bluuebunny/crossref_metadata_2025 huggingface dataset:
The consequences of expressing hsp70 in Drosophila cells at normal temperatures.
Authors: Feder J H , Rossi J M , Solomon J , Solomon N , Lindquist S
Published: June 2007

In Drosophila cells, regulatory mechanisms not only act to provide rapid induction of hsp70 during heat shock but also to prevent expression at normal temperatures. To determine whether expression of hsp70 is detrimental to cells growing at normal temperatures, we used heterologous promoters to force expression of the protein in tissue culture cells and in larval salivary glands. Initially, constitutive expression of hsp70 substantially reduces the rate of cell growth. With continued expression, however, growth rates recover. At the same time, the intracellular distribution of hsp70 changes. Immediately after induction, the protein is diffusely distributed throughout the cell, but as growth resumes it coalesces into discrete points of high concentration, which we term hsp70 granules. hsp70 granules are also observed both in wild-type Drosophila tissue culture cells and in salivary glands after extended periods of recovery from heat shock. The protein in these granules appears to be irreversibly inactivated. It cannot be dispersed with a second heat shock, and cells containing these granules do not show thermotolerance. Only partial overlap between hsp70 granules and lysosomes indicates that the granules form independently of lysosomes. We conclude that expression of hsp70 is detrimental to growth at normal temperatures. We suggest that the change in hsp70 distribution, from diffuse to granular, represents a mechanism for controlling the protein's activity by sequestration.
CIK1: a developmentally regulated spindle pole body-associated protein important for microtubule functions in Saccharomyces cerevisiae.
Authors: Page B D , Snyder M
Published: June 2007

A genetic screen was devised to identify genes important for spindle pole body (SPB) and/or microtubule functions. Four mutants defective in both nuclear fusion (karyogamy) and chromosome maintenance were isolated; these mutants termed cik (for chromosome instability and karyogamy) define three complementation groups. The CIK1 gene was cloned and characterized. Sequence analysis of the CIK1 gene predicts that the CIK1 protein is 594 amino acids in length and possesses a central 300-amino-acid coiled-coil domain. Two different CIK1-beta-galactosidase fusions localize to the SPB region in vegetative cells, and antibodies against the authentic protein detect CIK1 in the SPB region of alpha-factor-treated cells. Evaluation of cells deleted for CIK1 (cik1-delta) indicates that CIK1 is important for the formation or maintenance of a spindle apparatus. Longer and slightly more microtubule bundles are visible in cik1-delta strains than in wild type. Thus, CIK1 encodes a SPB-associated component that is important for proper organization of microtubule arrays and the establishment of a spindle during vegetative growth. Furthermore, the CIK1 gene is essential for karyogamy, and the level of the CIK1 protein at the SPB appears to be dramatically induced by alpha-factor treatment. These results indicate that molecular changes occur at the microtubule-organizing center (MTOC) as the yeast cell prepares for karyogamy and imply that specialization of the MTOC or its associated microtubules occurs in preparation for particular microtubule functions in the yeast life cycle.
The Drosophila spitz gene encodes a putative EGF-like growth factor involved in dorsal-ventral axis formation and neurogenesis.
Authors: Rutledge B J , Zhang K , Bier E , Jan Y N , Perrimon N
Published: June 2007

We describe the molecular characterization of the Drosophila gene spitz (spi), which encodes a putative 26-kD, EGF-like transmembrane protein that is structurally similar to TGF-alpha. Temporal and spatial expression patterns of spi transcripts indicate that spi is expressed throughout the embryo. Examination of mutant embryos reveals that spi is involved in a number of unrelated developmental choices, for example, dorsal-ventral axis formation, glial migration, sensory organ determination, and muscle development. We propose that spi may act as a ligand for cell-specific receptors, possibly rhomboid and/or the Drosophila EGF receptor homolog.
dorsal-twist interactions establish snail expression in the presumptive mesoderm of the Drosophila embryo.
Authors: Ip Y T , Park R E , Kosman D , Yazdanbakhsh K , Levine M
Published: June 2007

The first step in the differentiation of the Drosophila mesoderm is the activation of two regulatory genes, twist (twi) and snail (sna), in ventral regions of early embryos. sna is a transcriptional repressor that is uniformly expressed throughout the presumptive mesoderm. Its sharp lateral limits help to establish the boundary between the mesoderm and neuroectoderm. Genetic studies suggest that sna is a target of the dorsal (dl) morphogen, and this interaction provides a model for determining how a morphogen gradient establishes a sharp, on/off threshold response. We present evidence that dl and twi directly activate sna expression. Site-directed mutagenesis of dl- and twi-binding sites within defined regions of the sna promoter suggest that the two proteins (containing the Rel and helix-loop-helix domains, respectively) function multiplicatively to ensure strong, uniform expression of sna, particularly in ventral-lateral regions where there are diminishing amounts of dl. These results are consistent with the possibility that the sharp sna borders are formed by multiplying the shallow dl gradient and the steeper twi gradient.
Hlf, a novel hepatic bZIP protein, shows altered DNA-binding properties following fusion to E2A in t(17;19) acute lymphoblastic leukemia.
Authors: Hunger S P , Ohyashiki K , Toyama K , Cleary M L
Published: June 2007

Oncogenic conversion of transcription factors by chromosomal translocations is implicated in leukemogenesis. We report that the t(17;19) in acute lymphoblastic leukemia produces a chimeric transcription factor consisting of the amino-terminal portion of HLH proteins E12/E47 (products of the E2A gene) fused to the basic DNA-binding and leucine zipper dimerization motifs of a novel hepatic protein called hepatic leukemia factor (Hlf). Hlf, which is not normally transcribed in lymphoid cells, belongs to the recently described PAR subfamily of basic leucine zipper (bZIP) proteins, which also includes Dbp and Tef/Vbp. Wild-type Hlf is able to bind DNA specifically as a homodimer or as a heterodimer with other PAR factors. Structural alterations of the E2a-Hlf fusion protein markedly impair its ability to bind DNA as a homodimer compared with wild-type Hlf. However, E2a-Hlf can bind DNA as a heterodimer with other PAR proteins, suggesting a novel mechanism for leukemogenic conversion of a bZIP transcription factor.
v-ras and protein kinase C dedifferentiate thyroid cells by down-regulating nuclear cAMP-dependent protein kinase A.
Authors: Gallo A , Benusiglio E , Bonapace I M , Feliciello A , Cassano S , Garbi C , Musti A M , Gottesman M E , Avvedimento E V
Published: June 2007

Ras proteins are membrane-associated transducers of eternal stimuli to unknown intracellular targets. The constitutively activated v-ras oncogene induces dedifferentiation in thyroid cells. v-Ras appears to act by stimulating protein kinase C (PKC), which inhibits the nuclear migration of the catalytic subunit of the cAMP-dependent protein kinase A (PKA). Nuclear tissue-specific and housekeeping trans-acting factors that are dependent on phosphorylation by PKA are thus inactivated. Exclusion of the PKA subunit from the nucleus could represent a general mechanism for the pleiotropic effects of Ras and PKC on cellular growth and differentiation.
breathless, a Drosophila FGF receptor homolog, is essential for migration of tracheal and specific midline glial cells.
Authors: Klämbt C , Glazer L , Shilo B Z
Published: June 2007

A Drosophila homolog of the fibroblast growth factor (FGF) receptor was isolated and structurally characterized. After EMS mutagenesis or imprecise excisions of marked P elements inserted upstream to the gene, a phenotypic series of mutations in the locus was isolated. The mutants exhibit defects in the two embryonic tissues in which the receptor is expressed: the tracheal system and the midline. The tracheal cells fail to migrate in severe mutants and remain within the tracheal pits. Hypomorphic alleles exhibit partial migration of all tracheal branches; thus, the locus was termed breathless (btl). In the midline of the mutant embryos, the posterior pair of midline glial cells begins to migrate anteriorly, but fails to reach the posterior commissure. Abnormalities in cell migration appear to be a common denominator for the btl defects in these two disparate tissues. In hypomorphic mutants the cells exhibit partial migration but follow the normal tracts, suggesting that the presence of this receptor is essential for the ability of the migrating cells to recognize external guiding cues.
Stable synapsis of homologous DNA molecules mediated by the Escherichia coli RecA protein involves local exchange of DNA strands.
Authors: Adzuma K
Published: June 2007

Escherichia coli RecA protein promotes stable synapsis between a single-stranded DNA and a homologous duplex DNA, resulting in the formation of a complex of RecA with three DNA strands. To gain insight into the molecular interactions responsible for DNA synapsis, the base-pairing status within the synaptic complex was analyzed by using dimethylsulfate and potassium permanganate as probes. The results indicate that the original base pairs in the parental duplex are disrupted; one strand is displaced and the other strand appears to be involved in Watson-Crick base-pairing with the incoming single-stranded DNA. The state of base-pairing thus resembles that of the end products of strand exchange and not a canonical DNA triple helix involving non-Watson-Crick base-pairing. The results also indicate that this local strand exchange can occur without homology at the ends of the DNA substrates (i.e., when axial rotation of the product heteroduplex with respect to the axis of the parental duplex is obstructed). Taken together, these results suggest that exchange of DNA strands mediated by RecA occur at or before the stage of stable DNA synapsis by a process that does not require DNA rotation.
The Drosophila gene Hairless encodes a novel basic protein that controls alternative cell fates in adult sensory organ development.
Authors: Bang A G , Posakony J W
Published: June 2007

The mechanosensory bristles of adult Drosophila are composed of four cells that, in most cases, are progeny of a single sensory organ precursor (SOP) cell. Two sister cells in this lineage, the trichogen and tormogen, produce the external shaft and socket of the bristle, respectively. Loss-of-function mutations of Hairless (H) confer two distinct mutant phenotypes on adult bristles. The bristle loss phenotype results from the failure to specify and/or execute the SOP cell fate; the double socket phenotype results from the transformation of the trichogen (shaft) cell into a second tormogen (socket) cell. We have found that the H gene encodes a novel basic protein with a predicted molecular mass of 109 kD. Basal levels of expression of a transgene (P[Hs-H]) in which the H protein-coding region is under the control of the Hsp70 promoter are sufficient to provide full rescue of H mutant phenotypes. Heat shock treatment of P[Hs-H] transgenic animals as late larvae and early pupae produces a tormogen-to-trichogen (double shaft) cell fate transformation, as well as bristle multiplication and loss phenotypes very similar to those caused by loss-of-function mutations in the neurogenic gene Notch. Our results indicate that the SOP cell fate requires H to antagonize the activity of the neurogenic group of genes and that the expression of distinct cell fates by the trichogen/tormogen sister cell pair depends on an asymmetry in their levels of H+ activity or in their thresholds for response to H.
StuA is required for cell pattern formation in Aspergillus.
Authors: Miller K Y , Wu J , Miller B L
Published: June 2007

The stunted (stuA) gene product is required for the orderly differentiation and spatial organization of cell types of the Aspergillus nidulans conidiophore. Expression of the stuA gene is complex. Two transcripts, stuA alpha and stuA beta, are initiated from separate promoters. Transcription of both RNAs increases approximately 50-fold during the establishment of developmental competence. Induction-dependent transcriptional and post-transcriptional regulatory mechanisms further enhance expression 15-fold. Consistent with the latter observation, both transcripts have structural features characteristic of RNAs subject to translational control. Conidiophore morphogenesis requires regulatory interactions between the products of the stuA, bristle (brlA), and abacus (abaA) genes. Enhanced stunted expression is cell type specific and dependent on a functional BrlA protein. StuA affects the spatial localization of AbaA by acting directly, or indirectly, to repress abaA expression.
Each hypersensitive site of the human beta-globin locus control region confers a different developmental pattern of expression on the globin genes.
Authors: Fraser P , Pruzina S , Antoniou M , Grosveld F
Published: June 2007

We have tested the effect of the individual DNase I hypersensitive site (HS) regions of the globin locus control region (LCR) on the developmental expression pattern of the human gamma and beta genes in transgenic mice. The results show that HS3 is the most active site during the embryonic period. It is also the only site capable of high level expression of the gamma genes during fetal hematopoiesis, in a population of cells that are capable of expressing both the gamma and beta genes. Region HS4 shows the highest activity during the adult stage and expresses the gamma genes only at low levels during the embryonic period. HS2 drives equivalent levels of gamma or beta transgene expression throughout development. HS1 has a similar pattern to HS2, although the activity of HS1 is very low. From these results we conclude that the HS regions have distinct developmental specificities and suggest that in the complete LCR they interact with each other to form a larger complex which, in turn, interacts with the globin genes.
Role of the sigma 70 subunit of RNA polymerase in transcriptional activation by activator protein PhoB in Escherichia coli.
Authors: Makino K , Amemura M , Kim S K , Nakata A , Shinagawa H
Published: June 2007

Transcription of the genes belonging to the phosphate (pho) regulon in Escherichia coli, which are induced by phosphate starvation, requires the specific activator protein PhoB in addition to the RNA polymerase holoenzyme containing the major sigma-factor sigma 70. To study the mechanism of transcriptional activation and identify the subunit of RNA polymerase involved in specific interaction with PhoB, we attempted to isolate rpoA and rpoD mutants that are specifically defective in the expression of the pho genes. We isolated two rpoD mutants with such properties, but no rpoA mutant with similar properties. The rpoD mutations altered amino acids within and near the first helix of the putative helix-turn-helix (HTH) motif in the carboxy-terminal region of sigma 70. Activities of the pho promoters in vivo were severely reduced in these mutants, whereas those of the PhoB-independent promoters were affected only marginally at most. The reconstituted mutant RNA polymerase holoenzymes were severely defective in transcribing the pstS gene, one of the pho genes, whereas they were efficient in transcribing the PhoB-independent promoters. Phosphorylated PhoB, which binds to the pho promoters with high affinity, mediated the specific binding of the wild-type holoenzyme to the pstS promoter, but it did not mediate the binding of the mutant holoenzymes. These results suggest that PhoB promotes specific interaction between RNA polymerase and the pho promoters for transcriptional activation, and the first helix of the putative HTH motif plays an essential role in the interaction, probably by making direct contact with PhoB.
Elongation factor NusG interacts with termination factor rho to regulate termination and antitermination of transcription.
Authors: Li J , Mason S W , Greenblatt J
Published: June 2007

NusG is a transcriptional elongation factor in Escherichia coli that aids transcriptional antitermination by the phage lambda N protein. By using NusG affinity chromatography, we found that NusG binds directly and selectively to termination factor rho. NusG was shown previously to be needed for termination by rho in vivo, and we show here that NusG increases the efficiency of termination by rho at promoter-proximal sites in vitro. The rho026 mutation makes termination by rho less dependent on NusG. It also makes antitermination by N at rho-dependent terminators and the binding of rho to NusG temperature sensitive. Therefore, the interaction of NusG with rho is important both for rho-dependent termination and for antitermination by N at rho-dependent terminators.
Cooperative DNA binding of p53 with TFIID (TBP): a possible mechanism for transcriptional activation.
Authors: Chen X , Farmer G , Zhu H , Prywes R , Prives C
Published: June 2007

The p53 tumor-suppressor gene product, a sequence-specific DNA-binding protein, has been shown to act both as a transcriptional activator and repressor in vivo and in vitro. Consistent with its roles in regulating transcription are recent observations that p53 binds directly to the TATA box-binding protein (TBP) subunit of the basal transcription factor TFIID. Here, we show that p53 cooperates with either recombinant TBP or partially purified TFIID in binding to a DNA fragment containing both a specific p53-binding site (RGC) and a TATA box (RGC-TATA). Surprisingly, both TBP and TFIID also stimulate p53 binding to DNA containing a specific p53-binding site but lacking a TATA box. These data are supported by the observation that p53 and Drosophila TBP combinatorily activate transcription in vivo. Our results suggest that p53 activates transcription through the formation of a more stable p53-TFIID-promoter complex. We also examined whether p53 might affect the ability of TBP or TFIID to interact with DNA containing a TATA box but lacking a p53-binding site. Although p53 strongly inhibited the interaction of TBP with such DNA, it had virtually no effect on TFIID binding. Thus, transcriptional repression by p53 may require additional functions other than inhibiting TBP binding.
mRNA surveillance by the Caenorhabditis elegans smg genes.
Authors: Pulak R , Anderson P
Published: June 2007

mRNAs that contain premature stop codons are unstable in most eukaryotes, but the mechanism of their degradation is largely unknown. We demonstrate that functions of the six C. elegans smg genes are necessary for rapid turnover of nonsense mutant mRNAs of the unc-54 myosin heavy chain gene. Nonsense alleles of unc-54 express mRNAs that are unstable in smg(+) genetic backgrounds but have normal or near normal stability in smg(-) backgrounds. smg mutations also stabilize mRNA of unc-54(r293), a small deletion that removes the unc-54 polyadenylation site and expresses an aberrant mRNA. Most unc-54 nonsense mutations are recessive in both smg(+) and smg(-) genetic backgrounds. However, four specific alleles are recessive when smg(+) and dominant when smg(-). These smg-dependent dominant alleles express nonsense mutant polypeptides that disrupt thick filament and/or sarcomere assembly. All four alleles are predicted to express nonsense fragment polypeptides that contain most of the myosin globular head domain without an attached rod segment. By degrading messages that contain premature stop codons, the smg genes eliminate mRNAs that encode potentially toxic protein fragments. We propose that this system of mRNA turnover protects cells from their own errors of transcription, mRNA processing, or mRNA transport.
Multiple cis-acting signals for export of pre-U1 snRNA from the nucleus.
Authors: Terns M P , Dahlberg J E , Lund E
Published: June 2007

We have identified cis-acting sequences that promote nuclear export of pre-U1 RNA injected into Xenopus oocyte nuclei. At least three elements, the 5' m7G cap, the 3'-terminal stem-loop structure, and sequences in the 5'-terminal 124 nucleotides, contribute to efficient export of this RNA. Both the 5' and 3' export signals can function separately and do so independently of the cap structure. Experiments using hybrid RNAs indicate that the 5' and 3' export sequences of U1 RNA are sufficient to direct export of the heterologous, otherwise nonexportable, U6 RNA. The absence of comparable export signals in U6 RNA appears to be responsible for its retention in the nucleus. Stability of the pre-snRNAs in the nucleus depends on the presence of both a 5' cap structure and a 3' base-paired stem. The 5' m7G cap is neither sufficient nor necessary for nuclear export. The m7G cap by itself did not promote export of U6 RNA or nonspecific small RNAs. Moreover, substitution of this cap with either an AppG cap or gamma-mppG cap did not eliminate export of either full-length or a "minimal" U1 RNA (lacking most of the internal U1 RNA sequences), but it reduced the rate of export by about two to threefold. However, in the absence of the 3' stem-loop, substitution of the m7G cap led to a greater decrease in export rate, underscoring the cooperative action of the three different export elements of pre-U1 RNA. The m7G cap analog, m7GpppG, selectively destabilized pre-U1 RNA within the nucleus. Thus, nuclear components that recognize the 5' m7G cap may be important for both the stability and the export of pre-U1 RNA.
Four yeast spliceosomal proteins (PRP5, PRP9, PRP11, and PRP21) interact to promote U2 snRNP binding to pre-mRNA.
Authors: Ruby S W , Chang T H , Abelson J
Published: June 2007

We have analyzed the functions of several pre-mRNA processing (PRP) proteins in yeast spliceosome formation. Here, we show that PRP5 (a DEAD box helicase-like protein), PRP9, and PRP11 are each required for the U2 snRNP to bind to the pre-spliceosome during spliceosome assembly in vitro. Genetic analyses of their functions suggest that they and another protein, PRP21, act concertedly and/or interact physically with each other and with the stem-loop IIa of U2 snRNA to bind U2 snRNP to the pre-mRNA. Biochemical complementation experiments also indicate that the PRP9 and PRP11 proteins interact. The PRP9 and PRP11 proteins may be functioning similarly in yeast and mammalian cells. The requirement for ATP and the helicase-like PRP5 protein suggests that these factors might promote a conformational change (involving either the U1 or U2 snRNP) that is required for the association of U2 snRNP with the pre-mRNA.
Mutation of an RSV intronic element abolishes both U11/U12 snRNP binding and negative regulation of splicing.
Authors: Gontarek R R , McNally M T , Beemon K
Published: June 2007

A cis-acting negative regulator of splicing (NRS) within the gag gene of RSV is involved in control of the relative levels of spliced and unspliced viral mRNAs. Insertion of the NRS into the intron of an adenovirus pre-mRNA resulted in inhibition of splicing in vitro before the first cleavage step. Analyses of spliceosome assembly with this substrate showed that it formed large RNP complexes that did not migrate like mature spliceosomes on native gels. Affinity selection of the RNP complexes formed on NRS-containing pre-mRNAs showed an association with U11 and U12 snRNPs, as well as with the spliceosomal snRNPs. Immunoprecipitation with antisera specific for U1 and U2 snRNPS showed binding of both snRNPs to NRS RNA. A 7-nucleotide missense mutation in the NRS that prevented binding of U11 and U12 snRNPs impaired NRS activity in vivo, suggesting a functional role for U11 and U12 snRNPs in the inhibition of splicing mediated by the RSV NRS RNA.
Antineurogenic phenotypes induced by truncated Notch proteins indicate a role in signal transduction and may point to a novel function for Notch in nuclei.
Authors: Lieber T , Kidd S , Alcamo E , Corbin V , Young M W
Published: June 2007

Loss of any one of several neurogenic genes of Drosophila results in overproduction of embryonic neuroblasts at the expense of epidermoblasts. In this paper a variety of altered Notch proteins are expressed in transgenic flies. Dominant lethal, antineurogenic phenotypes were produced by expression of three classes of mutant proteins: (1) a protein comprised of the cytoplasmic domain of Notch and devoid of sequences permitting membrane association; (2) a transmembrane protein lacking the extracellular, lin12/Notch repeats; and (3) transmembrane proteins carrying amino acid substitutions replacing one or both extracellular cysteines thought to be involved in Notch dimerization. These Notch proteins not only suppress the neural hypertrophy observed in Notch- embryos, but also generate a phenotype in which elements of the embryonic nervous system are underproduced. Action of the intracellular cdc10 repeats appears to be essential for wild-type Notch function or for the antineurogenic activity of these proteins. The activities of the dominant, gain-of-function proteins indicate that Notch functions as a signal transducing receptor during ectoderm development. Production of antineurogenic Notch proteins in embryos deficient for the other neurogenic genes allowed functional dependencies to be established. Delta, mastermind, bigbrain, and neuralized appear to function in elaboration of a signal upstream of Notch. Genes of the Enhancer of split complex act after Notch. The cytoplasmic domain of Notch contains nuclear localization sequences that function in cultured cells, and one of the Notch antineurogenic proteins, the cytoplasmic domain, accumulates in nuclei in vivo.
The activity of the Escherichia coli transcription factor FNR is regulated by a change in oligomeric state.
Authors: Lazazzera B A , Bates D M , Kiley P J
Published: June 2007

The transcription factor FNR globally regulates gene expression in response to oxygen deprivation in Escherichia coli. To understand how oxygen deprivation activates FNR, a constitutively active FNR* mutant protein, DA154, was studied to determine how this mutant bypassed the normal regulation pathway. When purified from aerobically grown cells, the DA154 protein had a larger apparent native molecular mass and an increased affinity for a consensus FNR target site as compared with wild-type FNR prepared under identical conditions. These results suggested that FNR* DA154 may bypass the normal regulation pathway by converting FNR from an inactive monomer to an active dimer under aerobic conditions. To determine whether wild-type FNR is active as a dimer under anaerobic conditions, FNR mutants were isolated that inhibit the activity of wild-type FNR by forming mixed dimers (i.e., dominant-negative mutants). These dominant-negative FNR mutants were shown to have substitutions in the putative DNA-binding domain and to be defective in binding to a consensus FNR DNA target site in vitro. One representative dominant-negative mutant, EK209, was also shown to be unable to form mixed oligomers in vivo under aerobic conditions, suggesting that FNR may be monomeric in the inactive state. Taken together, these data have led us to propose that under anaerobic conditions FNR is a dimer that is active for DNA binding, and under aerobic conditions, FNR is inactivated by conversion to a monomer.
Ets-related protein PU.1 regulates expression of the immunoglobulin J-chain gene through a novel Ets-binding element.
Authors: Shin M K , Koshland M E
Published: June 2007

In a primary immune response, a signal from interleukin-2 (IL-2) activates transcription of the gene encoding the pentamer IgM joining component, the J chain. Recently, a bifunctional control element (JB) in the J-chain promoter has been identified. This finding was pursued in the present study by purifying and characterizing the nuclear protein (NF-JB) that mediates the positive regulatory activity of the JB element. The analyses revealed that NF-JB is identical to the Ets-related B-cell- and macrophage-specific transcriptional factor, PU.1, despite the fact that the JB site lacks the GGA core reported to be essential for binding by members of the Ets oncoprotein family. The two factors were found to be indistinguishable with respect to their DNA-binding characteristics, size, and peptide structure. Moreover, in transient transfection assays, PU.1 alone activated reporter constructs containing the JB cis-element, and the activation was shown to be dependent on a glutamine-rich sequence in the amino-terminal portion of PU.1. Finally, a dominant negative mutant of PU.1 was capable of suppressing the transcriptional activity of a 1.2-kb J-chain promoter sequence. These results establish an important role for PU.1 in the regulation of immunoglobulin J-chain gene expression and provide new insights into the function(s) of the Ets transcription factors in lymphoid cells.
The immunoglobulin mu enhancer core establishes local factor access in nuclear chromatin independent of transcriptional stimulation.
Authors: Jenuwein T , Forrester W C , Qiu R G , Grosschedl R
Published: June 2007

Factor access in chromatin has been proposed to be facilitated by transcriptional enhancers. With the aim of uncoupling factor access from transcriptional stimulation by protein-protein contacts, we analyzed the potential of enhancer fragments to confer accessibility upon a linked promoter for prokaryotic T7 RNA polymerase. Access to the T7 promoter in pre-B cells from transgenic mice was examined by transcribing chromatin of isolated nuclei with T7 RNA polymerase. A 95-bp immunoglobulin mu enhancer core element was necessary and sufficient to confer accessibility upon the T7 promoter independent of its chromosomal position. This enhancer-dependent factor access could be uncoupled from an active transcriptional state of the transgene and was not accompanied by the formation of pronounced DNase I hypersensitive sites. Additional mu enhancer sequences comprising previously identified matrix attachment regions and a cryptic promoter were required to induce DNase I hypersensitivity. Together, these data provide evidence that the 95-bp mu enhancer core can establish localized factor access in nuclear chromatin independent of detectable transcription by endogenous polymerases and suggest that multiple steps are involved in the alteration of chromatin structure.
Migrations of the Caenorhabditis elegans HSNs are regulated by egl-43, a gene encoding two zinc finger proteins.
Authors: Garriga G , Guenther C , Horvitz H R
Published: June 2007

During embryonic development, the two Caenorhabditis elegans HSN motor neurons migrate from their birthplace in the tail to positions near the middle of the embryo. Here, we demonstrate that of all cells that undergo long-range migrations, only the HSNs are affected in animals that lack function of the egl-43 gene. We also show that egl-43 function is required for normal development of phasmid neurons, which are sensory neurons located in the tail. The egl-43 gene encodes two proteins containing zinc finger motifs that are similar to the zinc fingers of the murine Evi-1 proto-oncoprotein. Our genetic and molecular results suggest that egl-43 encodes two transcription factors and acts to control HSN migration and phasmid neuron development, presumably by regulating other genes that function directly in these processes.
A switch from Myc:Max to Mad:Max heterocomplexes accompanies monocyte/macrophage differentiation.
Authors: Ayer D E , Eisenman R N
Published: June 2007

Mad is a basic-helix-loop-helix-zipper protein that heterodimerizes with Max in vitro. Mad:Max heterodimers recognize the same E-box-related DNA-binding sites as Myc:Max heterodimers. However, in transient transfection assays Myc and Mad influence transcription in opposite ways through interaction with Max; Myc activates while Mad represses transcription. Here, we demonstrate that Mad protein is induced rapidly upon differentiation of cells of the myeloid lineage. The Mad protein is synthesized in human cells as a 35-kD nuclear phosphoprotein with an extremely short half-life (t1/2 = 15-30 min) and can be detected in vivo in a complex with Max. In the undifferentiated U937 monocyte cell line Max was found complexed with Myc but not Mad. However, Mad:Max complexes began to accumulate as early as 2 hr after induction of macrophage differentiation with TPA. By 48 hr following TPA treatment only Mad:Max complexes were detectable. These data show that differentiation is accompanied by a change in the composition of Max heterocomplexes. We speculate that this switch in heterocomplexes results in a change in the transcriptional regulation of Myc:Max target genes required for cell proliferation.
Cooperative dimerization of paired class homeo domains on DNA.
Authors: Wilson D , Sheng G , Lecuit T , Dostatni N , Desplan C
Published: June 2007

Homeo domain-containing proteins mediate many transcriptional processes in eukaryotes. Because nearly all animal homeo proteins are believed to bind to short, highly related DNA sequences, the basis for their high specificity of action is not understood. We show that cooperative dimerization on palindromic DNA sequences can provide increased specificity to one of the three major classes of homeo domains, the Paired/Pax class. The 60-amino-acid homeo domains from this class contain sufficient information to bind cooperatively as homo- and heterodimers to palindromic DNA sequences; that is, the binding of one homeo domain molecule can increase the affinity of a second molecule by up to 300-fold. Different members of the Paired (Prd) class of homeo domains prefer different spacings between half-sites, as determined by the ninth amino acid residue of the recognition helix. In addition, this residue determines the identity of the base pairs at the center of the palindromic sites, as well as the magnitude of the cooperative interaction. The cooperative dimerization of homeo domains in the Prd class distinguishes them from other classes, whereas binding-site configuration and sequence specificity allow for distinctions within this class.
Extracellular cAMP is sufficient to restore developmental gene expression and morphogenesis in Dictyostelium cells lacking the aggregation adenylyl cyclase (ACA).
Authors: Pitt G S , Brandt R , Lin K C , Devreotes P N , Schaap P
Published: June 2007

Cell movement and cell-type-specific gene expression during Dictyostelium development are regulated by cAMP, which functions both as an extracellular hormone-like signal and an intracellular second messenger. Previous data indicated that aca- mutants, which lack adenylyl cyclase activity, fail to aggregate and do not express cell-type-specific genes. We show here that overexpression of ACG, a constitutively active adenylyl cyclase, which in wild-type cells is only expressed during spore germination, partially restores the coordination of cell movement and completely restores developmental gene expression. The aca- cells can also be induced to develop into viable spores by synergy with wild-type cells and, furthermore, form small but normal fruiting bodies, after a developmentally relevant regimen of stimulation with nanomolar cAMP pulses followed by micromolar cAMP concentrations. 2'-Deoxy cAMP, a cAMP analog that activates the cell-surface cAMP receptors but not cAMP-dependent protein kinase (PKA), also induces fruiting body formation as well as expression of prespore-specific and prestalk-enriched genes in aca- cells. Intracellular cAMP levels were not altered in aca- cells after stimulation with 2'-deoxy cAMP. Our data indicate that ACA is not required to provide intracellular cAMP for PKA activation but is essential to produce extracellular cAMP for coordination of cell movement during all stages of development and for induction of developmental gene expression.
Activity of Wnt-1 as a transmembrane protein.
Authors: Parkin N T , Kitajewski J , Varmus H E
Published: June 2007

The product of the Wnt-1 proto-oncogene is a cysteine-rich glycoprotein that plays a crucial role in the development of the vertebrate central nervous system. Wnt-1 protein is secreted but remains associated with the cell surface and extracellular matrix. The function of Wnt-1 in several different biological settings can be carried out by cells that receive the Wnt signal from adjacent cells. Ectopic expression of Wnt-1 in certain mammary gland cell lines, such as C57MG, causes morphological transformation; C57MG cells can also be transformed by a paracrine mechanism when mixed with other cell types secreting Wnt-1 protein. To ask whether Wnt-1 protein can function while bound to the cell of origin, a variety of cell types were programmed to produce chimeric proteins containing the complete sequence of mature Wnt-1 protein fused to part or all of the transmembrane protein CD4 or CD8. The chimeras were found at the cell surface of transfected cells and did not appear to be proteolytically processed. In autocrine and paracrine transformation assays with C57MG cells and in an axis induction assay in Xenopus laevis embryos, the Wnt-1/CD4 or CD8 fusions retained significant activity, as did a secreted chimera containing the CD8 extracellular domain but lacking the transmembrane domain. However, a chimera lacking a spacer between the Wnt-1 and the transmembrane domains was weakly active and only in autocrine transformation. These results show that tethering Wnt-1 to the cell surface still allows Wnt-1-mediated cell-to-cell signaling.
A single cDNA, hTFIIA/alpha, encodes both the p35 and p19 subunits of human TFIIA.
Authors: DeJong J , Roeder R G
Published: June 2007

TFIIA is a transcription factor that, by interacting with the TATA-binding subunit (TBP) of TFIID, modulates transcription initiation by RNA polymerase II in vitro. By use of a mobility shift assay, TFIIA was purified from HeLa cells as a complex of 35-, 19-, and 12-kD subunits. Oligonucleotides were used to isolate a human cDNA clone, hTFIIA/alpha, which encodes a 55-kD protein with homology to the product of the yeast gene TOA1. The open reading frame of hTFIIA/alpha contains peptide sequences obtained from both the p35 and p19 subunits of natural human TFIIA, and thus encodes these two subunits. Consistent with this, antiserum raised against the 55-kD hTFIIA/alpha-encoded protein reacted with both the p35 and p19 subunits of natural TFIIA, and the recombinant protein could functionally replace those subunits in a mobility shift assay with renatured p12. An efficient affinity purification for natural human TFIIA was suggested by the sequence of the hTFIIA/alpha protein and demonstrated biochemically. Finally, transcription from the adenovirus major late promoter was greatly reduced in nuclear extracts depleted with anti-TFIIA/alpha serum and was restored to original levels by the readdition of purified human TFIIA.
Isolation of a cDNA encoding the largest subunit of TFIIA reveals functions important for activated transcription.
Authors: Ma D , Watanabe H , Mermelstein F , Admon A , Oguri K , Sun X , Wada T , Imai T , Shiroya T , Reinberg D
Published: June 2007

Transcription factor IIA has been shown to interact with the TATA-binding protein and to act early during preinitiation complex formation. The human factor is composed of three subunits (alpha, beta, gamma). A human cDNA clone encoding the largest subunit of TFIIA (alpha) was isolated. The recombinant alpha polypeptide, together with the beta and gamma subunits, was capable of reconstituting TFIIA activity. Studies using antibodies raised against recombinant alpha polypeptide demonstrate that TFIIA can be an integral component of the preinitiation complex. We demonstrate that TFIIA not only interacts with TBP but also can associate with the TFIID complex. Functional assays establish that TFIIA has no apparent role in basal transcription but plays an important role in activation of transcription. Interestingly, amino acid sequence analyses of the beta-subunit demonstrate these residues to be entirely contained within the carboxyl terminus of the cDNA clone encoding the alpha-subunit.
Molecular characterization of the her-1 gene suggests a direct role in cell signaling during Caenorhabditis elegans sex determination.
Authors: Perry M D , Li W , Trent C , Robertson B , Fire A , Hageman J M , Wood W B
Published: June 2007

We have characterized two transcripts from the male-determining her-1 locus in Caenorhabditis elegans. The larger transcript, which appears more important for male development, is predicted to encode a novel 175-amino-acid, cysteine-rich polypeptide with an apparent amino-terminal signal sequence and potential cleavage and glycosylation sites. Expression of a full-length cDNA construct for the larger transcript driven by a body-wall-myosin promoter causes extensive masculinization of all sexually dimorphic tissues in XX (normally hermaphrodite) animals. This activity is dependent on the presence of the her-1 signal sequence or a substitute synthetic signal sequence in the encoded polypeptide. These results suggest that a secreted product of the her-1 gene dictates male development.
The regulation of empty spiracles by Abdominal-B mediates an abdominal segment identity function.
Authors: Jones B , McGinnis W
Published: June 2007

The empty spiracles (ems) homeo box gene is required for the development of the Drosophila larval filzkörper, which are structural specializations of the eighth abdominal segment. Filzkörper development is also dependent on the function of the homeotic selector gene Abdominal-B (Abd-B). Here, we show that ems is a downstream gene that is transcriptionally regulated by Abd-B proteins. This regulation is mediated by an Abd-B-dependent ems cis-regulatory element that in early- to mid-stage embryos is activated only in the eighth abdominal segment. Genetic epistasis tests suggest that both ems and Abd-B are required in combination for the specification of the filzkörper primordia. In a general sense, these results also provide evidence that the hierarchical level immediately downstream of the homeotic genes contains additional homeo domain transcription factors that define subsegmental domain identities.
The extremely conserved amino terminus of RAD6 ubiquitin-conjugating enzyme is essential for amino-end rule-dependent protein degradation.
Authors: Watkins J F , Sung P , Prakash S , Prakash L
Published: June 2007

The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme that is required for DNA repair, damage-induced mutagenesis, and sporulation. In addition, RAD6 mediates the multiubiquitination and degradation of amino-end rule protein substrates. The structure and function of RAD6 have been remarkably conserved during eukaryotic evolution. Here, we examine the role of the extremely conserved amino terminus, which has remained almost invariant among RAD6 homologs from yeast to human. We show that RAD6 is concentrated in the nucleus and that the amino-terminal deletion mutation, rad6 delta 1-9, does not alter the location of the protein. The amino-terminal domain, however, is essential for the multiubiquitination and degradation of amino-end rule substrates. In the rad6 delta 1-9 mutant, beta-galactosidase proteins bearing destabilizing amino-terminal residues become long lived, and purified rad6 delta 1-9 protein is ineffective in ubiquitin-protein ligase (E3)-dependent protein degradation in the proteolytic system derived from rabbit reticulocytes. The amino terminus is required for physical interaction of RAD6 with the yeast UBR1-encoded E3 enzyme, as the rad6 delta 1-9 protein is defective in this respect. The rad6 delta 1-9 mutant is defective in sporulation, shows reduced efficiency of DNA repair, but is proficient in UV mutagenesis. E3-dependent protein degradation by RAD6 could be essential for sporulation and could affect the efficiency of DNA repair.
CAR2, a prestalk cAMP receptor required for normal tip formation and late development of Dictyostelium discoideum.
Authors: Saxe C L , Ginsburg G T , Louis J M , Johnson R , Devreotes P N , Kimmel A R
Published: June 2007

Extracellular cAMP serves as a primary signaling molecule to regulate the development of Dictyostelium discoideum. It is required for chemotaxis, aggregation, cytodifferentiation, and morphogenetic movement. The receptors for cAMP are members of the family of cell-surface receptors that are linked to G proteins and characterized by seven putative transmembrane domains. Previously, we have isolated the gene for the cAMP receptor subtype 1 (CAR1) from Dictyostelium and suggested that several genes related to CAR1 were present in the genome. Here, we describe a family of cAMP receptor genes of Dictyostelium and the isolation and function of the gene for the cAMP receptor subtype 2, CAR2. CAR2 is structurally similar to CAR1. Overall, their transmembrane and loop domains are approximately 75% identical in amino acid sequence; however, their carboxyl termini are quite dissimilar; CAR2 possesses homopolymeric runs of histidines and asparagines that are absent from the corresponding region in CAR1. Although CAR1 is maximally expressed during the early stages of development, CAR2 is expressed only after cells have aggregated and, then, preferentially in prestalk cells. Transgenic Dictyostelium that have had their wild-type CAR2 gene replaced by a defective copy using homologous recombination proceed through early development but are detained at the tight mound stage. CAR2 may be required for cAMP-directed sorting of prestalk cells during pattern formation within the aggregation mound. Furthermore, although prestalk genes are expressed normally in aggregates that lack CAR2, they exhibit an enhanced expression of prespore-specific mRNA. Previously, we had shown that there was a requirement for CAR1 during early development. The present results demonstrate that the multiple responses of Dictyostelium to cAMP are regulated by distinct cAMP receptors that are encoded by unique genes.
The mouse Hox-1.3 gene is functionally equivalent to the Drosophila Sex combs reduced gene.
Authors: Zhao J J , Lazzarini R A , Pick L
Published: June 2007

To test whether the mouse Hox-1.3 gene is a cognate of the Drosophila Sex combs reduced (Scr) gene, we inserted a hsp 70-Hox-1.3 fusion gene into the Drosophila genome. Transgenic flies displayed Scr-like homeotic transformations after ectopic expression of Hox-1.3 induced by heat shock. In larvae, the thoracic segments T2 and T3 are transformed toward T1. In adults, head structures are dramatically disrupted, including transformation of antenna towards leg. Transformations are not the result of ectopic activation of the endogenous Scr gene. Rather, Hox-1.3 appears to directly regulate Scr target genes, as demonstrated by the ectopic activation of fork head by Hox-1.3. The results suggest that mouse Hox-1.3 cannot only substitute functionally for Drosophila Scr in the determination of external structures but also can participate in the regulatory hierarchy of insect organogenesis.
Induction of the Xenopus organizer: expression and regulation of Xnot, a novel FGF and activin-regulated homeo box gene.
Authors: von Dassow G , Schmidt J E , Kimelman D
Published: June 2007

We have searched for homeo box-containing genes expressed during gastrulation in Xenopus embryos with the expectation that analysis of the spatial and temporal expression of these genes will lead to greater understanding of the regionalization of the mesoderm. We describe the cloning and expression of Xnot, a novel homeo box-containing gene expressed primarily in the gastrula organizing region. We have studied the regulation of Xnot by signaling molecules involved in mesoderm induction and regionalization. Surprisingly, we found that FGF signaling is required for expression of Xnot in the gastrula organizing region, clearly implicating FGF in the induction of dorsal mesoderm. Furthermore, we found that Xnot is initially expressed throughout the embryo and that progressive translation of an unknown protein restricts expression of Xnot to the organizing region. Our results provide experimental evidence supporting the proposed division of Spemann's organizer into independently regulated organizing centers.