Overexpression of DR-nm23, a protein encoded by a member of the nm23 gene family, inhibits granulocyte differentiation and induces apoptosis in 32Dc13 myeloid cells.

Chronic myelogenous leukemia evolves in two clinically distinct stages: a chronic and a blast crisis phase. The molecular changes associated with chronic phase to blast crisis transition are largely unknown. We have identified a cDNA clone, DR-nm23, differentially expressed in a blast-crisis cDNA library, which has approximately 70% sequence similarity to the putative metastatic suppressor genes, nm23-H1 and nm23-H2. The deduced amino acid sequence similarity to the proteins encoded by these two latter genes is approximately 65% and includes domains and amino acid residues (the leucine zipper-like and the RGD domain, a serine and a histidine residue in the NH2- and in the COOH-terminal portion of the protein, respectively) postulated to be important for nm23 function. DR-nm23 mRNA is preferentially expressed at early stages of myeloid differentiation of highly purified CD34+ cells. Its constitutive expression in the myeloid precursor 32Dc13 cell line, which is growth-factor dependent for both proliferation and differentiation, results in inhibition of granulocytic differentiation induced by granulocyte colony-stimulating factor and causes apoptotic cell death. These results are consistent with a role for DR-nm23 in normal hematopoiesis and raise the possibility that its overexpression contributes to differentiation arrest, a feature of blastic transformation in chronic myelogenous leukemia.

Function of the p86 subunit of eukaryotic initiation factor (iso)4F as a microtubule-associated protein in plant cells.

The isozyme form of eukaryotic initiation factor 4F [eIF-(iso)4F] from wheat germ is composed of a p28 subunit that binds the 7-methylguanine cap of mRNA and a p86 subunit having unknown function. The p86 subunit was found to have limited sequence similarity to a kinesin-like protein encoded by the katA gene of Arabidopsis thaliana. Native wheat germ eIF-(iso)4F and bacterially expressed p86 subunit and p86-p28 complex bound to taxol-stabilized maize microtubules (MTs) in vitro. Binding saturation occurred at 1 mol of p86 per 5-6 mol of polymerized tubulin dimer, demonstrating a substoichiometric interaction of p86 with MTs. No evidence was found for a direct interaction of the p28 subunit with MTs. Unlike kinesin, cosedimentation of eIF-(iso)4F with MTs was neither reduced by MgATP nor enhanced by adenosine 5'-[gamma-imido]triphosphate. Both p86 subunit and p86-p28 complex induced the bundling of MTs in vitro. The p86 subunit was immunolocalized to the cytosol in root maize cells and existed in three forms: fine particles, coarse particles, and linear patches. Many coarse particles and linear patches were colocalized or closely associated with cortical MT bundles in interphase cells. The results indicate that the p86 subunit of eIF-(iso)4F is a MT-associated protein that may simultaneously link the translational machinery to the cytoskeleton and regulate MT disposition in plant cells.

Enhancers increase the probability but not the level of gene expression.

We have studied enhancer function in transient and stable expression assays in mammalian cells by using systems that distinguish expressing from nonexpressing cells. When expression is studied in this way, enhancers are found to increase the probability of a construct being active but not the level of expression per template. In stably integrated constructs, large differences in expression level are observed but these are not related to the presence of an enhancer. Together with earlier studies, these results suggest that enhancers act to affect a binary (on/off) switch in transcriptional activity. Although this idea challenges the widely accepted model of enhancer activity, it is consistent with much, if not all, experimental evidence on this subject. We hypothesize that enhancers act to increase the probability of forming a stably active template. When randomly integrated into the genome, enhancers may affect a metastable state of repression/activity, permitting expression in regions that would not permit activity of an isolated promoter.

The human plakoglobin gene localizes on chromosome 17q21 and is subjected to loss of heterozygosity in breast and ovarian cancers.

The gene encoding human plakoglobin was mapped to chromosome 17q12-q22. An intragenic restriction fragment length polymorphism was used to localize the plakoglobin gene distal to locus KRT10 and proximal to the marker D17S858. The plakoglobin gene colocalizes with the polymorphic 17q21 marker UM8 on the same cosmid insert. This subregion of chromosome 17 is known to be particularly subjected to genetic alterations in sporadic breast and ovarian tumors. We show loss of heterozygosity of the plakoglobin gene in breast and ovarian tumors. We have identified a low-frequency polymorphism in the plakoglobin coding sequence which results in an arginine to histidine substitution at amino acid position 142 of the protein, as well as a silent mutation at nucleotide position 332 of the coding sequence. This polymorphism allowed us to demonstrate an allelic association of plakoglobin with predisposition to familial breast and ovarian cancers. Our results, together with the present knowledge about the biological function of plakoglobin, suggest that plakoglobin might represent a putative tumor suppressor gene for breast and ovarian cancers.

PXA1, a possible Saccharomyces cerevisiae ortholog of the human adrenoleukodystrophy gene.

The adrenoleukodystrophy protein (ALDp) is an ATP-binding cassette (ABC) transporter in the human peroxisome membrane. It is defective in X chromosome-linked adrenoleukodystrophy (ALD), a neurodegenerative disorder with impaired peroxisomal oxidation of very long chain fatty acids. We report cloning and characterization of PXA1, a yeast gene encoding a protein (Pxa1p) exhibiting high similarity to ALDp. Disruption of PXA1 results in impaired growth on oleic acid and reduced ability to oxidize oleate. Pxa1p is peroxisome associated; however, in the PXA1 mutant yeast, as in ALD cells, peroxisomes are morphologically intact. Disruption of a second yeast gene, YKL741, which encodes a more distantly related ALDp homolog (Yk174p), in either wild-type or PXA1 mutant yeast, results in a growth phenotype identical to that of the PXA1 mutant. This result suggests that Yk1741p and Pxa1p may be subunits of the same transporter. Sequence analysis of Pxa1p, ALDp, and related ABC transporters reveals a possible fatty acid binding domain and a 14-amino acid EAA-like motif, previously described only in prokaryotes. Because of the similarities in sequence and function, we propose that Pxa1p is the Saccharomyces cerevisiae ortholog of ALDp.

PAK1, a gene that can regulate p53 activity in yeast.

The ability of p53 protein to activate transcription is central to its tumor-suppressor function. We describe a genetic selection in Saccharomyces cerevisiae which was used to isolate a mutant strain defective in p53-mediated transcriptional activation. The defect was partially corrected by overexpression of a yeast gene named PAK1 (p53 activating kinase), which localizes to the left arm of chromosome IX. PAK1 is predicted to encode an 810-aa protein with regions of strong similarity to previously described Ser/Thr-specific protein kinases. PAK1 sequences upstream of the coding region are characteristic of those regulating genes involved in cell cycle control. Expression of PAK1 was associated with an increased specific activity of p53 in DNA-binding assays accompanied by a corresponding increase in transactivation. Thus, PAK1 is the prototype for a class of genes that can regulate the activity of p53 in vivo, and the system described here should be useful in identifying other genes in this class.

Phosphorylation of the human leukemia inhibitory factor (LIF) receptor by mitogen-activated protein kinase and the regulation of LIF receptor function by heterologous receptor activation.

We used a bacterially expressed fusion protein containing the entire cytoplasmic domain of the human leukemia inhibitory factor (LIF) receptor to study its phosphorylation in response to LIF stimulation. The dose- and time-dependent relationships for phosphorylation of this construct in extracts of LIF-stimulated 3T3-L1 cells were superimposable with those for the stimulation of mitogen-activated protein kinase (MAPK). Indeed, phosphorylation of the cytoplasmic domain of the low-affinity LIF receptor alpha-subunit (LIFR) in Mono Q-fractionated, LIF-stimulated 3T3-L1 extracts occurred only in those fractions containing activated MAPK; Ser-1044 served as the major phosphorylation site in the human LIFR for MAPK both in agonist-stimulated 3T3-L1 lysates and by recombinant extracellular signal-regulated kinase 2 in vitro. Expression in rat H-35 hepatoma cells of LIFR or chimeric granulocyte-colony-stimulating factor receptor (G-CSFR)-LIFR mutants lacking Ser-1044 failed to affect cytokine-stimulated expression of a reporter gene under the control of the beta-fibrinogen gene promoter but eliminated the insulin-induced attenuation of cytokine-stimulated gene expression. Thus, our results identify the human LIFR as a substrate for MAPK and suggest a mechanism of heterologous receptor regulation of LIFR signaling occurring at Ser-1044.

Scavenger receptor A gene regulatory elements target gene expression to macrophages and to foam cells of atherosclerotic lesions.

Transcription of the macrophage scavenger receptor A gene is markedly upregulated during monocyte to macrophage differentiation. In these studies, we demonstrate that 291 bp of the proximal scavenger receptor promoter, in concert with a 400-bp upstream enhancer element, is sufficient to direct macrophage-specific expression of a human growth hormone reporter in transgenic mice. These regulatory elements, which contain binding sites for PU.1, AP-1, and cooperating ets-domain transcription factors, are also sufficient to mediate regulation of transgene expression during the in vitro differentiation of bone marrow progenitor cells in response to macrophage colony-stimulating factor. Mutation of the PU.1 binding site within the scavenger receptor promoter severely impairs transgene expression, consistent with a crucial role of PU.1 in regulating the expression of the scavenger receptor gene. The ability of the scavenger receptor promoter and enhancer to target gene expression to macrophages in vivo, including foam cells of atherosclerotic lesions, suggests that these regulatory elements will be of general utility in the study of macrophage differentiation and function by permitting specific modifications of macrophage gene expression.

Misexpression of the white (w) gene triggers male-male courtship in Drosophila.

We report here that the general ectopic expression of a tryptophan/guanine transmembrane transporter gene, white (w), induces male-male courtship in Drosophila. Activation of a hsp-70/miniwhite (mini-w) transgene in mature males results in a marked change in their sexual behavior such that they begin to vigorously court other mature males. In transformant populations containing equal numbers of both sexes, most males participate, thus forming male-male courtship chains, circles, and lariats. Mutations that ablate the w transgene function also abolish this inducible behavior. Female sexual behavior does not appear to be altered by ectopic w expression. By contrast, when exposed to an active homosexual courtship environment, non-transformant males alter their behavior and actively participate in the male-male chaining. These findings demonstrate that, in Drosophila, both genetic and environmental factors play a role in male sexual behavior.

Yeast histone H3 and H4 N termini function through different GAL1 regulatory elements to repress and activate transcription.

Previous work has shown that N-terminal deletions of yeast histone H3 cause a 2- to 4-fold increase in the induction of GAL1 and a number of other genes involved in galactose metabolism. In contrast, deletions at the H4 N terminus cause a 10- to 20-fold decrease in the induction of these same GAL genes. However, H3 and H4 N-terminal deletions each decrease PHO5 induction only 2- to 4-fold. To define the GAL1 gene regulatory elements through which the histone N termini activate or repress transcription, fusions were made between GAL1 and PHO5 promoter elements attached to a beta-galactosidase reporter gene. We show here that GAL1 hyperactivation caused by the H3 N-terminal deletion delta 4-15 is linked to the upstream activation sequence. Conversely, the relative decrease in GAL1 induction caused by the H4N-terminal deletion delta 4-28 is linked to the downstream promoter which contains the TATA element. These data indicate that the H3 N terminus is required for the repression of the GAL1 upstream element, whereas the H4N terminus is required for the activation of the GAL1 downstream promoter element.

Modulation of retinoblastoma gene in normal adult hematopoiesis: peak expression and functional role in advanced erythroid differentiation.

The retinoblastoma (RB) gene specifies a nuclear phosphoprotein (pRb 105), which is a prototype tumor suppressor inactivated in a variety of human tumors. Recent studies suggest that RB is also involved in embryonic development of murine central nervous and hematopoietic systems. We have investigated RB expression and function in human adult hematopoiesis--i.e., in liquid suspension culture of purified quiescent hematopoietic progenitor cells (HPCs) induced by growth factor stimulus to proliferation and unilinage differentiation/maturation through the erythroid or granulocytic lineage. In the initial HPC differentiation stages, the RB gene is gradually induced at the mRNA and protein level in both erythroid and granulopoietic cultures. In late HPC differentiation and then precursor maturation, RB gene expression is sustained in the erythroid lineage, whereas it is sharply downmodulated in the granulocytic series. Functional studies were performed by treatment of HPC differentiation culture with phosphorothioate antisense oligomer targeting Rb mRNA; coherent with the expression pattern, oligomer treatment of late HPCs causes a dose-dependent and selective inhibition of erythroid colony formation. These observations suggest that the RB gene plays an erythroid- and stage-specific functional role in normal adult hematopoiesis, particularly at the level of late erythroid HPCs.

Involvement of the ALL-1 gene in a solid tumor.

Translocations involving chromosome band 11q23, found in 5-10% of human acute leukemias, disrupt the ALL-1 gene. This gene is fused by reciprocal translocation with a variety of other genes in acute lymphoblastic and myelogenous leukemias, and it undergoes self-fusion in acute myeloid leukemias with normal karyotype or trisomy 11. Here we report an alteration of the ALL-1 gene in a gastric carcinoma cell line (Mgc80-3). Characterization of this rearrangement revealed a three-way complex translocation, involving chromosomes 1 and 11, resulting in a partial duplication of the ALL-1 gene. Sequencing of reverse transcription-PCR products and Northern blot analysis showed that only the partially duplicated ALL-1 gene was transcribed, producing an mRNA with exon 8 fused to exon 2. This report of ALL-1 gene rearrangement in a solid tumor suggests that ALL-1 plays a role in the pathogenesis of some solid malignancies. The absence of the normal transcript in this cell line, in association with the loss-of-heterozygosity studies on chromosome 11q23 seen in solid tumors, suggests that ALL-1 is involved in tumorigenesis by a loss-of-function mechanism.

Isolation and characterization of a MAGE gene family in the Xp21.3 region.

A human gene with strong homology to the MAGE gene family located in Xq27-qter has been isolated by using exon-trapping of cosmids in the Xp21.3 region. We have mapped and sequenced cDNA and genomic clones corresponding to this gene, MAGE-Xp, and shown that the last exon contains the open reading frame and is present in a minimum of five copies in a 30-kb interval. MAGE-Xp is expressed only in testis and, unlike the Xq27-qter MAGE genes, it is not expressed in any of 12 different tumor tissues tested. However, the gene and predicted protein structure are conserved, suggesting a similar function. MAGE-Xp is located in the 160-kb critical interval defined for the locus involved in sex determination within Xp21 and is 50 kb distal to the DAX-1 gene, which is responsible for X-chromosome-linked adrenal hypoplasia congenita.

Disruption of the gene for hsp30, an alpha-crystallin-related heat shock protein of Neurospora crassa, causes defects in thermotolerance.

The alpha-crystallin-related heat shock proteins are produced by all eukaryotes, but the role of these proteins in thermoprotection remains unclear. To investigate the function of one of these proteins, we disrupted expression of the single-copy hsp30 gene of Neurospora crassa, using repeat-induced point mutagenesis, and we generated and characterized mutant strains that were deficient in hsp30 synthesis. These strains could grow at high temperature and they acquired thermotolerance from a heat shock. However, the hsp30-defective strains proved to be extremely sensitive to the combined stresses of high temperature and carbohydrate limitation, enforced by the addition of a nonmetabolizable glucose analogue. Under these conditions, their survival was reduced by 90% compared with wild-type cells. This sensitive phenotype was reversed by reintroduction of a functional hsp30 gene into the mutant strains. The mutant cells contained mitochondria from which a 22-kDa protein was readily extracted with detergents, in contrast to its retention by the mitochondria of wild-type cells. Antibodies against hsp30 coimmunoprecipitated a protein also of approximately 22 kDa from wild-type cells. Results of this study suggest that hsp30 may be important for efficient carbohydrate utilization during high temperature stress and that it may interact with other mitochondrial membrane proteins and function as a protein chaperone.

The su(Hw) protein bound to gypsy sequences in one chromosome can repress enhancer-promoter interactions in the paired gene located in the other homolog.

The suppressor of Hairy-wing [su(Hw)] protein exerts a polar effect on gene expression by repressing the function of transcriptional enhancers located distally from the promoter with respect to the location of su(Hw) binding sequences. The directionality of this effect suggests that the su(Hw) protein specifically interferes with the basic mechanism of enhancer action. Moreover, mutations in modifier of mdg4 [mod(mdg4)] result in the repression of expression of a gene when the su(Hw) protein is bound to sequences in the copy of this gene located in the homologous chromosome. This effect is dependent on the presence of the su(Hw) binding region from the gypsy retrotransposon in at least one of the chromosomes and is enhanced by the presence of additional gypsy sequences in the other homology. This phenomenon is inhibited by chromosomal rearrangements that disrupt pairing, suggesting that close apposition between the two copies of the affected gene is important for trans repression of transcription. These results indicate that, in the absence of mod-(mdg4) product, the su(Hw) protein present in one chromosome can act in trans and inactivate enhancers located in the other homolog.