bluuebunny/crossref_metadata_2025 huggingface dataset:
csgA expression entrains Myxococcus xanthus development.
Authors: Li S , Lee B U , Shimkets L J
Published: June 2007

The development cycle of the myxobacterium Myxococcus xanthus consists of three partially overlapping morphological stages referred to as rippling, fruiting body formation, and sporulation, all of which are absent in csgA null mutants. The CsgA gene product is an extracellular protein, referred to as the C signal, which is essential for developmental cell-cell interactions. csgA expression increases throughout development, reaching its peak during sporulation. CsgA was made limiting for development by constructing nested deletions upstream from the csgA gene, which resulted in reduced csgA expression. Successively larger deletions resulted in termination of development at earlier and earlier stages, with rippling requiring approximately 20% maximum csgA expression, fruiting body formation requiring approximately 30% expression, and sporulation requiring 82% expression. Conversely, artificial induction of csgA also induced development provided nutrients were limiting. These results suggest that steady increases in CsgA over the course of development entrain the natural sequence of morphological events. The csgA upstream region appears to process information concerning the levels of nutrients, peptidoglycan components, and the B signal. In the absence of nutrients, a region extending 400 bp upstream from the start site of transcription was necessary for development and maximal csgA expression. In the presence of low levels of nutrients, a region extending approximately 930 bp upstream was essential for the same tasks. It appears that the upstream region extending from -400 to -930 stimulates csgA expression in the presence of excess carbon, nitrogen, and phosphate, thereby allowing development to go to completion.
Stable nucleosome positioning and complete repression by the yeast alpha 2 repressor are disrupted by amino-terminal mutations in histone H4.
Authors: Roth S Y , Shimizu M , Johnson L , Grunstein M , Simpson R T
Published: June 2007

Nucleosomes are positioned in the presence of the yeast repressor alpha 2 in minichromosomes containing the alpha 2 operator and on the promoters of a-cell-specific genes regulated by alpha 2. To investigate the possibility that alpha 2 directs nucleosome position through an interaction with a component of the core particle, we analyzed chromatin structures adjacent to the operator in alpha cells containing mutations in the amino-terminal region of histone H4. Deletion or point mutation of specific amino acids in histone H4 altered the location and/or stability of nucleosomes adjacent to the alpha 2 operator. These changes in chromatin structure were accompanied by partial derepression of a beta-galactosidase reporter construct under alpha 2 control, even though alpha 2 remained bound to its operator sequence. Our data suggest that complete repression by alpha 2 requires stable positioning of nucleosomes in promoter regions and this positioning involves the conserved amino-terminal region of histone H4.
DNA binding provides a signal for phosphorylation of the RNA polymerase II heptapeptide repeats.
Authors: Peterson S R , Dvir A , Anderson C W , Dynan W S
Published: June 2007

Isolated transcription complexes contain a protein kinase that phosphorylates the heptapeptide repeats of the carboxy-terminal domain (CTD) of the RNA polymerase II (RNAP II) large subunit in an apparently promoter-dependent manner. We now show that the essential features of this reaction can be reproduced in a reconstituted system containing three macromolecular components: a fusion protein consisting of the CTD of RNAP II fused to a heterologous DNA-binding domain, an activating DNA fragment containing the recognition sequence for the fusion protein, and a protein kinase that binds nonspecifically to DNA. This kinase closely resembles a previously known DNA-dependent protein kinase. Evidently, the association of the CTD with DNA provides a key signal for phosphorylation. There appears to be no absolute requirement for specific contacts with other DNA-bound transcription factors.
A conserved regulatory unit implicated in tissue-specific gene expression in Drosophila and man.
Authors: Falb D , Maniatis T
Published: June 2007

The Drosophila melanogaster alcohol dehydrogenase (Adh) gene is expressed in a specific set of tissues during larval development and in adults. Expression in the adult fat body is controlled by the Adh adult enhancer (AAE). Previous studies identified a negative regulatory element in the AAE and a protein that binds specifically to this sequence [adult enhancer factor-1 (AEF-1)]. Here, we show that the AEF-1-binding site in the AAE and in two other Drosophila fat body enhancers overlaps a sequence recognized by the mammalian transcription factor CCAAT/enhancer-binding protein (C/EBP). Remarkably, these two proteins also bind specifically to overlapping sites in a liver-specific regulatory element of the human Adh gene. Cotransfection experiments in mammalian cells reveal that C/EBP stimulates the activity of the AAE by 50-fold, and this activity can be suppressed by AEF-1. In addition, AEF-1 prevents C/EBP binding in vitro, and displaces prebound C/EBP. Thus, a tissue-specific regulatory unit consisting of one positive and one negative regulatory element has been conserved between Drosophila and man.
The erythroid-specific protein cGATA-1 mediates distal enhancer activity through a specialized beta-globin TATA box.
Authors: Fong T C , Emerson B M
Published: June 2007

The erythroid-specific protein cGATA-1 regulates the chick beta-globin gene through GATA sequences present at the canonical TATA location in the promoter as well as the distal 3' enhancer. We have analyzed beta-globin transcription in transfected erythroid cells and in erythroid extracts to determine whether cGATA-1 binding at -30 regulates promoter or enhancer activity. The interaction of both cGATA-1 and TFIID at different times with the -30 GATA site is required for efficient beta-globin expression in vivo, and the GATA enhancer site can functionally replace the TATA element in the beta-globin promoter. TFIID initiates transcription in vitro by complexing with adaptor proteins and displacing cGATA-1 from the -30 GATA site. Mutations that abolish TFIID binding to the -30 GATA box inactivate the promoter, whereas elimination of cGATA-1 binding to this site selectively diminishes enhancer-dependent transcription. We propose that interaction of cGATA-1 with the distal 3' enhancer and the specialized TATA box confers erythroid specificity to the initiation complex by mediating promoter-enhancer communication. Thus, one mechanism of action for tissue-specific proteins that recognizes noncanonical TATA motifs is to enable TFIID to be regulated by distal control elements. In this way, the initiation complex can be responsive to specific regulators that may not recognize a canonical TFIID-TATA structure.
Antisense CCAAT/enhancer-binding protein RNA suppresses coordinate gene expression and triglyceride accumulation during differentiation of 3T3-L1 preadipocytes.
Authors: Lin F T , Lane M D
Published: June 2007

Previous studies suggest that the CCAAT/enhancer-binding protein (C/EBP) functions in the coordinate expression of adipocyte genes during differentiation of 3T3-L1 preadipocytes. We sought to block expression of C/EBP selectively using a bovine papilloma virus (BPV) vector to direct transcription of a approximately 0.4-kb segment of C/EBP cDNA (in antisense orientation) containing translated sequence 5' to that encoding the basic and leucine zipper regions of the protein. Vector-directed expression of antisense C/EBP RNA in 3T3-L1 preadipocytes inhibited expression of C/EBP mRNA and protein, as well as several adipose-specific mRNAs, and also prevented cytoplasmic triglyceride accumulation. Rescue of the "adipocyte phenotype" was accomplished by transfection of cells expressing antisense RNA with a modified BPV vector that directs transcription of the complementary sense C/EBP RNA.
Transformation by FosB requires a trans-activation domain missing in FosB2 that can be substituted by heterologous activation domains.
Authors: Wisdom R , Yen J , Rashid D , Verma I M
Published: June 2007

Two functionally distinct proteins derived from the FosB gene by alternative splicing have recently been described. FosB protein transforms fibroblasts efficiently, whereas FosB2 protein, a carboxy-terminally truncated form of FosB, does not, despite the fact that both proteins can participate in high-affinity, sequence-specific DNA binding as part of a heterodimeric complex with c-Jun protein. We show here that the functional difference between these proteins is the result of the presence of a potent proline-rich transcriptional activation domain in the carboxy-terminal amino acids unique to FosB. This conclusion is supported by three lines of evidence: (1) Mutations in the carboxy-terminal region of FosB that impair transcriptional activation also reduce transforming potential, despite the fact that DNA binding as part of a complex with c-Jun is not affected; (2) the carboxy-terminal region unique to FosB functions as an activation domain when fused to the DNA-binding domain of GAL4; and (3) transforming potential can be conferred on FosB2 by fusing any of several different well-characterized trans-activation domains. These results identify an additional functional requirement for transformation by Fos proteins and have implications for the mechanism(s) of mitogenic signaling by the AP-1 transcription complex.
Developmental pattern of gene-specific DNA methylation in the mouse embryo and germ line.
Authors: Kafri T , Ariel M , Brandeis M , Shemer R , Urven L , McCarrey J , Cedar H , Razin A
Published: June 2007

Methylation patterns of specific genes have been studied by polymerase chain reaction and found to undergo dynamic changes in the germ line and early embryo. Some CpG sites are methylated in sperm DNA and unmodified in mature oocytes, indicating that the parental genomes have differential methylation profiles. These differences, however, are erased by a series of early embryonic demethylation and postblastula remodification events, which serve to reestablish the basic adult methylation pattern prior to organogenesis. During gametogenesis, all of these sites are unmethylated in primordial germ cells but eventually become remodified by 18.5 days postcoitum in both males and females. The final methylation profile of the mature germ cells is then formed by a multistep process of site-specific demethylation events. These results form a basis for the understanding of the biochemical mechanisms and role of DNA methylation in embryonic development.
A RAP1-interacting protein involved in transcriptional silencing and telomere length regulation.
Authors: Hardy C F , Sussel L , Shore D
Published: June 2007

The yeast RAP1 protein is a sequence-specific DNA-binding protein that functions as both a repressor and an activator of transcription. RAP1 is also involved in the regulation of telomere structure, where its binding sites are found within the terminal poly(C1-3A) sequences. Previous studies have indicated that the regulatory function of RAP1 is determined by the context of its binding site and, presumably, its interactions with other factors. Using the two-hybrid system, a genetic screen for the identification of protein-protein interactions, we have isolated a gene encoding a RAP1-interacting factor (RIF1). Strains carrying gene disruptions of RIF1 grow normally but are defective in transcriptional silencing and telomere length regulation, two phenotypes strikingly similar to those of silencing-defective rap1s mutants. Furthermore, hybrid proteins containing rap1s missense mutations are defective in an interaction with RIF1 in the two-hybrid system. Taken together, these data support the idea that the rap1s phenotypes are attributable to a failure to recruit RIF1 to silencers and telomeres and suggest that RIF1 is a cofactor or mediator for RAP1 in the establishment of a repressed chromatin state at these loci. By use of the two-hybrid system, we have isolated a mutation in RIF1 that partially restores the interaction with rap1s mutant proteins.
SR proteins: a conserved family of pre-mRNA splicing factors.
Authors: Zahler A M , Lane W S , Stolk J A , Roth M B
Published: June 2007

We demonstrate that four different proteins from calf thymus are able to restore splicing in the same splicing-deficient extract using several different pre-mRNA substrates. These proteins are members of a conserved family of proteins recognized by a monoclonal antibody that binds to active sites of RNA polymerase II transcription. We purified this family of nuclear phosphoproteins to apparent homogeneity by two salt precipitations. The family, called SR proteins for their serine- and arginine-rich carboxy-terminal domains, consists of at least five different proteins with molecular masses of 20, 30, 40, 55, and 75 kD. Microsequencing revealed that they are related but not identical. In four of the family members a repeated protein sequence that encompasses an RNA recognition motif was observed. We discuss the potential role of this highly conserved, functionally related set of proteins in pre-mRNA splicing.
Inhibition of muscle differentiation by the adenovirus E1a protein: repression of the transcriptional activating function of the HLH protein Myf-5.
Authors: Braun T , Bober E , Arnold H H
Published: June 2007

Myogenic differentiation can be inhibited by the adenovirus E1a protein in the rat L6 muscle cell line. The present investigation provides evidence that E1a interferes with the expression of myogenin and the activity of Myf-5, the two myogenic helix-loop-helix (HLH) proteins that are expressed in L6 muscle cells. In nuclei of E1a-expressing L6 cells, Myf-5 protein accumulates to normal or even elevated levels and shows no alterations of its ability to bind to the DNA-binding site (CANNTG). However, trans-activation of muscle-specific reporter genes by Myf-5 is strongly inhibited. The same inhibition by E1a can be shown for the other myogenic HLH proteins, MyoD, myogenin, and MRF4/Myf-6, that have been expressed in 10T1/2 fibroblasts. In contrast to the normal level of Myf-5 expression, synthesis of myogenin is entirely abolished in the differentiation-defective L6-E1a cells. Here, we demonstrate that the carboxy-terminal trans-activator domain and probably the basic-HLH (bHLH) region of Myf-5 constitute targets for the inhibition by E1a. The effect of E1a depends on its intact transforming regions but not on the transcriptional activator domain. Our data suggest that activation of myogenin gene expression and the establishment of the differentiated phenotype may require functional Myf-5. Expression of the Myf-5 gene, however, is apparently independent of auto- or cross-regulation by the myogenic HLH proteins.
The Drosophila pourquoi-pas?/wings-down zinc finger protein: oocyte nucleus localization and embryonic requirement.
Authors: Ségalat L , Perichon R , Bouly J P , Lepesant J A
Published: June 2007

The pourquoi-pas? (pqp) gene of Drosophila melanogaster encodes a Cys2/His2 zinc finger protein and is abundantly transcribed in adult ovaries. During oogenesis, we immunodetected the pqp protein in the nucleus of nurse cells at stages 1-6, in a spherical structure within the oocyte nucleus at stages 7-9, and uniformly distributed in the oocyte nucleus and in nurse cell nuclei at later stages. The pqp protein is also present at a lower level in the nuclei of follicle cells, embryos, and larvae. By means of a polymerase chain reaction (PCR) screen, we recovered three independent and phenotypeless P-element insertions at the pqp locus. In a second step, two excision-induced deletions of the pqp gene were isolated after mobilization of one of these P elements. The pqp mutants display zygotic (spread and drooping wings, cross-vein defects, extra bristles) and maternal (embryonic lethality) recessive phenotypes. The chromosomal position (98EF) of the pqp gene and the drooping wing phenotype of the pqp mutants agree with the hypothesis that the pqp gene is the wings down (wdn) gene for which T.H. Morgan isolated (and lost) mutants in the 1920s. This is the first reported occurrence of a zinc finger protein in the nucleus of the Drosophila oocyte.
Inactivation of the human beta-globin gene by targeted insertion into the beta-globin locus control region.
Authors: Kim C G , Epner E M , Forrester W C , Groudine M
Published: June 2007

The human beta-globin locus control region (LCR) is a complex regulatory element that controls the erythroid-specific expression of all cis-linked globin genes. The LCR is composed of five DNase I hypersensitive sites (HS) spanning 16 kb and located greater than 50 kb upstream of the beta-globin gene on chromosome 11. Constructs containing all or some of these HS have been shown to produce high-level erythroid-specific expression of linked genes in transgenic mice and transfected cells. In all transgenic and transfection experiments reported to date, however, the spatial relationships between the LCR and globin genes have been disrupted. We have used homologous recombination (HR) as an approach to gain insights into the potential interactions between the LCR and globin genes in their native locations. A hygromycin B resistance (hygro(R)) gene was inserted into the human beta-globin LCR on chromosome 11 in a mouse/human hybrid erythroid cell line that expresses the human beta-globin gene after the induction of differentiation. As a consequence of this targeted insertion, the beta-globin gene is transcriptionally inactive and not inducible. In contrast, the hygro(R) gene within the LCR is inducible, whereas randomly integrated hygro(R) genes are not inducible in these cells. The chromatin structure of the targeted locus is also altered. A new DNase I HS is present in the enhancer/promoter of the hygro(R) gene inserted into the LCR, whereas a HS normally present in the LCR 3' to the insertion is lost and the beta-globin gene promoter HS is not detectable. These results are consistent with the promoter/enhancer competition model for LCR function and globin gene switching.
Identification of a growth suppression domain within the retinoblastoma gene product.
Authors: Qin X Q , Chittenden T , Livingston D M , Kaelin W G
Published: June 2007

To date, all naturally occurring retinoblastoma susceptibility gene (RB) mutations known to be compatible with stable protein expression map to the T/E1A and cellular protein-binding region (the "pocket" domain). This domain extends from residue 379 to 792. When full-length RB and certain truncated forms were synthesized in human RB -/- cells, we found that the minimal region necessary for overt growth suppression extended from residue 379 to 928. A functional pocket domain and sequences extending from the carboxy-terminal boundary of the pocket to the carboxyl terminus of the protein were both necessary for growth suppression. Both sets of sequences were also required for E2F binding; hence, the two functions may be linked.
Interaction of murine ets-1 with GGA-binding sites establishes the ETS domain as a new DNA-binding motif.
Authors: Nye J A , Petersen J M , Gunther C V , Jonsen M D , Graves B J
Published: June 2007

The proto-oncogene ets-1 is the founding member of a new family of eukaryotic transcriptional regulators. Using deletion mutants of murine ets-1 cDNA expressed in Escherichia coli, we show that the DNA-binding domain corresponds closely to the ETS domain, an 85-amino-acid region that is conserved among ets family members. To investigate the specificity of DNA binding of the ETS domain, we mapped the DNA contacts of a monomeric Ets-1 fragment by chemical protection and interference assays. DNA backbone interactions span a 20-nucleotide region and are localized on one face of the helix. Close phosphate and base contacts are restricted to 10 central nucleotides. Contacts map to the major groove in the center of the site. Flanking minor groove interactions also are predicted. To determine the sequence preference in the close contact zone, we selected a pool of high-affinity binding sites using a purified Ets-1 carboxy-terminal fragment. Our Ets-1-selected consensus, 5'-A/GCCGGAA/TGT/C-3', differs from the binding consensus for the Drosophila ETS domain protein E74A, suggesting that specificity of action of ets family members is mediated by the ETS domain. Compared to other well-characterized classes of DNA-binding proteins, Ets-1 produces a unique pattern of DNA contacts. These studies demonstrate that the ETS domain proteins bind DNA in a novel manner.
Wild-type p53 mediates positive regulation of gene expression through a specific DNA sequence element.
Authors: Zambetti G P , Bargonetti J , Walker K , Prives C , Levine A J
Published: June 2007

It has been reported recently that the wild-type p53 gene product can positively regulate the expression of a test gene adjacent to the enhancer-promoter elements of the murine muscle-specific creatine kinase (MCK) gene. This discussion reports the identification of a wild-type p53 protein-specific DNA-binding element located within the p53-responsive region of the MCK enhancer-promoter element. This p53 protein/DNA-binding element has been defined by DNase I footprint analysis, which identified a 50-bp region. This 50-bp sequence was sufficient to confer wild-type p53 responsiveness on a heterologous minimal promoter. The mutant forms of p53 protein are much less capable of stimulating this DNA element. This study has identified the first example of a naturally occurring wild-type p53-specific DNA-binding element that is able to mediate positive regulation of a test gene. The results suggest a biological function in gene regulation for the wild-type p53 protein that is lost or altered in the mutant p53 proteins.
The Drosophila cellularization gene nullo produces a blastoderm-specific transcript whose levels respond to the nucleocytoplasmic ratio.
Authors: Rose L S , Wieschaus E
Published: June 2007

The initial development of the Drosophila embryo is characterized by rapid nuclear mitosis without cytokinesis. After 13 such mitoses, a coordinated cell division process called cellularization occurs, during which membranes simultaneously enclose each nucleus in a cell. Cellularization requires the establishment of a hexagonal network of actin and myosin filaments in the cortex of the embryo; the filaments are located on the cytoplasmic face of the invaginating membrane furrows. Zygotic expression of the nullo gene is essential for the maintenance of an intact actin-myosin network. We have cloned the nullo gene and present its sequence as well as a characterization of nullo transcript levels in wild-type and mutant embryos. The nullo gene encodes a predicted protein of 213 amino acids, a large proportion of which is basic. nullo transcripts are first detectable at nuclear cell cycle 11, peak in accumulation at the end of cycle 13, and disappear rapidly as cellularization begins. The gene does not appear to be expressed at any other time in the life of the organism. The normal accumulation of nullo transcripts does not require gene activity of other zygotic cellularization genes. The regulation of nullo RNA levels during cycle 14, however, is coupled to the nucleocytoplasmic ratio, which also controls the cessation of rapid, synchronous mitosis just before cellularization.
Order of action of components in the yeast pheromone response pathway revealed with a dominant allele of the STE11 kinase and the multiple phosphorylation of the STE7 kinase.
Authors: Cairns B R , Ramer S W , Kornberg R D
Published: June 2007

The signal transduction pathway that mediates the response of haploid yeast cells to peptide mating pheromones involves several components including the protein kinases STE7 and STE11. We have isolated and characterized a dominant allele of the STE11 gene and have demonstrated that expression of an amino-terminally truncated form of STE11 protein causes constitutive activation of the mating pathway. Expression of this dominant STE11 allele also restored mating ability to certain sterile strains. In conjunction with the results of others, our epistasis results establish the following order of action of pathway components: STE2, GPA1(SCG1), STE4, STE5, STE11, STE7, STE12. Transduction of the signal from STE11 to STE7 may involve phosphorylation because STE7 displays several phosphorylation forms, and STE7 is multiply phosphorylated in response to either pheromone or coexpression of dominant STE11 protein. Further signal propagation appears to require STE7 protein kinase activity, because a catalytically impaired STE7 mutant is defective in the mating response.
A tyrosyl-tRNA synthetase binds specifically to the group I intron catalytic core.
Authors: Guo Q , Lambowitz A M
Published: June 2007

The Neurospora CYT-18 protein, the mitochondrial tyrosyl-tRNA synthetase, functions in splicing group I introns in mitochondria. Here, we show that CYT-18 binds strongly to diverse group I introns that have minimal sequence homology and recognizes highly conserved structural features of the catalytic core of these introns. Inhibition experiments indicate that the intron RNA and tRNA(Tyr) compete for the same or overlapping binding sites in the CYT-18 protein. Considered together with functional analysis, our results indicate that the CYT-18 protein promotes splicing by binding to the intron core and stabilizing it in a conformation required for catalytic activity. Furthermore, the specific binding of the synthetase suggests that the group I intron catalytic core has structural similarities to tRNAs, which could reflect either convergent evolution or an evolutionary relationship between group I introns and tRNAs.
Cachexia and graft-vs.-host-disease-type skin changes in keratin promoter-driven TNF alpha transgenic mice.
Authors: Cheng J , Turksen K , Yu Q C , Schreiber H , Teng M , Fuchs E
Published: June 2007

Tumor necrosis factor alpha (TNF alpha) orchestrates a wide range of effects that combat severe infections in animals. At lower levels, TNF alpha plays an important protective role in stimulating chemotaxis and antimicrobial activity of neutrophils, macrophages, and eosinophils. During chronic illness, TNF alpha secretion can be elevated markedly, giving rise to cachexia, hemorrhage, necrosis and, ultimately, death. Although TNF alpha may mediate many of its effects through macrophages, 30% of TNF alpha injected into animals concentrates in the skin. In recent years, it has been shown that keratinocytes can be induced to synthesize TNF alpha. To explore the role of TNF alpha synthesis in keratinocytes, we used a keratin-14 (K14) promoter to target human TNF alpha expression in the epidermis and other stratified squamous epithelia of transgenic mice. Most mice expressing the K14-TNF alpha transgene stopped gaining weight within 1 week postbirth, and exhibited retarded hair growth. In the skin, adipose production was profoundly inhibited, whereas signs of fibrosis and immune infiltration were evident in the dermis. Over time, the epidermis exhibited an increased stratum corneum, as signs of necrosis began to appear in the skin. Within 3-5 weeks, the mice displayed features characteristic of cachexia and necrosis. Our results suggest that TNF alpha expression by keratinocytes not only plays a role in inflammatory and graft-versus-host-disease-like responses in the skin, but also in other tissues, apparently by virtue of stratified squamous epithelial-derived TNF alpha entering the bloodstream. Our results have enabled the first evaluation of many of the effects of TNF alpha in transgenic animals.
zeste, a nonessential gene, potently activates Ultrabithorax transcription in the Drosophila embryo.
Authors: Laney J D , Biggin M D
Published: June 2007

The GAGA, NTF-1, and zeste proteins have been purified previously from Drosophila embryo extracts and shown to activate the Ultrabithorax (Ubx) promoter in vitro. Here, differently mutated Ubx-promoter constructs containing binding sites for none, one, or all three of these transcription factors have been introduced into Drosophila by P-element transformation. Binding sites for each factor activate dramatically different patterns of transcription. In zeste mutant embryos, the activation by zeste protein-binding sites is essentially abolished. These genetic data, when considered with our earlier biochemical experiments, demonstrate that zeste directly and potently activates Ubx transcription in vivo. Surprisingly, previous genetic experiments indicate that zeste is a nonessential gene shown only to act in a dispensable regulatory process termed transvection. In our transgenic experiments, zeste is not activating transcription by transvection. We propose that the function of zeste in Drosophila is much broader than assumed previously, and that it is a member of a redundant system of transcription factors that regulate and maintain the expression of Ubx and other Drosophila genes.
The basis for the mechanistic bias for deletional over inversional V(D)J recombination.
Authors: Gauss G H , Lieber M R
Published: June 2007

V(D)J recombination between recognition sites in the genome is characterized by certain biases. At some loci, proximal sites undergo recombination substantially more frequently than distal ones. The joining of DH/JH is an example of this. Because the DH element bears signal sequences on each side, inversion would be expected as often as deletion in DH/JH recombination. However, the markedly favored outcome is deletion, entailing utilization of the closer recombination site. One model proposed to explain these biases is the tracking model in which the recombinase tracks from one site to the other. Here, we have directly tested for various types of tracking in V(D)J recombination and have found no indication that it occurs. In addition, we have created DH-JH minilocus substrates for analysis of the basis for the preference for deletion. We find that we can reproduce the deletional bias for the system. Moreover, by flipping the orientation of the D segment, we can reverse the bias such that the frequency of inversions can exceed the number of deletions. These results indicate (1) that there is no intrinsic topological preference in this reaction, and (2) that the sequence of the signal and coding ends determines the bias.
A protein-binding site in the c-myc promoter functions as a terminator of RNA polymerase II transcription.
Authors: Roberts S , Purton T , Bentley D L
Published: June 2007

Termination of transcription not only allows polymerases that have completed RNA synthesis to recycle, but it also has important functions in transcriptional regulation and in preventing promoter interference. The molecular basis for termination by RNA polymerase II (pol II) is unclear, however. We have identified a termination site in the promoter region of the c-myc gene, whose function correlates with DNA binding by a nuclear factor. When the c-myc gene was transcribed in injected Xenopus oocytes or a HeLa nuclear extract, a fraction of RNA initiated at the first promoter, P1, terminated at two positions, T1A and T1B, which flank the TATA box of the second promoter, P2. T1B is a T-rich sequence that resembles previously identified attenuation sites, but T1A appears to represent a different class of termination site. T1A is situated approximately 10 bases upstream of an element that overlaps the P2 TATA box. Mutagenesis of this element affected both the efficiency and the position at which termination occurred. A 28-base sequence including this element caused a low level of termination when inserted into the alpha-globin gene in either orientation. This sequence bound a factor called TBF I (terminator-binding factor), whose binding specificity correlated with T1A terminator function. We suggest that TBF I may function as a pol II termination factor.
Ras mediates translation initiation factor 4E-induced malignant transformation.
Authors: Lazaris-Karatzas A , Smith M R , Frederickson R M , Jaramillo M L , Liu Y L , Kung H F , Sonenberg N
Published: June 2007

Translation initiation factor eIF-4E binds to the eukaryotic mRNA 5' cap structure (m7 GpppN, where N is any nucleotide). eIF-4E is a limiting factor in translation and plays a key role in regulation of translation. We have shown previously that overexpression of eIF-4E in rodent fibroblasts results in tumorigenic transformation. eIF-4E also exhibits mitogenic activity when microinjected into serum-starved NIH-3T3 cells. To understand the mechanisms by which eIF-4E exerts its mitogenic property, we examined the involvement of the Ras signaling pathway in this activity. Here, we report that Ras is activated in eIF-4E-overexpressing cells, as the proportion of GTP-bound Ras is increased. Overexpression of the negative effector of cellular Ras, GTPase activating protein, causes reversion of the transformed phenotype. Furthermore, we show that neutralizing antibodies to Ras, or a dominant-negative mutant of Ras, inhibit the mitogenic activity of eIF-4E. We conclude that eIF-4E exerts its mitogenic and oncogenic activities by the activation of Ras.
Homeotic gene Antennapedia mRNA contains 5'-noncoding sequences that confer translational initiation by internal ribosome binding.
Authors: Oh S K , Scott M P , Sarnow P
Published: June 2007

The Antennapedia (Antp) homeotic gene of Drosophila melanogaster has two promoters, P1 and P2. The resulting Antp mRNAs contain 1512-nucleotide (P1) and 1727-nucleotide (P2) 5'-noncoding regions, composed of exons A, B, D, and E (P1) or exons C, D, and E (P2), respectively. Multiple AUG codons are present in exons A, B, and C. We have found that 252-nucleotide exon D, common to mRNAs from both transcription units and devoid of AUG codons, can mediate initiation of translation by internal ribosome binding in cultured cells. Many mRNAs in Drosophila contain long 5'-noncoding regions with apparently unused AUG codons, suggesting that internal ribosome binding may be a common mechanism of translational initiation, and possibly its regulation, in Drosophila.
Selective nuclear transport of the Drosophila morphogen dorsal can be established by a signaling pathway involving the transmembrane protein Toll and protein kinase A.
Authors: Norris J L , Manley J L
Published: June 2007

Establishment of dorsal-ventral polarity in the early Drosophila embryo requires a concentration gradient of the maternal morphogen dorsal (dl). This concentration gradient is established by selective nuclear transport of dl so that dl protein is present only in ventral nuclei. The activity of 11 genes is required for dl nuclear localization. One of these genes, Toll, encodes a transmembrane protein that appears to play the most direct role in regulating dl localization. We have examined the effects of Toll on dl in cotransfected Schneider cells to gain insight into the nature of the interaction between these proteins. We have found that Toll can enhance the nuclear localization of dl and, independently, the ability of dl to activate transcription once in the nucleus. We present evidence that the signaling pathway from Toll to dl involves protein kinase A (PKA) and that nuclear transport and activation of dl results from phosphorylation of dl by PKA. We discuss the significance of these results with respect both to Drosophila embryogenesis and to the regulation of the mammalian transcription factor NF-kappa B.
CLB5: a novel B cyclin from budding yeast with a role in S phase.
Authors: Epstein C B , Cross F R
Published: June 2007

Budding yeast strains have three CLN genes, which have limited cyclin homology. At least one of the three is required for cell cycle START. Four B cyclins are known in yeast; two have been shown to function in mitosis. We have discovered a fifth B-cyclin gene, called CLB5, which when cloned on a CEN plasmid can rescue strains deleted for all three CLN genes. CLB5 transcript abundance peaks in G1, coincident with the CLN2 transcript but earlier than the CLB2 transcript. CLB5 deletion does not cause lethality, either alone or in combination with other CLN or CLB deletions. However, strains deleted for CLB5 require more time to complete S phase, suggesting that CLB5 promotes some step in DNA synthesis. CLB5 is the only yeast cyclin whose deletion lengthens S phase. CLB5 may also have some role in promoting the G1/S transition, because cln1 cln2 strains require both CLN3 and CLB5 for viability on glycerol media and cln1,2,3- strains require CLB5 for rescue by the Drosophila melanogaster cdc2 gene. In conjunction with cln1,2,3- rescue by CLB5 overexpression and the coincident transcriptional regulation of CLB5 and CLN2, these observations are suggestive of partial functional redundancy between CLB5 and CLN genes.
The acidic activator GAL4-AH can stimulate polymerase II transcription by promoting assembly of a closed complex requiring TFIID and TFIIA.
Authors: Wang W , Gralla J D , Carey M
Published: June 2007

The assembly of activated RNA polymerase II (pol II) transcription complexes has been investigated by assaying whether pre-assembly of intermediate complexes reduces the extended time required for start-site melting. The results show that a closed complex requiring factors IIA, IID, and the acidic activator GAL4-AH forms in a rate-limiting step. This directs the templates into a productive assembly pathway. Factor TFIIB is then added rapidly, affording further protection against diversion into nonproductive pathways. These events are followed by a series of rapid steps in which the remaining general factors are assembled onto the template, which is then melted using the energy of ATP hydrolysis.
The dorsal gradient morphogen regulates stripes of rhomboid expression in the presumptive neuroectoderm of the Drosophila embryo.
Authors: Ip Y T , Park R E , Kosman D , Bier E , Levine M
Published: June 2007

rhomboid (rho) encodes a putative transmembrane receptor that is required for the differentiation of the ventral epidermis. It is initially expressed before the completion of cellularization in lateral stripes within the presumptive neuroectoderm. Here, we present evidence that the maternal morphogen dorsal (dl) acts in concert with basic helix-loop-helix (b-HLH) proteins, possibly including twist (twi), to activate rho in both lateral and ventral regions. Expression is blocked in ventral regions (the presumptive mesoderm) by snail (sna), which is also a direct target of the dl morphogen. A 300-bp region of the rho promoter (the NEE), which is sufficient for neuroectoderm expression, contains a cluster of dl and b-HLH activator sites that are closely linked to sna repressor sites. Mutations in these binding sites cause genetically predicted changes in the levels and limits of rho expression. In particular, the disruption of sna-binding sites causes a derepression of the pattern throughout ventral regions, providing evidence that sna is directly responsible for establishing the mesoderm/neuroectoderm boundary before gastrulation. The tight linkage of activator and repressor sites in the rho NEE is similar to the arrangement of binding sites observed in the even-skipped stripe 2 element, which is regulated by bicoid (bcd). This suggests that the dl and bcd morphogens use a similar mechanism to make stripes in the Drosophila embryo.
Concentration-dependent activities of the even-skipped protein in Drosophila embryos.
Authors: Manoukian A S , Krause H M
Published: June 2007

The Drosophila pair-rule gene even-skipped (eve) encodes a homeo-domain-containing protein (Eve) that is required for the development of both odd- and even-numbered parasegments. We have used a heat shock-inducible eve transgene to study the regulatory functions of Eve in vivo. Transcripts encoded by eight other segmentation genes were monitored for changes in distribution and abundance following short pulses of ectopic Eve expression. Two tiers of response times appeared to distinguish between genes that were direct [fushi tarazu (ftz), odd-skipped (odd), runt (run), paired, and wingless] and indirect [eve, hairy, and engrailed (en)] targets of Eve. Genes that appeared to be directly regulated by Eve were differentially repressed in a concentration-dependent fashion. Interestingly, the run and ftz genes could also be activated by Eve during a brief 20- to 30-min stage in development. The delayed actions upon the eve and en genes appeared to be mediated by run and odd. As in eve- embryos, these effects on segmentation gene expression patterns caused defects in both odd- and even-numbered parasegments. Four sequential phenotypes could be induced, each of which was attributable to the altered expression of a unique subset of target genes.
Conserved motifs in Fos and Jun define a new class of activation domain.
Authors: Sutherland J A , Cook A , Bannister A J , Kouzarides T
Published: June 2007

Fos and Jun form a tight heterodimeric complex that activates transcription by AP1 sites. We have recognized that two adjacent regions of the Jun A1 activation domain are conserved in the Fos protein, and we refer to these two homologous regions as homology box 1 (HOB1) and homology box 2 (HOB2). Using GAL4 chimeras, we show that the HOB1/HOB2 region of Fos and Jun is an independent activation domain in which HOB1 and HOB2 act cooperatively to activate transcription. This cooperativity is retained after the replacement of Fos HOB1 or HOB2 with the equivalent domain of Jun or when duplicated HOB1/HOB1 and HOB2/HOB2 combinations are generated. In the Fos protein, HOB1 or HOB2 can also cooperate with a distinct domain at the carboxyl terminus of the protein. Using the HOB2 consensus sequence as a guide, we identified a HOB2-containing activation domain in the CCAAT/enhancer binding protein (C/EBP) protein. This HOB2 motif can cooperate with as yet undefined sequences in C/EBP and will function even when linked to Jun HOB1. Thus, HOB1 and HOB2 represent inert "cooperating modules" that are combined to generate a functional activation domain. Each of these modules has the potential to cooperate with both distinct and identical domains. The presence of HOB-like modules in three different transcription factors indicates that the HOB motifs characterize a new class of activation domain. These motifs can be used now to identify other transcription factors with such modular characteristics.
Molecular genetics of aristaless, a prd-type homeo box gene involved in the morphogenesis of proximal and distal pattern elements in a subset of appendages in Drosophila.
Authors: Schneitz K , Spielmann P , Noll M
Published: June 2007

Viable aristaless (al) mutations of Drosophila affect pattern elements at both ends of the proximodistal axis in a subset of adult appendages. The al gene has been cloned and identified by P-element-mediated germ-line transformation with a genomic DNA fragment, which rescues a lethal mutation of al as well as aspects of the adult al phenotype. The al gene contains a prd-type homeo domain and a Pro/Gln-rich domain and, hence, probably encodes a transcription factor. Its transcript distribution in third-instar imaginal discs closely corresponds to the anlagen of the tissues that later become visibly affected in adult al mutants. The striking similarity of a bimodal al expression in different imaginal discs indicates that al is under the control of a "prepattern," which is shared at least among antennal, leg, and wing discs. The al gene is also transcribed during embryogenesis. Apart from a function in the ontogeny of specific larval head and tail organs, its embryonic transcript pattern suggests a possible role in early imaginal disc development.
Interactions between Xwnt-8 and Spemann organizer signaling pathways generate dorsoventral pattern in the embryonic mesoderm of Xenopus.
Authors: Christian J L , Moon R T
Published: June 2007

This study analyzes the hierarchy of signals that spatially restrict expression of Xenopus Xwnt-8 to mesodermal cells outside of the Spemann organizer field and examines the potential role that endogenous Xwnt-8 may play in dorsoventral patterning of the embryonic mesoderm. The effects of ectopic introduction of a Nieuwkoop center-like activity or of ectopic expression of goosecoid, on the distribution of endogenous Xwnt-8 transcripts were analyzed. The results of these studies are consistent with the hypothesis that maternally derived signals from the Nieuwkoop center function to positively regulate expression of the homeo box gene goosecoid in Spemann organizer cells, leading to a subsequent repression of Xwnt-8 expression in these cells. This exclusion of Xwnt-8 from cells of the organizer field may be important for normal dorsal development, in that ectopic expression of Xwnt-8 in organizer cells after the midblastula stage, by injection of plasmid DNA, ventralizes the fate of these cells. This is distinct from the previously observed dorsalizing effect of Xwnt-8 when expressed prior to the midblastula stage by injection of RNA. The effects of plasmid-derived Xwnt-8 on isolated blastula animal cap ectoderm were also analyzed. Expression of Xwnt-8 in animal pole ectoderm after the midblastula stage ventralizes the response of dorsal animal pole cells to activin and allows naive ectodermal cells to differentiate as ventral mesoderm in the absence of added growth factors. Collectively, these data are consistent with the hypothesis that Xwnt-8 plays a role in the mesodermal differentiation of ventral marginal zone cells during normal development. Furthermore, endogenous Xwnt-8 may ventralize the response of lateral mesodermal cells to dorsalizing signals from the organizer, thus contributing to the graded nature of the final body pattern.
SinI modulates the activity of SinR, a developmental switch protein of Bacillus subtilis, by protein-protein interaction.
Authors: Bai U , Mandic-Mulec I , Smith I
Published: June 2007

SinR, a 111-amino-acid DNA-binding protein, is a pleiotropic regulator of several late growth processes in Bacillus subtilis. It acts as a developmental switch, positively regulating genes for competence and motility and repressing aprE and stage II sporulation genes. It is encoded by the second gene in a two gene operon, but previous results have also indicated that these two genes are differently regulated. We show in this discussion that the product of sinI, the first open reading frame (ORF) of this operon, interferes with the function of SinR. In vivo experiments have demonstrated that overexpression of sinI results in phenotypes that are observed in cells with a null mutation of sinR. A chromosomal in-frame deletion of sinI gives rise to a phenotype associated with higher levels of SinR. Thus, SinI acts as an antagonist to SinR. In vitro experiments have shown that the interaction between these two proteins is a direct one. SinI prevents SinR from binding to its target sequence on aprE, and the two proteins form a complex that can be immunoprecipitated with antibodies to either SinR or SinI.
Sex-specific transcriptional regulation by the male and female doublesex proteins of Drosophila.
Authors: Coschigano K T , Wensink P C
Published: June 2007

The somatic sexual phenotype of Drosophila is regulated by the sexual differentiation pathway. Male (DSXM) and female (DSXF) proteins encoded by doublesex (dsx), a gene at the end of this pathway, bind to three sites within a 127-bp enhancer that directs sex- and tissue-specific transcription of Yolk protein genes. We describe mutagenesis of these binding sites and the resulting effects on DSXM and DSXF binding in vitro and on gene regulation in wild-type and dsx mutant flies. The results demonstrate that DSXM represses and DSXF activates transcription from the two strongest binding sites. Thus, the pathway regulates sex-specific transcription through the male and female dsx proteins that act directly on the target gene, but with opposite effects.