bluuebunny/crossref_metadata_2025 huggingface dataset:
A tertiary interaction in the Tetrahymena intron contributes to selection of the 5' splice site.
Authors: Downs W D , Cech T R
Published: June 2007

The utilization of cryptic splice sites has been observed in a number of RNA splicing reactions. In the self-splicing group I intron of Tetrahymena thermophila, point mutations of either A57 or A95 promote cleavage at two sites other than the normal 5' splice site, suggesting that these nucleotides are involved in a common tertiary interaction. These results are unusual since A57 and A95 are neither at nor near the 5' splice site in the sequence or secondary structure. Cleavage at the alternative sites appears to occur by intron cyclization, a reaction with well-established structural and mechanistic similarities to the first step of RNA self-splicing. Alternative docking of P1 (the helix containing the 5' splice site paired to the internal guide sequence of the intron) into the catalytic core accounts for cleavage at the cryptic reaction sites. We propose that the A57/A95 interaction, along with an element implicated previously (J1/2), provide structural connectivity from the reaction site in P1 to the catalytic core of the Tetrahymena intron. It seems likely that RNA splicing in general will require such tertiary interactions to position RNA helices.
Specificity determinants for the interaction of lambda repressor and P22 repressor dimers.
Authors: Whipple F W , Kuldell N H , Cheatham L A , Hochschild A
Published: June 2007

The related phage lambda and phage P22 repressors each bind cooperatively to adjacent and separated operator sites, an interaction that involves a pair of repressor dimers. The specificities of these interactions differ: Each dimer interacts with its own type but not with dimers of the heterologous repressor. The two repressors exhibit significant amino acid sequence homology in their carboxy-terminal domains, which are responsible for both dimer formation and the dimer-dimer interaction. Here, we identify a collection of amino acid substitutions that disrupt the protein-protein interaction of DNA-bound lambda repressor dimers and show that several of these substitutions have the same effect when introduced at the corresponding positions of P22 repressor. We use this information to construct a variant of the lambda repressor bearing only six non-wild-type amino acids that has a switched specificity; that is, it binds cooperatively with P22 repressor, but not with wild-type lambda repressor. These results identify a series of residues that determine the specificities of the two interactions.
mPPAR gamma 2: tissue-specific regulator of an adipocyte enhancer.
Authors: Tontonoz P , Hu E , Graves R A , Budavari A I , Spiegelman B M
Published: June 2007

Previously, we have isolated and characterized an enhancer from the 5'-flanking region of the adipocyte P2 (aP2) gene that directs high-level adipocyte-specific gene expression in both cultured cells and transgenic mice. The key regulator of this enhancer is a cell type-restricted nuclear factor termed ARF6. Target sequences for ARF6 in the aP2 enhancer exhibit homology to a direct repeat of hormone response elements (HREs) spaced by one nucleotide; this motif (DR-1) has been demonstrated previously to be the preferred binding site for heterodimers of the retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor (PPAR). We have cloned a novel member of the peroxisome proliferator-activated receptor family designated mPPAR gamma 2, and we demonstrate that a heterodimeric complex of mPPAR gamma 2 and RXR alpha constitute a functional ARF6 complex. Expression of mPPAR gamma 2 is induced very early during the differentiation of several cultured adipocyte cell lines and is strikingly adipose-specific in vivo. mPPAR gamma 2 and RXR alpha form heterodimers on ARF6-binding sites in vitro, and antiserum to RXR alpha specifically inhibits ARF6 activity in adipocyte nuclear extracts. Moreover, forced expression of mPPAR gamma 2 and RXR alpha activates the adipocyte-specific aP2 enhancer in cultured fibroblasts, and this activation is potentiated by peroxisome proliferators, fatty acids, and 9-cis retinoic acid. These results identify mPPAR gamma 2 as the first adipocyte-specific transcription factor and suggest mechanisms whereby fatty acids, peroxisome proliferators, 9-cis retinoic acid, and other lipids may regulate adipocyte gene expression and differentiation.
Regulation of the dorsal morphogen by the Toll and torso signaling pathways: a receptor tyrosine kinase selectively masks transcriptional repression.
Authors: Rusch J , Levine M
Published: June 2007

The dorsal (dl) nuclear gradient initiates the differentiation of the mesoderm, neuroectoderm, and dorsal ectoderm by activating and repressing gene expression in the early Drosophila embryo. This gradient is organized by a Toll signaling pathway that shares many common features with the mammalian IL-1 cytokine pathway. Here we present evidence that a second signaling pathway, controlled by the torso (tor) receptor tyrosine kinase, also modulates dl activity. Evidence is presented that the tor pathway selectively masks the ability of dl to repress gene expression but has only a slight effect on activation. Intracellular kinases that are thought to function downstream of tor, such as D-raf and the rolled MAP kinase, mediate this selective block in repression. Normally, the Toll and tor pathways are both active only at the embryonic poles, and consequently, target genes (zen and dpp) that are repressed in middle body regions are expressed at these sites. Constitutive activation of the tor pathway causes severe embryonic defects, including disruptions in gastrulation and mesoderm differentiation, as a result of misregulation of dl target genes. These results suggest that RTK signaling pathways can control gene expression by antirepression, and that multiple pathways can fine-tune the activities of a single transcription factor.
Apoptosis or retinoblastoma: alternative fates of photoreceptors expressing the HPV-16 E7 gene in the presence or absence of p53.
Authors: Howes K A , Ransom N , Papermaster D S , Lasudry J G , Albert D M , Windle J J
Published: June 2007

A transgenic mouse model for retinoblastoma was produced previously by directing SV40 T antigen expression to retinal photoreceptor cells using the promoter of the interstitial retinol-binding protein (IRBP) gene. This gene becomes active prior to the terminal differentiation of photoreceptors. Because T antigen-transforming activity is attributable, at least in part, to the inactivation of the retinoblastoma (pRb) and p53 tumor suppressor proteins, we addressed the role of p53 in the development of retinoblastoma in mice. Transgenic mice expressing HPV-16 E7 under the control of the IRBP promoter were generated to inactivate pRb in photoreceptors while leaving p53 intact. Rather than developing retinoblastomas, the retinas of these mice degenerate due to photoreceptor cell death at a time in development when photoreceptors are normally undergoing terminal differentiation. The dying cells exhibit the histological and ultrastructural features of apoptosis and contain fragmented DNA. p53 is required for the induction of apoptosis in this model, because mice expressing E7 in a p53 nullizygous background develop retinal tumors instead of undergoing retinal degeneration.
Xenopus embryos regulate the nuclear localization of XMyoD.
Authors: Rupp R A , Snider L , Weintraub H
Published: June 2007

Injection of Xenopus myoD mRNA into Xenopus embryos leads to only a modest activation of myogenic markers. In contrast, we show that injected mouse myoD mRNA leads to a potent activation. We postulate that XMyoD is under negative control in frog embryos, but because of slight sequence differences, mouse MyoD fails to see the negative signal. Whereas mMyoD is constitutively nuclear, XMyoD is largely cytoplasmic except in a region of the embryo that includes the location where mesoderm induction occurs; there, it is nuclear. At MBT, endogenous XmyoD mRNA is expressed ubiquitously in the frog embryo. Our results suggest that this expression would lead to cytoplasmic XMyoD protein. Among other events, muscle induction might remove this negative regulation, allow MyoD to enter the nucleus, and establish an autoregulatory loop that could commit cells to myogenesis.
The RSRF/MEF2 protein SL1 regulates cardiac muscle-specific transcription of a myosin light-chain gene in Xenopus embryos.
Authors: Chambers A E , Logan M , Kotecha S , Towers N , Sparrow D , Mohun T J
Published: June 2007

We have examined the role of two RSRF/MEF2 proteins in the onset of skeletal and cardiac muscle differentiation in early Xenopus embryos. In normal development, zygotic expression of SL1 (MEF2D) precedes that of SL2 (MEF2A) by several hours, but neither gene is expressed prior to the accumulation of MyoD and Myf5 transcripts in the somitic mesoderm. Ectopic expression of the myogenic factors in explants of presumptive ectoderm induces expression of both SL1 and SL2, whereas in reciprocal experiments, neither RSRF protein activates the endogenous myoD or Myf5 genes. We conclude that SL1 and SL2 lie downstream of these myogenic factors in the skeletal myogenic pathway. SL1 is distinguished from SL2 in being expressed in the presumptive heart region of the early tailbud embryo, prior to detection of any markers for cardiac muscle differentiation. Furthermore, ectopic SL1 induces the expression of an endogenous cardiac muscle-specific myosin light-chain (XMLC2) gene in cultured blastula animal pole explants, whereas SL2 has no comparable effect. These results demonstrate that in addition to a possible role in skeletal myogenesis, SL1 also acts in vivo as a regulator of cardiac muscle-specific transcription.
The GLABRA2 gene encodes a homeo domain protein required for normal trichome development in Arabidopsis.
Authors: Rerie W G , Feldmann K A , Marks M D
Published: June 2007

The GLABRA2 gene (GL2) is one of several genes known to have a role in trichome development in Arabidopsis. Mutations at this locus result in abnormal trichome expansion. We have identified several gl2 mutants from a T-DNA-mutagenized population of plants. The T-DNA insert in one of the mutant lines cosegregated with the recessive gl2 phenotype and thus served as a molecular tag to isolate genomic DNA at the putative GL2 locus. RFLP analysis of the segregating population and subsequent molecular complementation experiments established that the GL2 gene had been cloned. The predicted polypeptide from one of the ORFs contained on this fragment showed significant identity to the homeo domain sequence. The construction of a full-length cDNA by RT-PCR confirmed the presence of a homeo box in the GL2 gene and showed that it is substantially different from other recently cloned homeo box genes in plants. The expression pattern of GL2, as demonstrated by in situ hybridization, indicated that the gene is expressed in trichome progenitor cells and at stages associated with trichome development. This suggests that GL2 may regulate events required for the directional cell expansion observed during trichome formation.
The global transcriptional regulators, SSN6 and TUP1, play distinct roles in the establishment of a repressive chromatin structure.
Authors: Cooper J P , Roth S Y , Simpson R T
Published: June 2007

Repression of a-cell specific gene expression in yeast alpha cells requires MAT alpha 2 and MCM1, as well as two global repressors, SSN6 and TUP1. Previous studies demonstrated that nucleosomes positioned adjacent to the alpha 2/MCM1 operator in alpha cells directly contribute to repression. To investigate the possibility that SSN6 and TUP1 provide a link between MAT alpha 2/MCM1 and neighboring histones, nucleosome locations were examined in ssn6 and tup1 alpha cells. In both cases, nucleosome positions downstream of the operator were disrupted, and the severity of the disruption correlated with the degree of derepression. Nevertheless, the observed changes in chromatin structure were not dependent on transcription. Our data strongly indicate that SSN6 and TUP1 directly organize repressive regions of chromatin.
Internal tracts of telomeric DNA act as silencers in Saccharomyces cerevisiae.
Authors: Stavenhagen J B , Zakian V A
Published: June 2007

Telomeric position effect (TPE) refers to the ability of telomeres to repress the transcription of genes in their vicinity. Internal stretches of C1-3A DNA, the sequence found at Saccharomyces telomeres, also repressed transcription of nearby genes. This repression, hereafter called C1-3A-based silencing, was observed at several chromosomal loci, including on a circular chromosome. The magnitude of C1-3A-based silencing was increased by both proximity to a telomere and increased length of the C1-3A tracts. C1-3A-based silencing was affected by many of the same genes and conditions that influence TPE and acted in an orientation-independent manner. Thus, in yeast, an expanded array of a simple repetitive DNA, C1-3A, is sufficient to promote the assembly of a transcriptionally silent chromosomal domain.
The POP1 gene encodes a protein component common to the RNase MRP and RNase P ribonucleoproteins.
Authors: Lygerou Z , Mitchell P , Petfalski E , Séraphin B , Tollervey D
Published: June 2007

Two forms of the yeast 5.8S rRNA are generated from a large precursor by distinct processing pathways. Cleavage at site A3 is required for synthesis of the major, short form, designated 5.8S(S), but not for synthesis of the long form, 5.8S(L). To identify components required for A3 cleavage, a bank of temperature-sensitive lethal mutants was screened for those with a reduced ratio of 5.8S(S):5.8S(L). The pop1-1 mutation (for processing of precursor RNAs) shows this phenotype and also inhibits A3 cleavage. The pre-rRNA processing defect of pop1-1 strains is similar to that reported for mutations in the RNA component of RNase MRP; we show that a mutation in the RNase MRP RNA also inhibits cleavage at site A3. This is the first site shown to require RNase MRP for cleavage in vivo. The pop1-1 mutation also leads to a block in the processing of pre-tRNA that is identical to that reported for mutations in the RNA component of RNase P. The RNA components of both RNase MRP and RNase P are underaccumulated in pop1-1 strains at the nonpermissive temperature, and immunoprecipitation demonstrates that POP1p is a component of both ribonucleoproteins. The POP1 gene encodes a protein with a predicted molecular mass of 100.5 kD and is essential for viability. POP1p is the first protein component of the nuclear RNase P or RNase MRP for which the gene has been cloned.
Disruption of mesoderm and axis formation in fish by ectopic expression of activin variants: the role of maternal activin.
Authors: Wittbrodt J , Rosa F M
Published: June 2007

Formation of mesoderm in Xenopus embryos is the result of an induction event in which peptides such as FGF or activins have been implicated. It was recently demonstrated, by the ectopic expression of a truncated activin receptor, that activin receptor signaling pathways are involved in the processes of mesoderm and axis formation in vivo. However, this approach does not directly address the role of activin itself nor the involvement of activins in the formation of mesoderm in embryos from other vertebrates. In addition, activins are expressed maternally as a protein component of the egg as well as transcribed zygotically, and it is not clear which of the two forms is involved in mesoderm formation. To address those three issues, we analyzed the role of activins in the development of fish embryos by generating two activin dominant-negative variants. One of the variants behaves as an inhibitor of activin protein. The second variant was found to deplete the activin pool when cotranslated with wild-type activin. Injection of RNA encoding these variants into the two-cell embryo of the small teleost fish Oryzias latipes (Japanese medaka) demonstrates that only the maternally provided activin protein is required for mesoderm and axis formation in fish in vivo.
Differential expression of a new dominant agouti allele (Aiapy) is correlated with methylation state and is influenced by parental lineage.
Authors: Michaud E J , van Vugt M J , Bultman S J , Sweet H O , Davisson M T , Woychik R P
Published: June 2007

The agouti gene normally confers the wild-type coat color of mice. Dominant mutations at the agouti locus result in a pleiotropic syndrome that is characterized by excessive amounts of yellow pigment in the coat, obesity, a non-insulin-dependent diabetic-like condition, and the propensity to form a variety of tumors. Here, we describe a new dominant mutation at the agouti locus in which an intracisternal A-particle (IAP) has integrated in an antisense orientation immediately 5' of the first coding exon of the gene. This mutation, which we have named Aiapy, results in the ectopic expression of the agouti gene through the utilization of a cryptic promoter within the IAP 5' long terminal repeat (LTR). The coat color of Aiapy/-mice ranges from solid yellow to a pigment pattern that is similar to wild type (pseudoagouti), and the expressivity of this mutant phenotype varies with parental inheritance. Those offspring with a yellow coat ectopically express agouti mRNA at high levels and exhibit marked obesity, whereas pseudoagouti mice express agouti mRNA at a very low level and their weights do not differ from wild-type littermates. Data are presented to show that the differential expressivity of the Aiapy allele is correlated with the methylation status of the inserted IAP 5' LTR. These data further support the hypothesis that in dominant yellow mutations at the agouti locus, it is the ubiquitous expression of the wild-type agouti coding sequence that is responsible for the yellow coat color, obesity, diabetes, and tumorigenesis.
Autoregulatory control of E2F1 expression in response to positive and negative regulators of cell cycle progression.
Authors: Johnson D G , Ohtani K , Nevins J R
Published: June 2007

Both positive and negative signals govern the progression of cells from G1 into S phase, and a variety of data implicate the E2F transcription factor as a target for the action of one class of negative regulators, the Rb family of growth suppressors. We now find that the E2F1 gene, which encodes one of the components of E2F activity, is subject to autoregulatory control during progression from G0 to S phase and that this primarily reflects a negative control in G0 and early G1, a time when the majority of E2F activity exits as a complex with Rb family members. In addition, we find that deregulated expression of G1 cyclins in quiescent cells stimulates the E2F1 promoter and that this is augmented by coexpression of cyclin-dependent kinases in an E2F-dependent manner. We conclude that the E2F1 gene is a downstream target for G1 cyclin-dependent kinase activity, most likely as a consequence of phosphorylation of Rb family members, and that the autoregulation of E2F1 transcription may provide a sensitive switch for regulating the accumulation of E2F activity during the transition from G1 to S phase.
Multiple DNA elements are required for the growth regulation of the mouse E2F1 promoter.
Authors: Hsiao K M , McMahon S L , Farnham P J
Published: June 2007

To prepare for the DNA synthesis (S) phase of the cell cycle, transcription of many genes required for nucleotide biosynthesis increases. The promoters of several of these genes contain binding sites for the E2F family of transcription factors, and, in many cases, mutation of these sites abolishes growth-regulated transcription. The RNA levels of one family member, E2F1, increase about 15-fold at the G1/S-phase boundary and expression of E2F1 in quiescent cells activates transcription from some G1/S-phase-specific promoters, suggesting that E2F1 plays a critical role in preparing cells to enter S phase. To elucidate the signal transduction pathway leading to the activation of genes required for DNA synthesis, we are investigating the mechanism by which expression of E2F1 is regulated. To determine whether levels of E2F1 mRNA are controlled by changes in promoter activity, we have cloned and characterized the mouse E2F1 promoter. Sequence analysis revealed two sets of overlapping E2F-binding sites located between -12 and -40 relative to the transcription initiation site. We show that these sites bind cellular E2F and that an E2F1 promoter fragment can be activated up to 100-fold by coexpression of E2F proteins. We also show that the activity of this E2F1 promoter fragment increases approximately 80-fold at the G1/S-phase boundary and that this activation is, in part, regulated by G0-specific repression via the E2F sites. However, the E2F sites are not sufficient to mediate growth-regulated transcriptional activity; our results indicate that multiple DNA elements are required for transcription regulation of the E2F1 promoter at the G1/S-phase boundary.
A group III intron is formed from domains of two individual group II introns.
Authors: Hong L , Hallick R B
Published: June 2007

A 1352-nucleotide intron within the Euglena gracilis chloroplast ycf8 gene has been characterized as a complex twintron with overlapping internal introns and alternative splicing pathways. Partially spliced pre-mRNAs were characterized by a combination of cDNA cloning and sequencing, Northern hybridization, and S1 nuclease protection analyses. In the predominant pathway, two internal group II introns (601 and 392 nucleotides) are spliced from subdomain ID of an external group II intron (359 nucleotides). In an alternative pathway, following excision of the 601-nucleotide intron, splicing of a group III intron occurs. This group III intron is recruited from sequences of the external intron and the 392-nucleotide intron. This is the first evidence that a group III intron can be derived from portions of existing group II introns. The mechanism of group III intron formation may also be relevant to the evolution of nuclear introns from putative group II intron ancestors.
The cellular concentration of the sigma S subunit of RNA polymerase in Escherichia coli is controlled at the levels of transcription, translation, and protein stability.
Authors: Lange R , Hengge-Aronis R
Published: June 2007

The second vegetative sigma factor sigma S (encoded by the rpoS gene) is the master regulator in a complex regulatory network that governs the expression of many stationary phase-induced and osmotically regulated genes in Escherichia coli. Using a combination of gene-fusion technology and quantitative immunoblot, pulse-labeling, and immunoprecipitation analyses, we demonstrate here that rpoS/sigma S expression is not only transcriptionally controlled, but is also extensively regulated at the levels of translation and protein stability. rpoS transcription is inversely correlated with growth rate and is negatively controlled by cAMP-CRP. In complex medium rpoS transcription is stimulated during entry into stationary phase, whereas in minimal media, it is not significantly induced. rpoS translation is stimulated during transition into stationary phase as well as by an increase in medium osmolarity. A model involving mRNA secondary structure is suggested for this novel type of post-transcriptional growth phase-dependent and osmotic regulation. Furthermore, sigma S is a highly unstable protein in exponentially growing cells (with a half-life of 1.4 min), that is stabilized at the onset of starvation. When cells are grown in minimal glucose medium, translational induction and sigma S stabilization occur in a temporal order with the former being stimulated already in late exponential phase and the latter taking place at the onset of starvation. Although sigma S does not control its own transcription, it is apparently indirectly involved in a negative feedback control that operates on the post-transcriptional level. Our analysis also indicates that at least five different signals [cAMP, a growth rate-related signal (ppGpp?), a cell density signal, an osmotic signal, and a starvation signal] are involved in the control of all these processes that regulate rpoS/sigma S expression.
Cyclin D1-mediated inhibition of repair and replicative DNA synthesis in human fibroblasts.
Authors: Pagano M , Theodoras A M , Tam S W , Draetta G F
Published: June 2007

Cyclin D1 is a key regulator of the G1 phase of the cell cycle. Inhibition of cyclin D1 function results in cell cycle arrest, whereas unregulated expression of the protein accelerates G1. Cyclin D1 is localized to the nucleus during G1. We found that during repair DNA synthesis, subsequent to UV-induced DNA damage, G1 cells readily lost their cyclin D1 while the proliferating cell nuclear antigen (PCNA) tightly associated with nuclear structures. Microinjection of cyclin D1 antisense accelerated DNA repair, whereas overexpression of cyclin D1 prevented DNA repair and the relocation of PCNA after DNA damage. Coexpression of cyclin D1 with its primary catalytic subunit, Cdk4, or with Cdk2, also prevented repair. In contrast, coexpression of PCNA, which is also a cyclin D1-associated protein, restored the ability of cells to repair their DNA. Acute overexpression of cyclin D1 in fibroblasts prevented them from entering S phase. Again, these effects were abolished by coexpression of cyclin D1 together with PCNA, but not with Cdk4 or Cdk2. Altogether, these results indicate that down-regulation of cyclin D1 is necessary for PCNA relocation and repair DNA synthesis as well as for the start of DNA replication. Cyclin D1 appears to be an essential component of a G1-checkpoint.
Ectopic expression of the CCAAT/enhancer-binding protein alpha promotes the adipogenic program in a variety of mouse fibroblastic cells.
Authors: Freytag S O , Paielli D L , Gilbert J D
Published: June 2007

The CCAAT/enhancer-binding protein alpha (C/EBP alpha) has been implicated in the regulation of adipoblast differentiation. In this study we investigate the potential of C/EBP alpha to promote the adipogenic program in a variety of fibroblastic cells. Transduction of the C/EBP alpha gene into eight mouse fibroblastic cell lines by retroviruses and DNA transfection generates adipocyte colonies at variable frequencies. The most dramatic results are obtained with NIH-3T3 cells, in which the percentage of G418-resistant colonies that exhibit the adipocyte morphology is reproducibly > 50% when the C/EBP alpha gene is transduced by retroviruses. The ability to promote the adipogenic program requires the potent transcriptional activation domain of C/EBP alpha and is not observed with C/EBP beta. Paradoxically, in spite of its antimitogenic effects, clonal cell lines that stably express high amounts of C/EBP alpha can readily be generated. Stable expression of C/EBP alpha in BALB/c-3T3 cells dramatically enhances their ability to terminally differentiate into adipocytes. The results demonstrate that C/EBP alpha can efficiently promote the adipogenic program in a variety of mouse fibroblastic cells, including those that have little or no spontaneous capacity to undergo adipogenesis.
The POZ domain: a conserved protein-protein interaction motif.
Authors: Bardwell V J , Treisman R
Published: June 2007

We describe a novel zinc finger protein, ZID (zinc finger protein with interaction domain). At its amino terminus ZID contains a 120-amino-acid conserved motif present in a large family of proteins that includes both the otherwise unrelated zinc finger proteins, such as Ttk, GAGA, and ZF5, and a group of poxvirus proteins: We therefore refer to this domain as the POZ (poxvirus and zinc finger) domain. The POZ domains of ZID, Ttk, and GAGA act to inhibit the interaction of their associated finger regions with DNA. This inhibitory effect is not dependent on interactions with other proteins and does not appear dependent on specific interactions between the POZ domain and the finger region. The POZ domain acts as a specific protein-protein interaction domain: The POZ domains of ZID and Ttk can interact with themselves but not with each other, POZ domains from ZF5, or the viral protein SalF17R. However, the POZ domain of GAGA can interact efficiently with the POZ domain of Ttk. In transfection experiments, the ZID POZ domain inhibits DNA binding in NIH-3T3 cells and appears to localize the protein to discrete regions of the nucleus. We discuss the implications of multimerization for the function of POZ domain proteins.
Two homeo domain proteins bind with similar specificity to a wide range of DNA sites in Drosophila embryos.
Authors: Walter J , Dever C A , Biggin M D
Published: June 2007

We have used in vivo UV cross-linking to directly measure DNA binding by the homeo domain proteins even-skipped (eve) and fushi tarazu (ftz) in Drosophila embryos. Strikingly, these two proteins bind at uniformly high levels throughout the length of their genetically identified target genes and at lower, but significant, levels to genes that they are not expected to regulate. The data also suggest that these two proteins have very similar DNA-binding specificities in vivo. In contrast, a non-homeo domain transcription factor, zeste, is only detected on short DNA elements within a target promoter and not on other genes. These results are consistent with the in vitro properties of these various proteins, their respective concentrations in the nucleus, and with earlier predictions of how transcription factors bind DNA in vivo. We propose that these data favor the model that eve, ftz, and closely related homeo domain proteins act by directly regulating mostly the same target genes.
Coupling between genome translation and replication in an RNA virus.
Authors: Novak J E , Kirkegaard K
Published: June 2007

The replication of poliovirus RNA genomes containing amber mutations was studied to test whether viral proteins provided in trans could rescue the replication of an RNA genome that could not be completely translated itself. Mutants containing amber codons at different positions in the genome displayed vastly different abilities to be rescued by wild-type proteins provided by a helper genome. Amber-suppressing cell lines were used to ensure that the defects in the amber mutants arose from their failure to be translated, not from defects in RNA sequence or structure. An internal region of the poliovirus genome was identified whose translation is required in cis; failure to translate this region was shown to inhibit RNA replication. This coupling between translation and RNA replication could provide a late proofreading mechanism that enables poliovirus, and possibly many other RNA viruses, to prevent the replication of defective genomes.
Ras/MAP kinase-dependent and -independent signaling pathways target distinct ternary complex factors.
Authors: Hipskind R A , Büscher D , Nordheim A , Baccarini M
Published: June 2007

Transcriptional activation of the immediate early genes c-fos and egr-1 by extracellular signals appears to be mediated by ternary complex factors (TCFs). In BAC-1 macrophages, growth factor stimulation leads to the retardation of protein-DNA complexes containing distinct TCFs. One TCF is recognized by Elk-1 antisera, whereas the other is immunologically related to SAP-1. The appearance and decay of hyperphosphorylated TCF/Elk-1-containing complexes after stimulation coincide with the activation of mitogen-activated protein kinase (MAPK) and the induction and repression of c-fos and egr-1, whereas modified TCF/SAP-1-containing complexes decay more slowly. Suppression of MAPK activation in macrophages and fibroblasts correlates with the failure to induce TCF/Elk-1 hyperphosphorylation without blocking TCF/SAP-1 modification. Accordingly the modified Elk-1 complex is generated in vitro by activated MAPK, whereas that of SAP-1 is not. Expression of a dominant-negative Ras mutant (RasAsn17) in BAC-1 cells does not affect CSF-1-induced TCF/SAP-1 modification while suppressing TCF/Elk-1 phosphorylation. Neither PKC down-regulation by TPA nor inhibition of Gi proteins by pertussis toxin pretreatment influences CSF-1-induced signaling to TCFs. These data indicate the existence of two separate signaling pathways for the modification of distinct TCFs: one dependent on Ras and MAPK and converging on TCF/Elk-1, and the other targeting TCF/SAP-1 independently of Ras and MAPK.
The influenza virus NS1 protein: a novel inhibitor of pre-mRNA splicing.
Authors: Lu Y , Qian X Y , Krug R M
Published: June 2007

We have shown previously that the influenza virus NS1 protein inhibits the nuclear export of mRNAs. Here we demonstrate that the NS1 protein also regulates another post-transcriptional step: It inhibits pre-mRNA splicing both in vivo and in vitro. The mode by which the NS1 protein inhibits pre-mRNA splicing is novel. The pre-mRNA forms spliceosomes, but subsequent catalytic steps in splicing are inhibited. Affinity selection experiments establish that the NS1 protein is associated with the spliceosomes that are formed. The RNA-binding domain of the NS1 protein is required for the inhibition of splicing and for the interaction of the protein with spliceosomes. Because the NS1 protein is associated with U6 snRNA in influenza virus-infected cells as well as in splicing extracts from uninfected cells, it is likely that the NS1 protein also inhibits pre-mRNA splicing in infected cells. Surprisingly, the splicing of the viral ns1 mRNA, the very mRNA that encodes the NS1 protein, was resistant to inhibition by the NS1 protein. This resistance is conferred by sequences in ns1 mRNA.
Short-range repression permits multiple enhancers to function autonomously within a complex promoter.
Authors: Gray S , Szymanski P , Levine M
Published: June 2007

Transcriptional repressors play a key role in establishing localized patterns of gene expression in the early Drosophila embryo. Several different modes of repression have been implicated in previous studies, including competition and direct interference with the transcription complex. Here, we present evidence for "quenching," whereby activators and repressors co-occupy neighboring sites in a target promoter, but the repressor blocks the ability of the activator to contact the transcription complex. This study centers on a zinc finger repressor, snail (sna), which represses the expression of neuroectodermal regulatory genes in the presumptive mesoderm. We show that sna can mediate efficient repression when bound 50-100 bp from upstream activator sites. Repression does not depend on proximity of sna-binding sites to the transcription initiation site. sna is not a dedicated repressor but, instead, appears to block disparate activators. We discuss the importance of quenching as a means of permitting separate enhancers to function autonomously within a complex promoter.
Molecular and genetic characterization of GABP beta.
Authors: de la Brousse F C , Birkenmeier E H , King D S , Rowe L B , McKnight S L
Published: June 2007

This report outlines three observations relating to GABP beta, a polypeptide constituent of the heterotetrameric transcription factor GABP. Evidence is presented showing that the mouse genome encodes two highly related GABP beta polypeptides, designated GABP beta 1-1 and GABP beta 2-1. Genomic and cDNA copies of the newly defined Gabpb2 gene were cloned and characterized, providing the conceptually translated amino acid sequence of GABP beta 2-1. The genes encoding these two proteins, as well as GABP alpha, were mapped to three unlinked chromosomal loci. Although physically unlinked, the patterns of expression of the three genes were strikingly concordant. Finally, the molecular basis of GABP beta dimerization was resolved. Carboxy-terminal regions of the two GABP beta polypeptides, which mediate dimerization, bear highly related primary amino acid sequences. Both sequences are free of alpha-helix destabilizing residues and, when displayed on idealized alpha-helical projections, reveal marked amphipathy. Two observations indicate that these regions adopt an alpha-helical conformation and intertwine as coiled-coils. First, the dimer-forming region of GABP beta 2-1 can functionally replace the leucine zipper of a bZIP transcription factor. Second, a synthetic peptide corresponding to this region shows distinctive helical properties when examined by circular dichroism spectroscopy. Finally, evidence is presented showing that GABP beta 1-1 and GABP beta 2-1 can heterodimerize through this carboxy-terminal domain, but neither protein can heterodimerize via the dimer-forming region of the bZIP protein C/EBP beta.
Abnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice.
Authors: Soriano P
Published: June 2007

Platelet-derived growth factor, a major mitogen and chemoattractant for a number of cell types, is implicated in the processes of wound healing, tumorigenesis, and differentiation and is recognized by two receptors, alpha and beta. To begin understanding the role of these receptors in development, beta-receptor-deficient mice were generated by gene targeting in ES cells. Mutant mice are hemorrhagic, thrombocytopenic, and severely anemic, exhibit a defect in kidney glomeruli because of a lack of mesangial cells, and die at or shortly before birth. However, many cell types and tissues that express the receptor, including major blood vessels and the heart, appear normal in the absence of the receptor. These results indicate that whereas the beta receptor is essential in certain cell types during embryonic development, its broader role may be masked because of compensation by the alpha-subunit.
Sterol-resistant transcription in CHO cells caused by gene rearrangement that truncates SREBP-2.
Authors: Yang J , Sato R , Goldstein J L , Brown M S
Published: June 2007

Sterol-resistant CHO cells (SRD-1 cells) fail to repress sterol synthesis and LDL receptor gene transcription when incubated with 25-hydroxycholesterol. Here we trace the defect to a rearrangement in the gene encoding SREBP-2, a membrane-bound transcription factor that regulates cholesterol homeostasis. SREBP-2 is an 1139-amino acid protein that is bound to extranuclear membranes via a carboxy-terminal attachment domain. In sterol-depleted cells a protease liberates the amino-terminal fragment (approximately 480 amino acids). This fragment, which contains the transcriptional activation and bHLH-Zip domains, translocates to the nucleus. 25-Hydroxycholesterol abolishes protease activity and halts transcription. SRD-1 cells produce a soluble, truncated form of SREBP-2 (amino acids 1-460) that lacks the membrane attachment domain and activates transcription directly, bypassing the sterol-regulated proteolytic step. Although SRD-1 cells produce full-length SREBP-2 from the wild-type allele and a related transcription factor, SREBP-1, they fail to cleave both of these precursors, indicating that the truncated form of SREBP-2 down-regulates the protease through a form of end-product feedback inhibition. The current data provide genetic evidence for the previously proposed model in which cholesterol homeostasis is controlled by sterol-regulated proteolysis of a membrane-bound bHLH-Zip transcription factor.
Targeted disruption of the even-skipped gene, evx1, causes early postimplantation lethality of the mouse conceptus.
Authors: Spyropoulos D D , Capecchi M R
Published: June 2007

Implantation within the mammalian uterus elicits dramatic changes in the growth, differentiation, and morphogenesis of the conceptus. This process is interrupted in mice carrying a targeted disruption of the murine evx1 gene, a homolog of the Drosophila even-skipped (eve) gene. Upon implantation, presumptive evx1- homozygotes elicit a decidual response, invade the uterine epithelium, and attach to the basement membrane between uterine stroma and epithelium, but fail to differentiate extraembryonic tissues or to form egg cylinders prior to resorption. Retrograde analysis of embryo genotypes demonstrates that homozygotes could be isolated as free-floating blastocysts but not as gastrulating egg cylinders. Homozygous mutant blastocysts appeared normal and, when grown in vitro, attach, proliferate, and form trophoblastic giant cells surrounding a growing inner cell mass before rapidly degenerating. In situ hybridization analysis demonstrates evx1 gene expression within the visceral endoderm after implantation and prior to gastrulation, at a time in which the mutant phenotype is first detected.
U4/U5/U6 snRNP recognizes the 5' splice site in the absence of U2 snRNP.
Authors: Konforti B B , Konarska M M
Published: June 2007

Using an in vitro system in which a 5' splice site (5'SS) RNA oligo (AAG decreases GUAAGUAdT) is capable of inducing formation of U2/U4/U5/U6 snRNP complex we show that this oligo specifically binds to U4/U5/U6 snRNP and cross-links to U6 snRNA in the absence of U2 snRNP. Moreover, 5'SS RNA oligo bound to U4/U5/U6 snRNP is chased to U2/U4/U5/U6 snRNP complex upon addition of U2 snRNP. Recognition of the 5'SS by U4/U5/U6 snRNP correlates with the 5'SS consensus sequence. Unlike the interaction with U1 snRNP, this recognition depends largely on interactions other than RNA-RNA base pairing. Finally, the region of U6 snRNA required for this interaction with U4/U5/U6 snRNP is positioned upstream of stem I in the U4-U6 structure. We propose that the 5'SS-U4/U5/U6 snRNP complex is an intermediate in spliceosome assembly and that recognition of the 5'SS by U4/U5/U6 snRNP occurs after the 5'SS-U1 snRNA base pairing is disrupted but before the U4-U6 snRNA structure is destabilized.
The prespliceosome components SAP 49 and SAP 145 interact in a complex implicated in tethering U2 snRNP to the branch site.
Authors: Champion-Arnaud P , Reed R
Published: June 2007

The mammalian spliceosome-associated protein, SAP 49, is associated specifically with U2 snRNP and is the most efficiently UV cross-linked protein in the spliceosomal complexes A, B, and C. We show here that SAP 49 cross-links to a region in the pre-mRNA immediately upstream of the branchpoint sequence in the prespliceosomal complex A. In addition to the RNA-binding activity of SAP 49, we show that this protein interacts directly and highly specifically with another U2 snRNP-associated spliceosomal protein, SAP 145. We have isolated a cDNA-encoding SAP 49 and find that it contains two amino-terminal RNA-recognition motifs (RRMs), consistent with the observation that SAP 49 binds directly to pre-mRNA. The remainder of the protein is highly proline-glycine rich (39% proline and 17% glycine). Unexpectedly, the SAP 49-SAP 145 protein-protein interaction requires the amino-terminus of SAP 49 that contains the two RRMs. The observation that SAP 49 and SAP 145 interact directly with both U2 snRNP and the pre-mRNA suggests that this protein complex plays a role in tethering U2 snRNP to the branch site.
Combined deficiencies of Src, Fyn, and Yes tyrosine kinases in mutant mice.
Authors: Stein P L , Vogel H , Soriano P
Published: June 2007

Three members of the Src family of tyrosine kinases, src, fyn, and yes, are broadly expressed throughout mouse development. Mutations in the c-src and fyn genes were shown previously to lead to restricted nonoverlapping phenotypes only in a subset of cells in which these kinases are expressed. In this work we show that a mutation in the yes gene does not lead to an overt phenotype. Except for brain, the level or distribution of related kinases is not altered in major tissues. To gain further insight into the possibility that these kinases compensate for each other, animals deficient in multiple src-kinases were generated. Whereas most of the src/fyn or src/yes double mutants die perinatally, a substantial proportion of fyn/yes double mutants are viable but undergo degenerative renal changes leading to diffuse segmental glomerulosclerosis. Taken together, these data are consistent with the hypothesis that, at least in some cells, these kinases are able to compensate for the loss of the other related kinases.
Two independent and interactive DNA-binding subdomains of the Pax6 paired domain are regulated by alternative splicing.
Authors: Epstein J A , Glaser T , Cai J , Jepeal L , Walton D S , Maas R L
Published: June 2007

Vertebrate Pax proteins share a conserved 128-amino-acid DNA-binding motif, the paired domain. The PAX6 gene, which is mutated in the murine Small eye and human aniridia developmental defects, also encodes a second protein with a 14-amino-acid insertion in the paired domain. This protein, which arises by alternative mRNA splicing, exhibits unique DNA-binding properties. Unlike other paired domains, which bind DNA predominantly by their amino termini, the extended Pax6 paired domain interacts with DNA exclusively through its carboxyl terminus. This property can be stimulated by deletion of 30 amino-terminal residues from the Pax6 or Pax2 paired domains. Thus, the insertion acts as a molecular toggle to unmask the DNA-binding potential of the carboxyl terminus. The functional nonequivalence of the two Pax6 proteins is underscored by a T-->C mutation at position -3 of the alternative splice acceptor site that changes the ratio of the two isoforms and causes a distinct human ocular syndrome.
An env-like protein encoded by a Drosophila retroelement: evidence that gypsy is an infectious retrovirus.
Authors: Song S U , Gerasimova T , Kurkulos M , Boeke J D , Corces V G
Published: June 2007

The gypsy element of Drosophila differs from most LTR retrotransposons in containing a third open reading frame that resembles retroviral env genes. The protein encoded by ORF3 is glycosylated and processed, like all retroviral envelope proteins. The protein is expressed at high levels in fly strains in which gypsy elements are active. In these strains the protein is found primarily in viral particles. When larvae of fly strains in which gypsy is normally inactive are exposed to sucrose gradient fractions containing these particles, a high level of gypsy insertion activity is observed in their progeny. Thus, gypsy has the expected properties of an insect retrovirus.
The cAMP receptor CAR4 regulates axial patterning and cellular differentiation during late development of Dictyostelium.
Authors: Louis J M , Ginsburg G T , Kimmel A R
Published: June 2007

Pseudoplasmodia of developing Dictyostelium are organized with anteroposterior polarity. We have isolated CAR4, the gene for a new cell-surface, G protein-linked cAMP receptor. CAR4 mRNA is initially expressed during tip elongation and continues to accumulate into culmination. CAR4 is maximally expressed in pseudoplasmodia anteriors which are centers for extracellular cAMP signaling and for organization of cellular patterning. Although car4 null cells progress unperturbed through early development, they exhibit major patterning aberrations as the anteroposterior axis becomes established. Prestalk gene expression is significantly reduced in car4 nulls, whereas prespore-specific markers are overexpressed and detected in zones normally restricted to prestalk cells. Patterning defects are similarly apparent in terminally differentiated fruiting bodies. Our results show that cAMP signaling is required for pattern formation and cellular differentiation during late Dictyostelium development.
Structure-function analysis of the TBP-binding protein Dr1 reveals a mechanism for repression of class II gene transcription.
Authors: Yeung K C , Inostroza J A , Mermelstein F H , Kannabiran C , Reinberg D
Published: June 2007

Dr1, a repressor of class II genes, regulates transcription by a novel mechanism. Biochemical analyses reveal that Dr1 directly interacts with the multiprotein TFIID complex. By use of the yeast two-hybrid system, we demonstrate that the association of Dr1 with the TATA-binding protein (TBP) subunit of TFIID occurs in vivo. In addition, Dr1 can repress transcription from TATA-containing as well as TATA-less promoters in transient transfection assays. Importantly, Dr1-mediated repression can be reversed by overexpression of TBP in vivo. By use of diverse approaches, we mapped two distinct domains in Dr1 required for repression. One domain is essential for the Dr1-TBP interaction, and the second is rich in alanine residues. The TBP-binding domain of Dr1 cannot be replaced by a heterologous DNA-binding domain in mediating repression. We demonstrate that some, but not all, transcriptional activators can reverse Dr1-mediated repression in vivo.