bluuebunny/crossref_metadata_2025 huggingface dataset:
The carboxy-terminal tail of the homeo domain protein alpha 2 is required for function with a second homeo domain protein.
Authors: Mak A , Johnson A D
Published: June 2007

The homeo domain protein alpha 2 from Saccharomyces cerevisiae has two roles in the a/alpha cell: With MCM1, alpha 2 turns off transcription of a-specific genes; with a1 (a second homeo domain protein), alpha 2 represses transcription of haploid-specific genes. From the carboxy-terminal side of the alpha 2 homeo domain extends an unstructured 22-amino-acid residue tail. In this paper we show that the carboxy-terminal tail of alpha 2 is required for formation of a stable a1/alpha 2-operator complex and is thus required for a1/alpha 2-mediated repression of transcription. In contrast, the tail is dispensable for alpha 2/MCM1-mediated repression. These results indicate that a short, unstructured tail mediates the interaction between two homeo domain proteins.
Establishment of ventral cell fates in the Drosophila embryonic ectoderm requires DER, the EGF receptor homolog.
Authors: Raz E , Shilo B Z
Published: June 2007

The embryonic ectoderm in Drosophila displays a highly organized arrangement of specific structures along the dorsal-ventral axis. To establish this characteristic design, cells must receive instructive cues regarding their position. We present evidence that during stages 8-9 of embryonic development, the Drosophila EGF receptor homolog (DER) is essential for determining the identity of cells within the ventral ectoderm. In the absence of DER activity at this phase, alterations in cell fate are observed: Ventral cells acquire more dorsal fates, as visualized by the expression profile of specific markers. The ventralizing effect of DER appears to function later than that of the dorsalizing dpp pathway, and the spatial overlap between them is minimal. A model for the determination of cell fates along the dorsal-ventral axis involving the two pathways is presented. Some aspects of the mutant ectodermal and CNS phenotypes of the DER locus (faint little ball, flb) resemble the phenotype of mutations from the spitz group. Synergistic interactions between flb and spitz or Star mutations suggest that these genes participate in a common signaling pathway.
Spatial and temporal phosphorylation of a transcriptional activator regulates pole-specific gene expression in Caulobacter.
Authors: Wingrove J A , Mangan E K , Gober J W
Published: June 2007

Polar localization of proteins in the Caulobacter predivisional cell results in the formation of two distinct progeny cells, a motile swarmer cell and a sessile stalked cell. The transcription of several flagellar promoters is localized to the swarmer pole of the predivisional cell. We present evidence that the product of the flbD gene is the transcriptional activator of these promoters. We show that FlbD is distributed in all cell types and in both poles of the predivisional cell. We also demonstrate that FlbD can be phosphorylated, and that a FlbD kinase activity is under cell cycle control. Cells expressing a FlbD mutant that should activate transcription in the absence of phosphorylation, exhibited an alteration in the temporal pattern of flagellin transcription. Furthermore, predivisional cells expressing the mutant FlbD failed to polarly localize flagellin synthesis. We propose that the phosphorylation of FlbD is restricted to the swarmer compartment of the predivisional cell, and serves as the control point for regulating the spatial transcription of flagellar promoters.
Replication-coupled chromatin assembly is required for the repression of basal transcription in vivo.
Authors: Almouzni G , Wolffe A P
Published: June 2007

The chromatin assembly process coupled to DNA synthesis in the Xenopus oocyte nucleus is significantly more repressive toward basal transcription than chromatin assembly on duplex DNA. We show that chromatin assembly concurrent with DNA synthesis over the promoter region itself is causal for repression. However, the trans-activator Gal4-VP16 both relieves repression and activates transcription regardless of the chromatin assembly pathway. This activation is independent of whether Gal4-VP16 addition occurs before or after chromatin assembly. We propose that replication-coupled chromatin assembly represents a general mechanism to direct the efficient repression of basal transcription. However transcription induction by a specific activator, Gal4-VP16, occurs independent of this chromatin-mediated repression.
Cell cycle-regulated nuclear localization of MCM2 and MCM3, which are required for the initiation of DNA synthesis at chromosomal replication origins in yeast.
Authors: Yan H , Merchant A M , Tye B K
Published: June 2007

MCM2 and MCM3 are two genetically interacting and structurally related proteins essential for growth in Saccharomyces cerevisiae. Mutants defective in these proteins affect the stability of minichromosomes in general, but the severity of the defect is dependent on the autonomously replicating sequence (ARS) that drives the replication of that plasmid. In this paper we show by two-dimensional gel electrophoresis that the initiation of DNA synthesis at chromosomal replication origins is also reduced in frequency in these mutants. We show further that the nuclear and subnuclear localizations of the MCM2 and MCM3 proteins are temporally regulated with respect to the cell cycle. These proteins enter the nucleus at the end of mitosis, persist there throughout G1 phase, and disappear from it at the beginning of S phase. Once inside the nucleus, a fraction of the MCM2 and MCM3 proteins becomes tightly associated with DNA. The association of MCM2 and MCM3 with chromatin presumably leads to the initiation of DNA synthesis, and their subsequent disappearance from the nucleus presumably prevents reinitiation of DNA synthesis at replication origins. This temporally and spatially restricted localization of MCM2 and MCM3 in the nucleus may serve to ensure that DNA replication occurs once and only once per cell cycle.
Oligomerization of NTRC at the glnA enhancer is required for transcriptional activation.
Authors: Porter S C , North A K , Wedel A B , Kustu S
Published: June 2007

To activate transcription of the glnA gene, the dimeric NTRC protein (nitrogen regulatory protein C) of enteric bacteria binds to an enhancer located approximately 100 bp upstream of the promoter. The enhancer is composed of two binding sites for NTRC that are three turns of the DNA helix apart. One role of the enhancer is to tether NTRC in high local concentration near the promoter to allow for its frequent interaction with sigma 54 holoenzyme by DNA looping. We have found that a second role of the enhancer is to ensure oligomerization of NTRC into a complex of at least two dimers that is required for transcriptional activation. Formation of this complex is greatly facilitated by a protein-protein interaction between NTRC dimers that is increased when the protein is phosphorylated.
Factors (TAFs) required for activated transcription interact with TATA box-binding protein conserved core domain.
Authors: Zhou Q , Boyer T G , Berk A J
Published: June 2007

TFIID is a multisubunit protein containing the TATA box-binding polypeptide (TBP) and associated factors (TFIID-TAFs) required for activated transcription by RNA polymerase II. TBPs from different eukaryotes contain a highly conserved carboxy-terminal domain and very divergent amino-terminal domains. Earlier studies proposed that the amino-terminal domains of metazoan TBPs are required for activated transcription. However, we report that a human TFIID complex containing an amino-terminal truncated TBP contains all the major TFIID-TAFs and supports in vitro transcriptional stimulation by different classes of activation domains and from a TATA-less promoter. Protein blotting experiments revealed direct interactions between the conserved domain of TBP and the two largest TAFs. The results suggest a model for the interaction of TFIID-TAFs with TBP.
Jun family members are controlled by a calcium-regulated, cyclosporin A-sensitive signaling pathway in activated T lymphocytes.
Authors: Ullman K S , Northrop J P , Admon A , Crabtree G R
Published: June 2007

The octamer-binding transcription factor Oct-1 is involved in a wide variety of cellular processes but appears to lack a strong transcriptional activation domain, suggesting that it functions in the context of other proteins. We demonstrated previously that Oct-1, in association with a 40-kD protein, OAP40, contributes to the induction of interleukin-2 (IL-2), an early activation gene and major growth factor for T lymphocytes. Here we report that amino acid sequences obtained from purified OAP40 are identical to regions within JunD and c-Jun. We demonstrate that each of these Jun family members can participate in a complex that includes Oct-1 and a regulatory element in the IL-2 enhancer. In transient transfections, both JunD and c-Jun can contribute to activation-specific transcription mediated by this antigen receptor response element. These studies reveal a role, distinct from AP-1 activity, for Jun family members that is controlled by a calcium-triggered, cyclosporin A-sensitive mechanism.
Rescue of the tail defect of Brachyury mice.
Authors: Stott D , Kispert A , Herrmann B G
Published: June 2007

The mouse Brachyury (T) gene is required for normal development of axial structures. Embryos homozygous for the T mutation show severe deficiencies in mesoderm formation. They lack the notochord and allantois, have abnormal somites, and die at approximately 10 days postcoitum probably as a result of the allantois defect. Mice heterozygous for the T mutation exhibit a variable short-tailed phenotype. The T gene has been cloned and shown to be expressed in the tissues most strongly affected by the mutation. In this paper, we show that a single-copy transgene representing the wild-type T allele is able to rescue the T-associated tail phenotype. In addition, we show that increasing dosage of the T gene in Tc/+ mice causes an increased extension of the axis. These data show the correlation of the level of T product with the extension of the anteroposterior axis, directly demonstrating the involvement of the T product in this process.
Transgenic mice as a model to study the role of TGF-beta-related molecules in hair follicles.
Authors: Blessing M , Nanney L B , King L E , Jones C M , Hogan B L
Published: June 2007

There is increasing evidence that members of the TGF-beta superfamily are important regulators of epithelial growth and differentiation in vivo. Here, transgenic mice have been used to study the role of the TGF-beta-related growth factors BMP-2 and BMP-4 in hair and whisker development. In the mature hair follicle, BMP-2 transcripts are normally seen only in precortex cells at the base of the hair shaft. In the transgenic mice reported here, BMP-4, a closely related molecule, has been ectopically expressed in the outer root sheath of hair and whisker follicles using an expression vector based on the bovine cytokeratin IV* promoter. In response to transgene expression, both outer root sheath cells below the stem cell compartment and hair matrix cells around the dermal papilla cease proliferation. In addition, the expression pattern of cytokeratin markers is disturbed in some transgenic hair follicles. These results support a model in which members of the TGF-beta superfamily play an active role in the inhibiton of cell proliferation and the onset of expression of trichocyte-specific genes that take place when cells leave the matrix of the follicle and differentiate into shaft cells.
Homology with Saccharomyces cerevisiae RNA14 suggests that phenotypic suppression in Drosophila melanogaster by suppressor of forked occurs at the level of RNA stability.
Authors: Mitchelson A , Simonelig M , Williams C , O'Hare K
Published: June 2007

The suppressor of forked [su(f)] locus of Drosophila melanogaster encodes at least one cell-autonomous vital function. Mutations at su(f) can affect the expression of unlinked genes where retroviral-like transposable elements are inserted. Changes in phenotype are correlated with changes in mRNA profiles, indicating that su(f) affects the production and/or stability of mRNAs. We have cloned the su(f) gene by P-element transposon tagging. Alterations in the DNA map of eight lethal alleles were detected in a 4.3-kb region. P-element-mediated transformation using a fragment including this interval rescued all aspects of the su(f) mutant phenotype. The gene is transcribed to produce a major 2.6-kb RNA and minor RNAs of 1.3 and 2.9 kb, which are present throughout development, being most abundant in embryos, pupae, and adult females. The major predicted gene product is an 84- kD protein that is homologous to RNA14 of Saccharomyces cerevisiae, a vital gene where mutation affects mRNA stability. This suggests that phenotypic modification by su(f) occurs at the level of RNA stability.
Identification and targeted gene disruption of cAR3, a cAMP receptor subtype expressed during multicellular stages of Dictyostelium development.
Authors: Johnson R L , Saxe C L , Gollop R , Kimmel A R , Devreotes P N
Published: June 2007

Extracellular cAMP acts through cell-surface receptors to coordinate the developmental program of Dictyostelium. A cAMP receptor (cAR1), which is expressed during early aggregation, has been cloned and sequenced previously. We have identified a new receptor subtype, cAR3, that has approximately 56% and 69% amino acid identity with cAR1 and cAR2, respectively. cAR1, cAR2, or cAR3 expressed from plasmid in growing Dictyostelium cells can be photoaffinity labeled with 8-N3[32P]cAMP and phosphorylated when stimulated with cAMP. cAR3 RNA was not present during growth but appeared during late aggregation. Its expression peaked at 9 hr and then fell to a reduced level that was maintained until culmination. The expression of cAR3 protein followed a similar pattern, but with a 3-hr lag, and reached a maximum at the mound stage. In contrast, cAR1 protein was expressed predominantly during early aggregation and at low levels during later stages. At their respective peaks of expression, there were approximately 5 x 10(3) cAR3 sites per cell compared with approximately 7 x 10(4) cAR2 sites per cell. The cAR3 gene was disrupted by homologous recombination in several different parental cell lines. Surprisingly, the car3- cell lines display no obvious phenotype.
Stages in the second reaction of pre-mRNA splicing: the final step is ATP independent.
Authors: Horowitz D S , Abelson J
Published: June 2007

We have analyzed pre-mRNA splicing in yeast extracts immunodepleted of the PRP18 protein. We find that while the first step of splicing (cleavage at the 5' splice site, and generation of the exon 1 and lariat intermediates) is unaffected by the absence of PRP18, the second step of splicing (excision of the lariat intron and formation of mRNA) is substantially slower in the absence of PRP18. The splicing intermediates that are formed in the absence of PRP18 can be rapidly chased into products by the addition of purified PRP18 protein. This chasing is not dependent on ATP, implying that ATP is not required during the second cleavage-and-ligation reaction. This result suggests that there are ordered stages within the second step of splicing and that PRP18 acts late in the second step, perhaps during the catalytic step. The ATP independence also supports the idea that this reaction proceeds by a transesterification mechanism.
The role of exon sequences in splice site selection.
Authors: Watakabe A , Tanaka K , Shimura Y
Published: June 2007

Using mouse immunoglobulin mu (IgM) pre-mRNA as the model substrate for in vitro splicing, we have explored the role of exon sequences in splicing. We have found that deletion of the 5' portion of exon M2 of the IgM gene abolishes the splicing of its immediately upstream intron. Splicing was restored when a purine-rich sequence found within the deleted region was reinserted into the deletion construct. This M2 exon sequence was able to stimulate the splicing of a heterologous intron of the Drosophila doublesex pre-mRNA that contains a suboptimal 3' splice site sequence. These results show that the IgM M2 exon sequence functions as a splicing enhancer. We found that the assembly of the early splicing complex is stimulated by the M2 exon sequence. In vitro competition experiments show that this stimulatory effect is mediated by the interaction of some trans-acting factors. Our results suggest that the U1 snRNP is one such factor. We propose that recognition of an enhancer exon sequence by the components of splicing machinery plays a vital role in the selection of splice sites, not only for the IgM pre-mRNA but for other pre-mRNAs. We designate such a sequence as exon recognition sequence (ERS).
Mutation of twins encoding a regulator of protein phosphatase 2A leads to pattern duplication in Drosophila imaginal discs.
Authors: Uemura T , Shiomi K , Togashi S , Takeichi M
Published: June 2007

The Drosophila gene twins was identified through a P-element-induced mutation that caused overgrowth in posterior regions of the wing imaginal disc. Analyses using position-specific markers showed that the inactivation of this locus induced the formation of extra wing blade anlagen in the posterior compartment of the disc. The duplication was mirror symmetrical, and the line of the symmetry did not correspond to any of the known compartment borders. We isolated the twins gene and found that it encoded one of the regulatory subunits of protein phosphatase 2A (PP2A). These results suggest a novel aspect of physiological roles of protein dephosphorylation; that is, the control of PP2A activity is crucial for specification of tissue patterns.
JunB differs from c-Jun in its DNA-binding and dimerization domains, and represses c-Jun by formation of inactive heterodimers.
Authors: Deng T , Karin M
Published: June 2007

JunB differs considerably from c-Jun in its ability to activate AP-1-responsive genes and induce oncogenic transformation. We demonstrate that the decreased ability of JunB to activate gene expression is the result of a small number of amino acid changes between its DNA-binding and dimerization motifs and the corresponding regions of c-Jun. These changes lead to a 10-fold decrease in the DNA-binding activity of JunB. JunB can be converted into a c-Jun-like activator by substituting four amino acids in its DNA-binding and dimerization motifs with the corresponding c-Jun sequences. JunB can also attenuate trans-activation by c-Jun, an activity mediated by its leucine zipper. This ability depends on two glycine residues that decrease the stability of the JunB leucine zipper, resulting in decreased homodimerization and increased heterodimerization. These results illustrate how small changes in primary structure, including chemically conservative changes, can result in functional divergence of two highly related transcriptional regulators.
SWI5 instability may be necessary but is not sufficient for asymmetric HO expression in yeast.
Authors: Tebb G , Moll T , Dowzer C , Nasmyth K
Published: June 2007

Homothallic haploid yeast cells divide to produce a mother cell that switches mating type and a daughter cell that does not. This pattern is the result of HO endonuclease transcription exclusively in mother cells, and there only transiently in late G1 as cells undergo Start. SWI5 encodes an HO transcription factor that is expressed during the S, G2, and M phases of the cell cycle. The lack of synthesis of SWI5 during G1 is essential to prevent HO transcription in daughter cells. Thus, HO must be activated by SWI5 protein synthesized in the previous cell cycle if it is to be properly regulated. SWI5 is inherited by both mother and daughter cells, and we show here that most of it is rapidly degraded during early G1. More stable mutant SWI5 proteins cause daughter cells to switch mating type, suggesting that SWI5 destruction is necessary to prevent HO expression in daughters. We show further that mother cells can still express HO when stimulated to undergo Start after arrest in early G1 for several hours. We propose that a small fraction of the SWI5 protein inherited by mother cells is extremely stable and that the crucial difference between mothers and daughters with regard to HO transcription is their differential ability to sequester SWI5 in a stable form, possibly as a component of transcription complexes on the HO promoter.
Wild-type p53 mediates apoptosis by E1A, which is inhibited by E1B.
Authors: Debbas M , White E
Published: June 2007

Transformation of primary rodent cells by the adenovirus E1A and E1B oncogenes is a two-step process, where E1A-dependent induction of proliferation is coupled to E1B-dependent suppression of programmed cell death (apoptosis). The E1B gene encodes two distinct transforming proteins, the 19K and 55K proteins, both of which independently cooperate with E1A. E1B 19K or 55K protein, or the human Bcl-2 protein, functions to suppress apoptosis and thereby permits transformation with E1A. The E1B 55K protein blocks p53 tumor suppressor protein function, indicating that p53 may mediate apoptosis by E1A. In the mutant conformation, p53 blocked induction of apoptosis by E1A and efficiently cooperated with E1A to transform primary cells. When p53 was returned to the wild-type conformation, E1A+p53 transformants underwent cell death by apoptosis. This induction of apoptosis by conformational shift of p53 from the mutant to the wild-type form was inhibited by expression of the E1B 19K protein. Thus, the p53 protein may function as a tumor suppressor by initiating a cell suicide response to deregulation of growth control by E1A. E1B 19K and 55K proteins provide separate mechanisms that disable the cell suicide pathway of p53.
Oct-2, although not required for early B-cell development, is critical for later B-cell maturation and for postnatal survival.
Authors: Corcoran L M , Karvelas M , Nossal G J , Ye Z S , Jacks T , Baltimore D
Published: June 2007

Oct-2, a POU homeo domain transcription factor, is believed to stimulate B-cell-restricted expression of immunoglobulin genes through binding sites in immunoglobulin gene promoters and enhancers. To determine whether Oct-2 is required for B-cell development or function, or has other developmental roles, the gene was disrupted by homologous recombination. Oct-2-l- mice develop normally but die within hours of birth for undetermined reasons. Mutants contain normal numbers of B-cell precursors but are somewhat deficient in IgM+ B cells. These B cells have a marked defect in their capacity to secrete immunoglobulin upon mitogenic stimulation in vitro. Thus, Oct-2 is not required for the generation of immunoglobulin-bearing B cells but is crucial for their maturation to immunoglobulin-secreting cells and for another undetermined organismal function.
GHF-1-promoter-targeted immortalization of a somatotropic progenitor cell results in dwarfism in transgenic mice.
Authors: Lew D , Brady H , Klausing K , Yaginuma K , Theill L E , Stauber C , Karin M , Mellon P L
Published: June 2007

During pituitary development, the homeo domain protein GHF-1 is required for generation of somatotropes and lactotropes and for growth hormone (GH) and prolactin (PRL) gene expression. GHF-1 mRNA is detectable several days before the emergence of GH- or PRL-expressing cells, suggesting the existence of a somatotropic progenitor cell in which GHF-1 transcription is first activated. We have immortalized this cell type by using the GHF-1 regulatory region to target SV40 T-antigen (Tag) tumorigenesis in transgenic mice. The GHF-Tag transgene caused developmental entrapment of somatotropic progenitor cells that express GHF-1 but not GH or PRL, resulting in dwarfism. Immortalized cell lines derived from a transgenic pituitary tumor maintain the characteristics of the somato/lactotropic progenitor in that they express GHF-1 mRNA and protein yet fail to activate GH or PRL transcription. Using these cells, we identified an enhancer that activates GHF-1 transcription at this early stage of development yet is inactive in cells representing later developmental stages of the somatotropic lineage or in other cell types. These experiments not only demonstrate the potential for immortalization of developmental progenitor cells using the regulatory regions from cell type-specific transcription factor genes but illustrate the power of such model systems in the study of developmental control.
The interplay between multiple enhancer and silencer elements defines the pattern of decapentaplegic expression.
Authors: Huang J D , Schwyter D H , Shirokawa J M , Courey A J
Published: June 2007

The product of the zygotically active decapentaplegic (dpp) gene appears to function as a morphogen that specifies positional information in the dorsal half of the Drosophila embryo. The dorsal-specific transcription of dpp is the key step in establishing a morphogen gradient. We demonstrate here that multiple regions within the second intron of the gene cooperate with one another to generate the wild-type level and pattern of dpp transcription. These regions contain both generalized enhancer elements as well as ventral-specific repressor elements. Placed within the context of heterologous promoters, the intron retains its ability to direct general activation and ventral repression. The ventral specific repression of dpp transcription is directly mediated by binding sites for the dorsal (dl) morphogen in the repressor elements. In contrast with the zerknüllt (zen) ventral repressor element, which contains a few high-affinity dl-binding sites, dpp contains multiple relatively low-affinity sites that function together to bring about complete ventral repression. Because dpp and zen have nearly coincident early expression domains, these results indicate that the same boundary of repression can be specified by dl-binding sites of different affinity. We discuss the possibility that unknown factors interact with dl protein to determine the domain of dl-mediated repression.
LYAR, a novel nucleolar protein with zinc finger DNA-binding motifs, is involved in cell growth regulation.
Authors: Su L , Hershberger R J , Weissman I L
Published: June 2007

A cDNA encoding a novel zinc finger protein has been isolated from a mouse T-cell leukemia line on the basis of its expression of a Ly-1 epitope in a lambda gt11 library. The putative gene was mapped on mouse chromosome 1, closely linked to Idh-1, but not linked to the Ly-1 (CD5) gene. The cDNA is therefore named Ly-1 antibody reactive clone (LYAR). The putative polypeptide encoded by the cDNA consists of 388 amino acids with a zinc finger motif and three copies of nuclear localization signals. Antibodies raised against a LYAR fusion protein reacted with a protein of 45 kD on Western blots and by immunoprecipitation. Immunolocalization indicated that LYAR was present predominantly in the nucleoli. The LYAR mRNA was not detected in brain, thymus, bone marrow, liver, heart, and muscle. Low levels of LYAR mRNA were detected in kidney and spleen. However, the LYAR gene was expressed at very high levels in immature spermatocytes in testis. The LYAR mRNA is present at high levels in early embryos and preferentially in fetal liver and fetal thymus. A number of B- and T-cell leukemic lines expressed LYAR at high levels, although it was not detectable in bone marrow and thymus. During radiation-induced T-cell leukemogenesis, high levels of LYAR were expressed in preleukemic thymocytes and in acute T leukemia cells. Fibroblast cells overexpressing the LYAR cDNA from a retrovirus vector, though not phenotypically transformed in vitro, had increased ability to form tumors in nu/nu mice. Therefore, LYAR may function as a novel nucleolar oncoprotein to regulate cell growth.
Myb and NF-M: combinatorial activators of myeloid genes in heterologous cell types.
Authors: Ness S A , Kowenz-Leutz E , Casini T , Graf T , Leutz A
Published: June 2007

The c-Myb transcription factor regulates the differentiation of immature erythroid, lymphoid, and myeloid cells, although only the latter cells become transformed by the v-myb oncogene. These are also the only cells that express the Myb-regulated gene mim-1, suggesting that Myb requires tissue-specific, cooperating factors to activate such genes. Here, we investigated the tissue-specific regulation of the mim-1 promoter and found that it not only contains binding sites for Myb but also for NF-M, a myeloid-specific transcription factor that probably corresponds to mammalian C/EBP beta. Both types of binding sites were found to be required for full activity of the promoter. Remarkably, ectopic coexpression of Myb and NF-M proteins in erythroid cells or fibroblasts was sufficient to induce endogenous markers of myeloid differentiation, like the mim-1 and lysozyme genes. Our results indicate that c-Myb and NF-M proteins act as a bipartite, combinatorial signal that regulates the expression of myeloid-specific genes, even in heterologous cell types.
Overexpression of the maize homeo box gene, KNOTTED-1, causes a switch from determinate to indeterminate cell fates.
Authors: Sinha N R , Williams R E , Hake S
Published: June 2007

The KNOTTED-1 (KN1) locus of maize is defined by dominant mutations that affect leaf cell fates. Transposon tagging led to the isolation of the gene and the discovery that KN1 encodes a homeo domain. Immunolocalization studies showed that in wild-type maize plants, KN1 protein is present in nuclei of apical meristems and immature shoot axes but is down-regulated as lateral organs, such as leaves, are initiated. The protein is not immunohistochemically detectable in wild-type leaves at any stage. In developing leaves of plants carrying the dominant Kn1 mutation, temporally and spatially restricted ectopic expression of KN1 causes the mutant phenotype. To better understand the function of KN1 in plant development, we sought to determine the phenotype of plants in which KN1 was constitutively expressed. We find that tobacco plants transformed with the KN1 cDNA driven by the CaMV 35S promoter have a dramatically altered phenotype. The phenotypes are variable and depend on the level of KN1 protein. Plants expressing moderate levels of KN1 are reduced in stature with rumpled or lobed leaves. Plants with relatively high levels of KN1 lack apical dominance and are severely dwarfed in overall height and leaf size. Small shoots originate from the surface of these diminutive leaves. On the basis of phenotypes in maize and tobacco, we propose that the KN1 homeo box gene plays a role in determining cell fate. The consequences of KN1 overexpression appear to depend on the concentration of KN1 and the timing of its expression during organogenesis.
The segment identity functions of Ultrabithorax are contained within its homeo domain and carboxy-terminal sequences.
Authors: Chan S K , Mann R S
Published: June 2007

Using an in vivo assay for segment identity, the structural differences that distinguish two Drosophila homeotic selector proteins, Ultrabithorax (Ubx) and Antennapedia (Antp), have been investigated. There are at least two independent parts of Ubx and Antp that contribute to their functional specificities: (1) their homeo domains and (2) residues carboxy-terminal to their homeo domains (C-tails). In the absence of any C-tail, differences in 5 homeo domain amino acids are sufficient to distinguish between the functions of Ubx and Antp. Two of these are at the amino terminus of the homeo domain and could contact DNA directly. A three dimensional model suggests that the other 3 homeo domain residues and the C-tails are unlikely to contact DNA. In addition, we demonstrate that the assay used to measure the segment identity functions of Ubx and Antp is independent of any homeotic selector gene normally active in thoracic and abdominal segments. Therefore, it is likely that this assay measures the coordinate regulation of many downstream target genes. This expectation is confirmed for at least one Ubx target gene, Distal-less.
GAL4 disrupts a repressing nucleosome during activation of GAL1 transcription in vivo.
Authors: Axelrod J D , Reagan M S , Majors J
Published: June 2007

Photofootprinting in vivo of GAL1 reveals an activation-dependent pattern between the UASG and the TATA box, in a sequence not required for transcriptional activation by GAL4. The pattern results from a nucleosome whose position depends on sequences within the UASG. In the wild-type gene, activation by GAL4 and derivatives disrupts this nucleosome. This activity is independent of interactions with DNA-bound core transcription factors and is proportional to the strength of the activator. Presence of the nucleosome correlates with low basal transcription levels under various conditions, suggesting a role in limiting basal expression. We propose a role for the GAL4 activation domain in displacing a nucleosome and suggest that this is part of the mechanism by which GAL4 activates transcription in vivo.
Multiple extracellular signals govern the production of a morphogenetic protein involved in aerial mycelium formation by Streptomyces coelicolor.
Authors: Willey J , Schwedock J , Losick R
Published: June 2007

The formation of an aerial mycelium by the filamentous bacterium Streptomyces coelicolor is determined in part by a small morphogenetic protein called SapB. A collection of representative bald (bld) mutants, which are blocked in aerial mycelium formation, are all defective in the production of this protein and regain the capacity to undergo morphological differentiation when SapB is supplied exogenously. We now report that most of the bld mutants are rescued for SapB production and aerial mycelium formation when grown near certain other bld mutants. Extracellular complementation experiments of this kind indicate that morphological differentiation is governed by a hierarchical cascade of at least four kinds of intercellular signals. At least one such signal is present in conditioned medium. It is resistant to boiling and protease treatment, and it remains effective even when diluted up to eightfold in fresh medium.
Phosphorylation of tobacco mosaic virus cell-to-cell movement protein by a developmentally regulated plant cell wall-associated protein kinase.
Authors: Citovsky V , McLean B G , Zupan J R , Zambryski P
Published: June 2007

In host plants, cell-to-cell spread of tobacco mosaic virus (TMV) presumably occurs through intercellular connections, the plasmodesmata. TMV movement is mediated by a specific virus-encoded single-strand nucleic acid-binding protein, P30. The mechanism by which P30 operates is largely unknown. Here, we demonstrate that P30 expressed in transgenic plants is a phosphoprotein. We have developed an assay for in vitro phosphorylation of purified P30 by plant cell wall fractions and have localized the phosphorylation sites to amino acid residues Ser-258, Thr-261, and Ser-265. Interestingly, the P30 phosphorylation sites do not correspond to any known consensus phosphorylation sites for protein kinases. While P30 binding to single-stranded DNA (ssDNA) was shown to involve Thr-261, phosphorylation of this residue does not appear to play a role in binding activity. The protein kinase activity contained in the cell wall fractions was developmentally regulated, expressed predominantly in leaves. Within a leaf, this protein kinase activity increased with leaf maturation and correlated with the reported development of secondary plasmodesmata, sites of P30 accumulation. We suggest that phosphorylation may represent a mechanism for the host plant to sequester P30 following its localization to cell walls.
A functional association between the 5' and 3' splice site is established in the earliest prespliceosome complex (E) in mammals.
Authors: Michaud S , Reed R
Published: June 2007

The earliest detectable mammalian prespliceosome complex (E) contains the non-snRNP splicing factor U2AF, U1 snRNP, and several spliceosome-associated proteins (SAPs). We show that specific complexes, designated E3' and E5', assemble independently on RNAs containing only a 3' or 5' splice site, respectively. U2AF is enriched in E3', whereas U1 snRNP is enriched in E5'. Using a highly sensitive substrate-competition assay, we show that both the 5' splice site and the pyrimidine tract at the 3' splice site are required for efficient E complex assembly on intact pre-mRNA. We conclude that the 5' and 3' splice sites are associated functionally as early as E complex by either direct or indirect interactions between U1 snRNP and U2AF. Our observations predict that E complex assembly is a major control point for establishing splice site selection in both constitutively and alternatively spliced pre-mRNAs.
Negative regulation of mitosis by the fission yeast protein phosphatase ppa2.
Authors: Kinoshita N , Yamano H , Niwa H , Yoshida T , Yanagida M
Published: June 2007

To understand the role of the type 2A-like protein phosphatase in the cell division cycle, we investigated the mutant phenotypes obtained when the fission yeast ppa1+ and ppa2+ phosphatase genes (which encode polypeptides with approximately 80% identity to mammalian type 2A phosphatases) were either deleted or overexpressed. We also investigated the in vivo effect of okadaic acid, an inhibitor of protein serine/threonine phosphatases, on cell division. We show that ppa2+ interacts genetically with the cell cell regulators cdc25+ and wee1+, as a ppa2 deletion is lethal when combined with wee1-50 but partially suppresses the conditional lethality of cdc25-22 mutation. Evidence that ppa2+ negatively controls the entry into mitosis, possibly through the regulation of cdc2 tyrosine phosphorylation, is presented. ppa2 phosphatase is abundant in the cytoplasm, in contrast to the type 1-like phosphatase dis2, which is enriched in the nucleus. Overproduced ppa1 or ppa2 proteins accumulate in the cytoplasm near the nuclear periphery, and cells arrest in interphase. Okadaic acid-treated cells, like a ppa2 deletion, are short in length and display protein hyperphosphorylation. Cytokinesis is also inhibited, producing binucleated cells. We show that ppa2 is the genetic locus controlling okadaic acid sensitivity. The ppa2 deletion reveals the same hyperphosphorylated proteins as okadaic acid. When a strain deleted for ppa2 is treated with okadaic acid, cell size is reduced further to that of wee1-50 mutant strain or overexpressing the cdc25+ gene product, suggesting functional relationship of ppa2 with the cdc25 tyrosine phosphatase and/or the wee1 kinase in cell cycle control.
Replication initiates at a confined region during DNA amplification in Sciara DNA puff II/9A.
Authors: Liang C , Spitzer J D , Smith H S , Gerbi S A
Published: June 2007

Two independent two-dimensional (2D) gel methods were used to map an origin of replication that is developmentally regulated by the steroid hormone ecdysone, namely an origin for DNA puff amplification in the fungus fly Sciara coprophila. Initiation of replication was found to occur within a small region of no larger than 6 kb by use of the neutral/neutral 2D gel method. Neutral/alkaline 2D gel analyses support the results of the neutral/neutral 2D gels and further define within the origin region an approximately 1-kb area where the majority of replication initiates. This is the first example of an origin of replication in multicellular eukaryotes that has been mapped by 2D gels to such a small defined region. Moreover, replication can be seen by the neutral/alkaline 2D gel method to proceed bidirectionally outward from this replication origin region. These data are consistent with an onion-skin mechanism whereby multiple rounds of DNA replication initiate at a specific origin of replication for Sciara DNA puff amplification.
Ectopic expression of a conditional GATA-2/estrogen receptor chimera arrests erythroid differentiation in a hormone-dependent manner.
Authors: Briegel K , Lim K C , Plank C , Beug H , Engel J D , Zenke M
Published: June 2007

The GATA factors are a family of transcriptional regulatory proteins in eukaryotes that share extensive homology in their DNA-binding domains. One enigmatic aspect of GATA factor expression is that several GATA proteins, which ostensibly share the same DNA-binding site specificity, are coexpressed in erythroid cells. To elucidate the roles of individual GATA factors in erythropoiesis, conditional alleles of GATA-1, GATA-2, and GATA-3 were prepared by fusing each of the factors to the hormone-binding domain of the human estrogen receptor (ER). These GATA/ER chimeric factors were shown to be hormone-inducible trans-activating proteins in transient transfection assays. When stably introduced into primary erythroblasts or conditionally transformed erythroid progenitors cells, exogenous GATA-2/ER promoted proliferation and inhibited terminal differentiation in an estrogen-dependent manner. These phenotypic effects are specifically attributable to the action of ectopically expressed GATA-2/ER because erythroblasts expressing exogenous GATA-2 are constitutively arrested in differentiation and because erythroid progenitors expressing either Gal/ER or GATA-3/ER do not display a hormone-responsive block in differentiation. Thus, the GATA-2 transcription factor appears to play a role in regulating the self-renewal capacity of early erythroid progenitor cells.
Initial organization of the Drosophila dorsoventral axis depends on an RNA-binding protein encoded by the squid gene.
Authors: Kelley R L
Published: June 2007

Females mutant for the newly identified squid (sqd) gene are sterile and lay eggs that display only dorsal structures. The resulting embryos are also dorsalized even if fertilized by wild-type sperm. The gene acts midway through oogenesis at about the time dorsoventral (D/V) axis is established within the growing egg chamber. The sqd gene encodes at least three distinct proteins generated by alternative RNA processing that are members of a well-characterized family of RNA-binding proteins. At least one Sqd isoform is essential in somatic tissues. The ventralizing mutations gurken (grk), torpedo (tor), and cornichon are all epistatic to sqd. Strong alleles of grk and top can act as dominant suppressors of sqd dorsalization. A model of D/V axis formation is presented postulating that squid is needed to organize a concentration gradient of a morphogen originating in the germinal vesicle.
Products of the unc-52 gene in Caenorhabditis elegans are homologous to the core protein of the mammalian basement membrane heparan sulfate proteoglycan.
Authors: Rogalski T M , Williams B D , Mullen G P , Moerman D G
Published: June 2007

Mutations in the unc-52 gene of Caenorhabditis elegans affect attachment of the myofilament lattice to the muscle cell membrane. Here, we demonstrate that the unc-52 gene encodes a nematode homolog of perlecan, the mammalian basement membrane heparan sulfate proteoglycan. The longest potential open reading frame of this gene encodes a 2482-amino-acid protein with a signal peptide and four domains. The first domain is unique to the unc-52 polypeptide, whereas the three remaining domains contain sequences found in the LDL receptor (domain II) laminin (domain III) and N-CAM (domain IV). We have identified three alternatively spliced transcripts that encode different carboxy-terminal sequences. The two larger transcripts encode proteins containing all or part of domain IV, whereas the smaller transcript encodes a shortened polypeptide that completely lacks domain IV. We have determined that the disorganized muscle phenotype observed in unc-52(st196) animals is caused by the insertion of a Tc1 transposon into domain IV. Two monoclonal antibodies that recognize an extracellular component of all contractile tissues in C. elegans fail to stain embryos homozygous for a lethal unc-52 allele. We have mapped the epitopes recognized by both monoclonal antibodies to a region of domain IV in the unc-52-encoded protein sequence.
A switch in translation mediated by an antisense RNA.
Authors: Ranade K , Poteete A R
Published: June 2007

Antisense RNAs regulate expression of target genes in a variety of ways--transcription termination, translation initiation, and mRNA stability. We describe a case in which the target gene encodes two polypeptides, and antisense RNA causes a switch in its translation by selectively inhibiting synthesis of one of the polypeptides. Bacteriophage P22 is a temperate Salmonella phage; in the prophage state it expresses only a handful of its genes. One of these genes, sieB, aborts the lytic development of some phages. P22 itself is insensitive to the lethal effect of SieB because it harbors a determinant called esc. We show that the sieB gene encodes two polypeptides--SieB, which is the exclusion protein, and Esc, which is a truncated version of SieB that inhibits its action. Superinfecting P22 synthesizes an antisense RNA, sas, that inhibits synthesis of SieB but allows continued synthesis of Esc, thus allowing P22 to bypass SieB-mediated exclusion. This translational switch induced by sas RNA is essential to vegetatively developing P22; a mutation that prevents this switch causes P22 to commit SieB-mediated suicide. Finally, we show that P22's Esc allows it to circumvent the SieB-mediated exclusion system of bacteriophage lambda.
Subunit rearrangement of the cyclin-dependent kinases is associated with cellular transformation.
Authors: Xiong Y , Zhang H , Beach D
Published: June 2007

In normal human diploid fibroblasts, cyclins of the A, B, and D classes each associate with cyclin-dependent kinases (CDKs), proliferating cell nuclear antigen (PCNA), and p21, thereby forming multiple independent quaternary complexes. Upon transformation of diploid fibroblasts with the DNA tumor virus SV40, or its transforming tumor antigen (T), the cyclin D/p21/CDK/PCNA complexes are disrupted. In transformed cells, CDK4 totally dissociates from cyclin D, PCNA, and p21 and, instead, associates exclusively with a polypeptide of 16 kD (p16). Quaternary complexes containing cyclins A or B1 and p21/CDK/PCNA also undergo subunit rearrangement in transformed cells. Both PCNA and p21 are no longer associated with CDC2-cyclin B1 binary complexes. Cyclin A complexes no longer contain p21, and a new 19-kD polypeptide (p19) is found in association with cyclin A. The pattern of subunit rearrangement of cyclin-CDK complexes in SV40-transformed cells is also shared in those containing adeno- or papilloma viral oncoproteins. Rearrangement also occurs in p53-deficient cells derived from Li-Fraumeni patients that carry no known DNA tumor virus. These findings suggest a mechanism by which oncogenic proteins alter the cell cycle of transformed cells.
A turnover pathway for both stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation.
Authors: Decker C J , Parker R
Published: June 2007

To determine pathways of mRNA turnover in yeast, we have followed the poly(A) tail removal and degradation of a pulse of newly synthesized transcripts from four different genes. Before decay of both stable and unstable mRNAs initiated, there was a temporal lag during which the poly(A) tail was deadenylated to an oligo(A) length. Altering the deadenylation rate of an mRNA led to a corresponding change in the length of this lag. The rate of deadenylation and the stability of the oligo(A) species varied between mRNAs, explaining the differences in mRNA half-lives. To examine how the transcript body was degraded following deadenylation, we used the strategy of inserting strong RNA secondary structures, which can slow exonucleolytic digestion and thereby trap decay intermediates, into the 3' UTR of mRNAs. Fragments lacking the 5' portion of two different mRNAs accumulated after deadenylation as full-length mRNA levels decreased. Therefore, these results define an mRNA decay pathway in which deadenylation leads to either internal cleavage or decapping followed by 5'-->3' exonucleolytic degradation of the mRNA.