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Presents a letter to the editor which reports that X-linked inhibitors of apoptosis protein-deficiency leads to delayed lobuloalveolar development in the mammary gland.

The article reviews the known characteristics of mammary stem and progenitor cells by considering the hierarchy of epithelial cell types in the mammary gland of a mouse model. It attempts to provide insight into the cells of origin in the different subtypes of breast cancer as well as the nature of cancer-propagating cells. Also discussed are the steroid receptor status of mammary stem and progenitor cells and the pathways that have been implicated in regulating their self-renewal and differentiation

LIM-only protein 4 (LMO4) is strongly linked to the progression of breast cancer. Although the mechanisms underlying this phenomenon are not well understood, a role is emerging for LMO4 in regulation of the cell cycle. We determined the solution structure of LMO4 in complex with CtIP (C-terminal binding protein interacting protein)/RBBP8, a tumour suppressor protein that is involved in cell cycle progression, DNA repair and transcriptional regulation. Our data reveal that CtIP and the essential LMO cofactor LDB1 (LIM-domain binding protein 1) bind to the same face on LMO4 and cannot simultaneously bind to LMO4. We hypothesise that overexpression of LMO4 may disrupt some of the normal tumour suppressor activities of CtIP, thereby contributing to breast cancer progression., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

LMO4 belongs to a family of LIM-only transcriptional regulators, the first two members of which are oncoproteins in acute T cell leukemia. We have explored a role for LMO4, initially described as a human breast tumor autoantigen, in developing mammary epithelium and breast oncogenesis. Lmo4 was expressed predominantly in the lobuloalveoli of the mammary gland during pregnancy. Consistent with a role in proliferation, forced expression of this gene inhibited differentiation of mammary epithelial cells. Overexpression of LMO4 mRNA was observed in 5 of 10 human breast cancer cell lines. Moreover, in situ hybridization analysis of 177 primary invasive breast carcinomas revealed overexpression of LMO4 in 56% of specimens. Immunohistochemistry confirmed overexpression in a high percentage (62%) of tumors. These studies imply a role for LMO4 in maintaining proliferation of mammary epithelium and suggest that deregulation of this gene may contribute to breast tumorigenesis.

The nuclear LIM-only (LMO) transcription factors LMO2 and LMO4 play important roles in both normal and leukemic T-cell development. LIM domains are cysteine/histidine-rich domains that contain two structural zinc ions and that function as protein-protein adaptors; members of the LMO family each contain two closely spaced LIM domains. These LMO proteins all bind with high affinity to the nuclear protein LIM domain binding protein 1 (ldb1). The LMO-ldb1 interaction is mediated through the N-terminal LIM domain (LIM1) of LMO proteins and a 38-residue region towards the C-terminus of ldb1 [ldb1(LID)]. Unfortunately, recombinant forms of LMO2 and LMO4 have limited solubility and stability, effectively preventing structural analysis. Therefore, we have designed and constructed a fusion protein in which ldb1(LID) and LIM1 of LMO2 can form an intramolecular complex. The engineered protein, FLIN2 (fusion of the LIM interacting domain of ldb1 and the N-terminal LIM domain of LMO2) has been expressed and purified in milligram quantities. FLIN2 is monomeric, contains significant levels of secondary structure and yields a sharp and well-dispersed one-dimensional (1)H NMR spectrum. The analogous LMO4 protein, FLIN4, has almost identical properties. These data suggest that we will be able to obtain high-resolution structural information about the LMO-ldb1 interactions.

The nuclear LIM domain protein LMO2, a T cell oncoprotein, is essential for embryonic erythropoiesis. LIM-only proteins are presumed to act primarily through protein-protein interactions. We, and others, have identified a widely expressed protein, Ldb1, whose C-terminal 76-residues are sufficient to mediate interaction with LMO2. In murine erythroleukemia cells, the endogenous Lbd1 and LMO2 proteins exist in a stable complex, whose binding affinity appears greater than that between LMO2 and the bHLH transcription factor SCL. However, Ldb1, LMO2, and SCL/E12 can assemble as a multiprotein complex on a consensus SCL binding site. Like LMO2, the Ldb1 gene is expressed in fetal liver and erythroid cell lines. Forced expression of Ldb1 in G1ER proerythroblast cells inhibited cellular maturation, a finding compatible with the decrease in Ldb1 gene expression that normally occurs during erythroid differentiation. Overexpression of the LMO2 gene also inhibited erythroid differentiation. Our studies demonstrate a function for Ldb1 in hemopoietic cells and suggest that one role of the Ldb1/LMO2 complex is to maintain erythroid precursors in an immature state.

The hematopoietic transcription factor GATA-1 is essential for development of the erythroid and megakaryocytic lineages. Using the conserved zinc finger DNA-binding domain of GATA-1 in the yeast two-hybrid system, we have identified a novel, multitype zinc finger protein, Friend of GATA-1 (FOG), which binds GATA-1 but not a functionally inactive mutant lacking the amino (N) finger. FOG is coexpressed with GATA-1 during embryonic development and in erythroid and megakaryocytic cells. Furthermore, FOG and GATA-1 synergistically activate transcription from a hematopoietic-specific regulatory region and cooperate during both erythroid and megakaryocytic cell differentiation. These findings indicate that FOG acts as a cofactor for GATA-1 and provide a paradigm for the regulation of cell type-specific gene expression by GATA transcription factors.

GATA-1, a transcription factor of the 'zinc-finger' family, is required for the development of mature erythroid cells and is also highly expressed in the megakaryocytic and mast cell lineages. The helix-loop-helix gene SCL (or TAL) is expressed in the same three hematopoietic lineages as GATA-1. To explore the role of GATA-1 and SCL in hematopoietic differentiation, we introduced a new expression vector bearing each gene into the early myeloid cell line 416B, which could originally differentiate in vivo along the megakaryocytic and granulocytic lineages. Enforced expression of SCL at high levels did not provoke differentiation, but GATA-1 induced the appearance of megakaryocytes as assessed by morphology, the presence of acetylcholinesterase and a polyploid DNA content. Although GATA-1 is thought to stimulate its own transcription in erythrocytes, expression of the endogenous gene was not increased in the megakaryocytic lines; hence GATA-1 may not be autoregulatory in this lineage. Megakaryocytic differentiation was accompanied by a marked decrease in the myeloid surface marker Mac-1. The absence of mast cell or erythroid differentiation suggests that GATA-1 may not be sufficient to provoke maturation along these lineages or that these pathways are impeded in 416B cells. These results demonstrate that a member of the GATA gene family can act as an important regulator of megakaryocytic differentiation.

Full-length double-stranded cDNA was prepared from purified circular RNA of two new Australian field isolates of citrus exocortis viroid (CEV) using two synthetic oligodeoxynucleotide primers. The cDNA was then cloned into the phage vector M13mp9 for sequence analysis. Sequencing of nine cDNA clones of isolate CEV-DE30 and eleven cDNA clones of isolate CEV-J indicated that both isolates consisted of a mixture of viroid species and led to the discovery of eleven new sequence variants of CEV. These new variants, together with the six reported previously, form two classes of sequence which differ by a minimum of 26 nucleotides in a total of 370 to 375 residues. These two classes correlate with two biologically distinct groups when propagated on tomato plants where one produces severe symptoms and the other gives rise to mild symptoms. Two regions of the native structure of CEV, comprising 18% of the total residues, differ between the sequence variants of mild and severe isolates. Whether or not both of these regions are essential for the variation in pathogenicity has yet to be determined.

The nucleotide sequences of two new Australian isolates of citrus exocortis viroid (CEV), CEV-DE25 and CEV-DE26, have been determined and compared with the previously sequenced Australian isolate, CEV-A (J. E. Visvader, A. R. Gould, G. E. Breuning, and R. H. Symons. FEBS Lett.137, 288-292 (1982).) and a Californian isolate, CEV-C (H. J. Gross, G. Krupp, H. Domdey, M. Raba, P. Jank, C. Lossow, H. Alberty, K. Ramm, and H. L. Sanger. Eur. J. Biochem.121, 249-257 (1982).). Sequencing of recombinant DNA clones prepared from purified CEV-A showed sequence heterogeneity at four nucleotide residues which was not observed by direct RNA sequencing. The data indicated that purified CEV-A contained minor amounts of at least one other CEV species designated CEV-AM. All isolates contain 371 residues. In comparison with the sequence of CEV-A, four residues are different in CEV-C and CEV-DE25 while CEV-DE26 has 27 nucleotide differences which include exchanges, insertions, and deletions. Most of the nucleotide differences in CEV-C, CEV-DE25, and CEV-DE26 alter the proposed secondary structure relative to CEV-A. No nucleotide differences were found to occur in the highly conserved central region previously shown to be present in the predicted native structures of CEV, potato spindle tuber viroid, chrysanthemum stunt viroid, and coconut cadang-cadang viroid.

In order to investigate the mechanism of replication of viroids and virusoids, we have compared the replication intermediates of three members of each group in nucleic acid extracts of infected plants. Viroids were avocado sunblotch viroid (ASBV), citrus exocortis viroid (CEV) and coconut cadang cadang viroid (CCCV). Virusoids were from velvet tobacco mottle virus (VTMoV), solanum nodiflorum mottle virus (SNMV) and lucerne transient streak virus (LTSV). Analysis of intermediates was by the Northern hybridization technique with single-strand DNA and RNA probes prepared from recombinant DNA clones. The results obtained are discussed in terms of current models of viroid and virusoid replication.The plus RNA species consisted of an oligomeric series up to decamers based on the unit of full-length viroid or virusoid, which was always the major component, except for CEV where only monomer and dimer species were found. In the case of ASBV and the virusoids of VTMoV and SNMV, a minor, multimeric series of components (X-bands) was superimposed on the main oligomeric series.The complementary minus species proved more difficult to detect and characterise, with each viroid and virusoid exhibiting a unique pattern on Northern hybridization. However, they all had greater than unit-length minus species. In addition, minus species analogous to the plus X-bands were found in ASBV and CEV. The experimental difficulties encountered in this work are discussed in terms of the problem of detecting minus species by Northern analysis in the presence of excess complementary plus species.

Monomeric cDNA clones of citrus exocortis viroid (CEV) were constructed in the plasmid vector pSP6-4 and the infectivity of the clones plus in vitro-synthesized RNA transcripts determined by inoculation onto tomato seedlings. Infectivity was dependent on the site of the viroid molecule used for cloning and the orientation of the cDNA insert. Only the plus BamHI cDNA clone was infectious and produced progeny viroid with wild-type sequence at the region corresponding to the BamHI cloning site. Infectivity correlated with the terminal repetition of 11 nucleotides of viroid sequence, 5'GGATCCCCGGG 3', in the vector adjacent to the insert. The 11-nucleotide sequence lies within the highly conserved central region of viroids. Site-directed mutagenesis of a single nucleotide in the repeat at the 5'-end of the CEV insert to 5' GGATCCCC(T,A)GG 3' gave two point mutants. The two mutant CEV inserts, when excised from the vector, were not infectious. However, plasmid DNA and RNA transcripts from non-excised mutant CEV inserts were infectious. The progeny of one of these clones was examined and contained wild-type sequence. It was concluded that in vivo processing of longer-than-unit-length CEV occurs at one of three adjacent sites in the 11 nucleotide sequence and that the G nucleotide at position 97 is important for viroid replication.

Viroids are infectious, circular RNA molecules of 246 to 375 nucleotides found in plants. Virusoids are of similar size and structure but they are dependent on, and encapsidated in, a helper virus. A rolling circle mechanism of replication is considered to account for the presence of greater-than-unit-length plus and minus RNAs of both viroids and virusoids found in infected plants. An essential feature of this mechanism is the specific processing or cleavage of high molecular weight intermediates to produce linear monomers which are then ligated to circular monomers. We have investigated the putative processing cleavage reactions using in vitro-synthesized RNA transcripts of dimeric cDNA clones of the 247-nucleotide avocado sunblotch viroid (ASBV) and of partial cDNA clones of the 324-nucleotide virusoid of lucerne transient streak virus (vLTSV). In both cases, there is a specific, non-enzymic, self-cleavage of plus as well as minus transcripts. The plus and minus sites of cleavage are in neighbouring parts of ASBV and of vLTSV and highly conserved two-dimensional structures can be drawn around the cleavage sites as well as around the putative or demonstrated cleavage sites of precursors of the virusoids of three other viruses and of the linear satellite RNA of tobacco ringspot virus. The results also indicate that the sole function of about one-third of the ASBV and vLTSV molecules is provision of sequences that allow the formation of the self-cleavage structures of both 'plus' and 'minus' RNA precursors during the replication cycle. Similar self-cleavage of 'plus' RNA transcripts of a dimeric cDNA clone of citrus exocortis virus (CEV) was not observed. However, the putative processing site for CEV precursors was located within three nucleotides by site-directed mutagenesis. No two-dimensional structures similar to those found for ASBV and vLTSV were found around the processing site. It is possible that a different type of self-cleavage or enzymic processing event occurs during the replication cycle of CEV and related viroids.