at 213 days of lactation required a combination of prolactin, insulin and hydrocortisone. Transcripts of LLPA, LLPB and b -lactoglobulin (TBLG) were localized to the same cells by in situ hybridization. A substantial level of alveolar maturation is required for expression of the LLP genes, unlike TBLG, which is expressed in immature alveoli. We hypothesize that the temporal expression of the LLPB and LLPA genes may be regulated both by endocrine stimuli and factors intrinsic to the mammary gland. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Late lactation protein; Mammary gland; Autocrine regulation; Milk composition; Marsupial 1. Introduction Macropodid marsupials such as the tammar wallaby (Macropus eugenii) have a unique lactation compared to eutherian species. After a short 26.5-day gestation they give birth to an altricial neonate and during the subsequent lactation of 300–350 days all the major components of the milk change significantly, presumably to provide appropri- ate nutrition for the extensive physiological development of the pouch young (Tyndale-Biscoe and Janssens, 1988). In contrast, eutherians give birth to a more developed young (Renfree, 1993), lactate for less time and do not change the composition of their milk during lactation (Green, 1984). Lactation in the tammar wallaby can be divided into four phases: phases 1, 2A, 2B and 3 (Tyndale-Biscoe and Renfree, 1987; Tyndale-Biscoe and Janssens, 1988). Phase 1 encom- passes pregnancy and lactogenesis at parturition. Phase 2 commences at lactogenesis and is divided into phase 2A (days 0–100) when the young is permanently attached to the teat, and phase 2B (days 100–200) when the young is intermittently attached to the teat but permanently in the pouch. The young is entirely dependent on milk during phase 2 of lactation. Phase 3 (days 200–340) commences when the young first exits the pouch and consumes herbage in addition to milk. During the last 100–150 days of lactation the young develops the metabolic capacity to become nutri- tionally independent of milk (Tyndale-Biscoe and Renfree, 1987). Gene 283 (2002) 287–297 0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S0378-1119(01)00883-6 www.elsevier.com/locate/gene Abbreviations: aa, amino acid(s); a-lac, a-lactalbumin; BLG, b-lactoglo- bulin; bp, base pair(s); cDNA, DNA complementary to RNA; cpm, counts per minute; DIG, digoxigenin; ELP, early lactation protein; F, hydrocorti- sone; HPLC, high-performance liquid chromatography; I, insulin; LLP, late lactation protein; PRL, prolactin; SA, serum albumin; SDS, sodium dodecyl sulfate; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; SEM, stan- dard error of the mean; SSC, 0.15 M NaCl/0.015 M Na 3 citrate, pH 7.6; VEGP, von Ebner’s gland protein; WAP, whey acidic protein * Corresponding author. Present address: Room 204, Terrill Hall, Depart- ment of Animal Science, University of Vermont, Burlington, VT 05405, USA. Tel.: 11-802-656-5894; fax: 11-802-656-8196. E-mail address: trottj@mac.com (J.F. Trott). 1 These authors contributed equally to this work. 2 Present address: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.