bluuebunny/crossref_metadata_2025 huggingface dataset:
The LIM domain-containing homeo box gene Xlim-1 is expressed specifically in the organizer region of Xenopus gastrula embryos.
Authors: Taira M , Jamrich M , Good P J , Dawid I B
Published: June 2007

A novel cysteine-rich motif, named LIM, has been identified in the homeo box genes lin-11, Isl-1, and mec-3; the mec-3 and lin-11 genes determine cell lineages in Caenorhabditis elegans. We isolated LIM class homeo box genes from Xenopus laevis that are closely related to lin-11 and mec-3 in the LIM and homeo domains. This paper deals with one of these genes, Xlim-1. Xlim-1 mRNA is found at low abundance in the unfertilized egg, has a major expression phase at the gastrula stage, decreases, and rises again during the tadpole stage. In adult tissues the brain shows the highest abundance, by far, of Xlim-1 mRNA. The maternal and late expression phases of the Xlim-1 gene suggest that it has multiple functions at different stages of the Xenopus life cycle. In the gastrula embryo, Xlim-1 mRNA is localized in the dorsal lip and the dorsal mesoderm, that is, in the region of Spemann's organizer. Explant experiments showed that Xlim-1 mRNA is induced by the mesoderm-inducer activin A and by retinoic acid, which is not a mesoderm inducer but affects patterning during Xenopus embryogenesis; application of activin A and retinoic acid together results in synergistic induction. The structure, inducibility, and localized expression in the organizer of the Xlim-1 gene suggest that it has a role in establishing body pattern during gastrulation.
The scalloped gene encodes a novel, evolutionarily conserved transcription factor required for sensory organ differentiation in Drosophila.
Authors: Campbell S , Inamdar M , Rodrigues V , Raghavan V , Palazzolo M , Chovnick A
Published: June 2007

The scalloped (sd) gene of Drosophila melanogaster was initially characterized by mutants affecting structures on the wing of the adult fly. The sequence of a cDNA clone of the gene reveals a predicted protein sequence homologous to that of a human transcriptional enhancer factor, TEF-1 (68% identity). The homology includes a sequence motif, the TEA domain, that was shown previously to be a DNA-binding domain of TEF-1. An sd enhancer trap strain expresses the reporter gene in a subset of neuroblasts in the central nervous system and in the peripheral sense organs of the embryo. The reporter gene is later expressed in specific regions of the imaginal discs, including regions of the wing disc destined to become structures defective in viable sd mutants. Later still, expression in the adult brain is restricted to subsets of cells, some in regions involved in the processing of gustatory information. These observations indicate that the sd gene encodes a transcription factor that functions in the regulation of cell-specific gene expression during Drosophila development, particularly in the differentiation of the nervous system.
A density-sensing factor controls development in Dictyostelium.
Authors: Jain R , Yuen I S , Taphouse C R , Gomer R H
Published: June 2007

For an unknown reason, several genes expressed during Dictyostelium development are regulated by cell density. This is mediated by an 80-kD glycoprotein, conditioned medium factor (CMF), which is slowly secreted and simultaneously sensed by starved cells. To examine further this eukaryotic cell density-sensing mechanism, we have isolated a cDNA encoding CMF. The derived amino acid sequence of CMF shows no obvious similarity to any known protein and thus may represent a new class of eukaryotic intercellular signal. CMF antisense transformants do not aggregate, whereas normal development is restored by the addition of purified CMF protein. This suggests that CMF might synchronize the onset of development in Dictyostelium by triggering aggregation when a majority of the cells in a given area have starved, as signaled by CMF secretion.
A Drosophila CREB/ATF transcriptional activator binds to both fat body- and liver-specific regulatory elements.
Authors: Abel T , Bhatt R , Maniatis T
Published: June 2007

We have identified a Drosophila transcription factor that binds to fat body-specific enhancers of alcohol dehydrogenase (Adh) and yolk protein genes. DNA sequence analysis of cDNA clones encoding this protein, box B-binding factor-2 (BBF-2), indicates that it is a member of the CREB/ATF family of transcriptional regulatory proteins. A number of observations suggest that BBF-2 is involved in fat body-specific expression: Mutations that disrupt BBF-2 binding to two different Adh fat body enhancers in vitro decrease the activity of these enhancers in transgenic flies. BBF-2 mRNA is present in all cell types examined, and the protein is present in cells that express ADH. Finally, BBF-2 is a transcriptional activator in Drosophila tissue culture cells. Remarkably, BBF-2 also binds specifically to regulatory elements required for liver-specific expression of the human Adh and rat tyrosine aminotransferase genes. Thus, BBF-2 and the DNA sequence to which it binds may be important components of a tissue-specific regulatory mechanism conserved between Drosophila and man.
Molecular cloning and characterization of PEA3, a new member of the Ets oncogene family that is differentially expressed in mouse embryonic cells.
Authors: Xin J H , Cowie A , Lachance P , Hassell J A
Published: June 2007

The PEA3 motif, first recognized in the polyomavirus enhancer, is an oncogene, serum growth factor, and phorbol ester-responsive element. An activity capable of binding to this sequence, termed PEA3 (polyomavirus enhancer activator 3), was identified in mouse 3T6 cell nuclear extracts. We have cloned cDNAs that encode PEA3 from a mouse FM3A cell cDNA library. A continuous open reading frame in the longest cDNA predicts a 555-amino-acid protein with a calculated molecular mass of 61 kD. Recombinant PEA3 binds to DNA with the same sequence specificity as that endogenous to FM3A cells and activates transcription through the PEA3 motif in HeLa cells. Deletion mapping of the protein revealed that the DNA-binding domain is located within a stretch of 102 amino acids near the carboxyl terminus. This region shares extensive sequence similarity with the ETS domain, a conserved protein sequence common to all ets gene family members. PEA3 is encoded by a 2.4-kb mRNA that is expressed to differing extents in fibroblastic and epithelial cell lines but not in hematopoietic cell lines. In the mouse, PEA3 expression is highly restricted; only the epididymis and the brain contain readily detectable amounts of its mRNA. Interestingly, the amount of PEA3 mRNA is down-regulated during retinoic acid-induced differentiation of mouse embryonic cell lines. These findings suggest that PEA3 plays a regulatory role during mouse embryogenesis.
The Rex system of bacteriophage lambda: tolerance and altruistic cell death.
Authors: Parma D H , Snyder M , Sobolevski S , Nawroz M , Brody E , Gold L
Published: June 2007

The rexA and rexB genes of bacteriophage lambda encode a two-component system that aborts lytic growth of bacterial viruses. Rex exclusion is characterized by termination of macromolecular synthesis, loss of active transport, the hydrolysis of ATP, and cell death. By analogy to colicins E1 and K, these results can be explained by depolarization of the cytoplasmic membrane. We have fractionated cells to determine the intracellular location of the RexB protein and made RexB-alkaline phosphatase fusions to analyze its membrane topology. The RexB protein appears to be a polytopic transmembrane protein. We suggest that RexB proteins form ion channels that, in response to lytic growth of bacteriophages, depolarize the cytoplasmic membrane. The Rex system requires a mechanism to prevent lambda itself from being excluded during lytic growth. We have determined that overexpression of RexB in lambda lysogens prevents the exclusion of both T4 rII mutants and lambda ren mutants. We suspect that overexpression of RexB is the basis for preventing self-exclusion following the induction of a lambda lysogen and that RexB overexpression is accomplished through transcriptional regulation.
Sequence requirements for efficient translational frameshifting in the Escherichia coli dnaX gene and the role of an unstable interaction between tRNA(Lys) and an AAG lysine codon.
Authors: Tsuchihashi Z , Brown P O
Published: June 2007

Synthesis of the gamma-subunit of DNA polymerase III holoenzyme depends on precise and efficient translational frameshifting to the -1 frame at a specific site in the dnaX gene of Escherichia coli. In vitro mutagenesis of this frameshift site demonstrated the importance of an A AAA AAG heptanucleotide sequence, which allows two adjacent tRNAs to retain a stable interaction with mRNA after they slip to the -1 position. The AAG lysine codon present in the 3' half of this heptanucleotide was a key element for highly efficient frameshifting. A tRNA(Lys) with a CUU anticodon, which has a strong affinity for AAG lysine codons, is present in eukaryotic cells but absent in E. coli. Expression in E. coli of a mutant tRNA(Lys) with a CUU anticodon specifically inhibited the frameshifting at the AAG codon, suggesting that the absence of this tRNA in E. coli contributes to the efficiency of the dnaX frameshift.
A novel, activin-inducible, blastopore lip-specific gene of Xenopus laevis contains a fork head DNA-binding domain.
Authors: Dirksen M L , Jamrich M
Published: June 2007

The organizer region, or dorsal blastopore lip, plays a central role in the initiation of gastrulation and the formation of the body axis during Xenopus development. A similar process can also be induced in ectodermal explants by activin or by injection of activin mRNA into embryos. We have searched early embryo-specific cDNA libraries for genes containing the fork head box sequence that encodes a DNA-binding domain similar to that of the Drosophila homeotic gene fork head and rat hepatocyte nuclear factor HFN3 beta. These genes were subsequently tested for expression in the organizer region of blastula/gastrula-stage embryos as well as inducibility by activin. Our effort resulted in the isolation of a gene, XFKH1, that is primarily expressed in the dorsal blastopore lip of early gastrulae and is inducible by activin. At later stages it is expressed in the notochord and neural floor plate. Because of its spatial and temporal expression pattern, as well as its inducibility by activin, this gene is a good candidate to have a regulatory function in the initial processes of axis formation in Xenopus laevis embryos.
The Viviparous-1 gene and abscisic acid activate the C1 regulatory gene for anthocyanin biosynthesis during seed maturation in maize.
Authors: Hattori T , Vasil V , Rosenkrans L , Hannah L C , McCarty D R , Vasil I K
Published: June 2007

The Viviparous-1 (Vp1) gene is required for expression of the C1 regulatory gene of the anthocyanin pathway in the developing maize seed. We show that VP1 overexpression and the hormone, abscisic acid (ABA), activate a reporter gene driven by the C1 promoter in maize protoplasts. Cis-acting sequences essential for these responses were localized. Mutation of a conserved sequence in the C1 promoter abolishes both ABA regulation and VP1 trans-activation. An adjacent 5-bp deletion blocks ABA regulation but not VP1 trans-activation. The latter mutant reconstructs the promoter of c1-p, an allele that is expressed during seed germination but not during seed maturation. We suggest that VP1 activates C1 specifically during maturation by interacting with one or more ABA-regulated transcription factors.
The functional domains of the Drosophila morphogen dorsal: evidence from the analysis of mutants.
Authors: Isoda K , Roth S , Nüsslein-Volhard C
Published: June 2007

The dorsal (dl) protein is a member of the Rel family of transcription factors. It is distributed in a nuclear concentration gradient along the dorsoventral axis of Drosophila embryos and activates or represses a discrete set of zygotic genes in a concentration-dependent manner. The nuclear uptake of the dl protein is stimulated by products of the dorsal group genes but inhibited by the cactus (cact) product. To analyze the functional domains of the dl protein, we sequenced 11 dl alleles and studied their interaction with cact. Four of these alleles were found to result in carboxy-terminal truncations of the protein. A deletion of 80 carboxy-terminal amino acids abolishes the ability of dl protein to activate the expression of mesodermal genes. Larger deletions also affect the repressor function of dl. However, a protein consisting only of the Rel homologous region still acts as a weak repressor of zerknüllt transcription. A missense mutation in the presumptive DNA-binding domain causes a complete lack-of-function phenotype in trans to a deficiency but exerts a dominant-negative effect in trans to a wild-type copy of dl. These and genetic data with the alleles that produce truncated proteins indicate that dl oligomerizes. The proteins truncated at the carboxy-terminal end show increased levels of nuclear uptake dorsally, but they still respond to the cact-mediated inhibition of nuclear transport. Therefore, carboxy-terminal sequences influence the cytoplasmic retention, although a domain of dl-cact interaction residues in the amino-terminal portion.
Control of c-myc mRNA half-life in vitro by a protein capable of binding to a coding region stability determinant.
Authors: Bernstein P L , Herrick D J , Prokipcak R D , Ross J
Published: June 2007

Polysome-associated c-myc mRNA is degraded relatively rapidly in cells and in an in vitro mRNA decay system containing extracts from cultured mammalian cells. Using this system, a competition/screening assay was devised to search for factors that bind to specific regions of polysome-associated c-myc mRNA and thereby alter its half-life. mRNA stability was first assayed in reactions containing exogenous competitor RNAs corresponding to portions of c-myc mRNA itself. The addition of a 182-nucleotide sense strand fragment from the carboxy-terminal portion of the c-myc-coding region destabilized c-myc mRNA by at least eightfold. This RNA fragment had no effect on the stability of other mRNAs tested. Moreover, c-myc mRNA was not destabilized in reactions containing unrelated competitor RNAs or sense strand RNA from the c-myc 5' region. Polysome-associated globin mRNA containing the c-myc-coding region segment in-frame was also destabilized in vitro by the 182-nucleotide RNA. As determined by UV-cross-linking experiments, the 182-nucleotide RNA fragment was recognized by and bound to an approximately 75-kD polysome-associated protein. On the basis of these data plus Northern blotting analyses of c-myc mRNA decay products, we suggest that the approximately 75-kD protein is normally bound to a c-myc-coding region determinant and protects that region of the mRNA from endonuclease attack. Possible links between the protective protein, translation, ribosome pausing, and c-myc mRNA turnover are discussed.
HIV-1 Tat acts as a processivity factor in vitro in conjunction with cellular elongation factors.
Authors: Kato H , Sumimoto H , Pognonec P , Chen C H , Rosen C A , Roeder R G
Published: June 2007

The HIV-1 trans-activator Tat increases the rate of transcription from the HIV-1 LTR promoter through the stem-loop-containing TAR RNA. To analyze the mechanisms of Tat action, a cell-free trans-activation system with no preincubation has been developed. Recombinant Tat specifically increased the level of a long runoff transcript but not a promoter-proximal transcript in a TAR-dependent fashion. These observations and the result of pulse-chase experiments support strongly the hypothesis that Tat enhances the ability of RNA polymerase to elongate over longer distances. Increased levels of the purified cellular factor TFIIF, essential for initiation and also implicated in elongation of transcription, obviated trans-activation by Tat by increasing the basal (Tat-independent) activity. However, another elongation factor, ATN/TFIIS, showed synergistic activation with Tat. An antiserum against a recombinant form of the large subunit of TFIIF (RAP 74) preferentially suppressed the activated level of transcription exerted by Tat. We propose the hypothesis that Tat acts as a processivity factor on RNA polymerase II in an analogous manner to TFIIF.
Fos and Jun repress transcriptional activation by myogenin and MyoD: the amino terminus of Jun can mediate repression.
Authors: Li L , Chambard J C , Karin M , Olson E N
Published: June 2007

Myogenin and MyoD belong to a family of muscle-specific helix-loop-helix (HLH) proteins that have the potential to activate muscle-specific genes in nonmyogenic cells. Peptide growth factors can block the ability of myogenin and MyoD to activate their target genes. Here, we show that the growth factor-inducible proto-oncogenes c-fos, c-jun, and junB mimic the effects of exogenous growth factors and suppress trans-activation of the muscle creatine kinase (MCK) enhancer by myogenin and MyoD. In contrast, JunD, which shares DNA-binding specificity with JunB and c-Jun but is expressed constitutively in muscle cells, is an inefficient inhibitor of the trans-activating capacity of myogenin and MyoD. Transcriptional repression by Fos and Jun is specific to myogenic HLH proteins and is not observed with the widely expressed HLH protein E47, which recognizes the same DNA sequence. Repression of the MCK enhancer by Fos and Jun is targeted at the myogenin and MyoD DNA recognition sequence and can be mediated by the amino terminus of c-Jun. Comparison of several myogenin mutants for their responsiveness to Fos and Jun shows that repression is directed at the basic-HLH region. These results indicate that members of the Jun family can be distinguished on the basis of their effects on muscle-specific transcription and suggest there is cross talk between transcription factors that control myogenesis and those involved in cell proliferation.
I-Rel: a novel rel-related protein that inhibits NF-kappa B transcriptional activity.
Authors: Ruben S M , Klement J F , Coleman T A , Maher M , Chen C H , Rosen C A
Published: June 2007

The NF-kappa B transcription factor complex is comprised of two subunits, p50 and p65, that share significant homology to the rel oncogene. We have isolated a cDNA encoding a novel 66-kD rel-related protein, designated I-Rel. Unlike other rel-related proteins, I-Rel does not interact with DNA. I-Rel forms heterodimers with p50, however, and greatly attenuates its DNA-binding activity--an effect probably resulting from the presence of a domain inhibitory to DNA binding present within the 121 amino-terminal residues of I-Rel. In contrast, I-Rel does not associate with p65. Transfection experiments demonstrate that I-Rel suppresses NF-kappa B-induced transcription, probably through its association with p50. Expression of I-Rel mRNA is induced by mitogenic stimulation and accumulates after the appearance of p50 transcripts. Our findings suggest that p50 and I-Rel are components of a feedback pathway where expression of I-Rel may modulate indirectly the expression of genes responsive to the NF-kappa B transcription factor complex.
Independent modes of transcriptional activation by the p50 and p65 subunits of NF-kappa B.
Authors: Fujita T , Nolan G P , Ghosh S , Baltimore D
Published: June 2007

Recombinant subunits of the transcription factor NF-kappa B, p50 and p65, were analyzed both for binding to various kappa B motifs and in vitro activation. The subunits preferentially form a heterodimer that activates transcription. Although p50 and p65 bind DNA individually as homodimers and are structurally related, their activation mechanisms are distinct. p65 activates transcription by its unique carboxy-terminal activation domain. (p50)2 displays higher affinity DNA binding than (p65)2 for many distinct kappa B motifs and provides strong transcriptional activation only when adopting a chymotrypsin-resistant conformation induced by certain kappa B motifs but not others. Thus, (p50)2 acts as a positive regulator in vitro, consistent with its isolation as a putative constitutive regulator of MHC class I genes. Both subunits of NF-kappa B, therefore, contribute independently to provide regulation at given kappa B motifs.
Analysis of a scrambled gene: the gene encoding alpha-telomere-binding protein in Oxytricha nova.
Authors: Mitcham J L , Lynn A J , Prescott D M
Published: June 2007

Following cell mating in ciliates, a copy of the micronuclear genome is processed into a new macronucleus through massive cutting, reordering, splicing, elimination, and amplification of the DNA. DNA processing includes the deletion of short interrupting elements called internal eliminated sequences (IESs), followed by the splicing of the remaining segments, known as macronuclear destined sequences (MDSs). The MDSs in some micronuclear genes, such as actin I, are scrambled and must be reordered during IES removal and MDS splicing to yield a functional gene. Here, we describe the cloning, sequencing, and characterization of a different scrambling pattern for the gene that encodes the alpha subunit of the telomere-binding protein of Oxytricha nova. The micronuclear gene is made up of 14 MDSs in the scrambled order 1-3-5-7-9-11-2-4-6-8-10-12-13-14. Only the scrambled version is present in the micronucleus, and only the unscrambled version is present in the macronucleus. We propose that unscrambling occurs by homologous recombination guided by pairs of direct repeats at MDS-IES junctions. The patterned array of scrambling may be a clue to the origin of scrambling in this gene.
Polar localization of a bacterial chemoreceptor.
Authors: Alley M R , Maddock J R , Shapiro L
Published: June 2007

The bacterial chemotaxis signal transducer MCP is an integral membrane receptor protein. The chemoreceptor is localized at the flagellum-bearing pole of Caulobacter crescentus swarmer cells. Amino-terminal sequences of the MCP target the protein to the membrane while the carboxy-terminal portion of the protein is responsible for polar localization. The C. crescentus and Escherichia coli MCPs have highly conserved carboxy-terminal domains, and when an E. coli MCP is expressed in C. crescentus, it is targeted to the swarmer cell progeny. These results suggest that subcellular localization of a prokaryotic protein involves interaction of specific regions of the protein with unique cell sites that contain either localized binding proteins or a specific secretory apparatus.
Immortalization of conditionally transformed chicken cells: loss of normal p53 expression is an early step that is independent of cell transformation.
Authors: Ulrich E , Boehmelt G , Bird A , Beug H
Published: June 2007

Clones of mortal chicken fibroblasts and erythroblasts transformed by temperature-sensitive v-src and v-erb B oncoproteins have been developed into immortal cell lines that retain the conditional transformed phenotype. The expressions of two tumor suppressor genes, the retinoblastoma (Rb) gene and the p53 gene, were investigated during senescence, crisis, and cell line establishment. In temperature-sensitive (ts)-v-erb B erythroblasts and ts-v-src fibroblasts (as well as in v-myc macrophages), loss of p53 mRNA or expression of a mutated p53 gene invariably occurred in the early phase of immortalization. In contrast, expression of the Rb gene was unchanged at all stages of immortalization. Inactivation of the original temperature-sensitive oncogene led to loss of the transformed phenotype in fibroblasts and to differentiation in erythroblasts, even in lines that were immortal and lacked p53. The results demonstrate that the process of immortalization is distinct from cell transformation, probably requiring different mutational events.
The Drosophila sloppy paired locus encodes two proteins involved in segmentation that show homology to mammalian transcription factors.
Authors: Grossniklaus U , Pearson R K , Gehring W J
Published: June 2007

The sloppy paired locus is involved in the establishment of the metameric body plan of the Drosophila embryo. We have cloned the sloppy paired locus by P-element-mediated enhancer detection. The locus is composed of two genes, slp1 and slp2, that are structurally and functionally related. They belong to a novel class of putative transcription factors containing a fork head domain that has also been found in mammalian hepatocyte transcription factors. The spatial expression patterns of the two transcripts are very similar, suggesting common regulation of the two genes. We recovered additional sloppy paired alleles by remobilization of an enhancer detector transposon. Genetic analysis suggests that both genes contribute to the segmentation phenotype that has characteristics of both, pair-rule and segment polarity genes, and that they interact functionally. The two genes appear to share an enhancer element situated upstream of slp1 that acts on both the proximal slp1 promoter and the distal slp2 promoter.
Disruption of the sporulation-specific gene spiA in Dictyostelium discoideum leads to spore instability.
Authors: Richardson D L , Loomis W F
Published: June 2007

The spiA gene of Dictyostelium is expressed specifically in prespore cells and spores during culmination, the final stage of development during which prespore and prestalk cells undergo terminal differentiation to form spores and stalk. We have used homologous recombination to delete this gene and have characterized the resulting phenotype. The spiA- strains develop normally and produce spores that are indistinguishable from those of wild-type strains by transmission and scanning electron microscopy. Mutant spores have normal viability when assayed soon after the completion of development, but, as the spiA- spores age, they lose viability more rapidly than those of the spiA+ parent. The drop in viability is more pronounced when spores are submerged in dilute buffer at a concentration that does not allow germination; after 11 days submerged, the viability of spiA- spores is 10(5)-fold reduced, whereas that of the parent is decreased only 10-fold. Reinserting an intact copy of the spiA gene into a spiA- strain restores the stability of its spores. The product of the spiA gene, Dd31, was identified on Western blots as a 30-kD protein using an antibody raised against a fusion protein containing a portion of the coding sequence. Dd31 is associated with the inner face of spore coat fragments in a detergent-resistant manner. This location is consistent with its observed role in maintaining stability of the spores.
Mapping functional specificity in the Dfd and Ubx homeo domains.
Authors: Lin L , McGinnis W
Published: June 2007

To define homeo domain subregions that are important for embryonic targeting specificity of homeotic proteins, we generated a series of Deformed/Ultrabithorax chimeric genes in which parts of the Deformed homeo box region were substituted with Ultrabithorax sequences. Chimeric coding regions were attached to heat shock promoters and introduced into the Drosophila genome by P-element transformation. After heat-induced ectopic expression in embryos, we examined the cuticular phenotypes induced by the resulting chimeric proteins. We also tested the ability of the chimeric proteins to regulate transcription units that are normal targets of Deformed and Ultrabithorax. Our results indicate that specific amino acid residues at the amino end of the Ultrabithorax homeo domain are required to specifically regulate Antennapedia transcription; and in the context of a Deformed protein, these amino-end residues are sufficient to switch from Deformed- to Ultrabithorax-like targeting specificity. Although residues in the amino end of the homeo domain are also important in determining a Deformed-like targeting specificity, other regions of the Deformed homeo domain are also required for full activity.
Control of 4-8S RNA transcription at the midblastula transition in Xenopus laevis embryos.
Authors: Lund E , Dahlberg J E
Published: June 2007

Transcription of Xenopus laevis U1 snRNA genes is subject to a precise program with respect both to the timing of activation at the midblastula transition (MBT) and to the relative levels of the two embryonic U1 RNAs (xU1b1 and b2) that are made. Here, we demonstrate that exogenous xU1b genes injected into developing X. laevis embryos come under the same controls as the endogenous genes. Injected U1 genes, unlike exogenous RNA polymerase III genes, remain quiescent until MBT and their activation at MBT requires protein synthesis during the early cleavage stages. Significantly, the onset of 4-8S RNA transcription occurs at the normal time, even when the DNA content of the embryo has been increased by injection of exogenous DNA or reduced through cleavage arrest, indicating that transcriptional activation at MBT is independent of the ratio of DNA (nucleus) to cytoplasm. In cleavage-arrested (coenocytic) embryos, the reduced level of DNA at MBT results both in a decrease in snRNA and tRNA synthesis (reflecting the lower gene dosage) and in a prolonged synthesis of large amounts of unusual RNA polymerase III transcripts, OAX RNAs. In normally cleaving embryos, small amounts of these unstable OAX RNAs (encoded by satellite I DNA) are synthesized only briefly at MBT. Our demonstration that RNA and DNA metabolism is aberrant in cleavage-arrested embryos requires reevaluation of previous experiments on transcriptional activation that utilized such coenocytic embryos.
Nonsense mutations inhibit splicing of MVM RNA in cis when they interrupt the reading frame of either exon of the final spliced product.
Authors: Naeger L K , Schoborg R V , Zhao Q , Tullis G E , Pintel D J
Published: June 2007

mRNAs R1 and R2 of the autonomous parvovirus minute virus of mice (MVM), which encode the viral nonstructural proteins NS1 and NS2, respectively, are processed in an ordered splicing pathway in which R2 is generated from mature spliced R1. Introduction of translation termination signals into these genes alters the processing of these RNAs; there is a significant (up to fourfold) increase in the accumulated steady-state levels of R1 relative to R2, when compared with wild-type levels, although the total accumulated levels of R1 plus R2 remain the same. The increase in accumulated R1 relative to R2 in mutant infected or transfected murine cells is independent of RNA stability and transport and decreases, in a polar manner, with the distance of the inserted termination signal from the shared initiation codon for NS1 and NS2 at nucleotide 260. The increased ratio of R1 to R2 is a consequence of the artificially introduced translation termination signals acting in cis rather than in the absence of a functional viral gene product. These mutations have an effect when they interrupt previously open reading frames in either exon of the spliced product R2. Nonsense mutations that are located in the second exon of R2 inhibit splicing of R1 to R2 only when they interrupt an open reading frame (ORF) that has the potential, after normal splicing, to be joined in-frame with the initiating AUG. These results suggest that nonsense mutations inhibit splicing of R1 to R2 by influencing the mechanism by which exons are defined in murine cells.
Distinct molecular signals for nuclear import of the nucleolar snRNA, U3.
Authors: Baserga S J , Gilmore-Hebert M , Yang X W
Published: June 2007

Export to the cytoplasm of U3 RNA transcribed from a rat U3 gene injected into the nucleus of Xenopus oocytes indicates that the biogenesis of U3 RNA, like that of the previously studied Sm-precipitable nucleoplasmic snRNAs (U1, U2, U4, and U5), includes a cytoplasmic phase. The regulation of import of the U3 snRNA into the nucleus has been analyzed by injection of synthetic human U3 transcripts into the cytoplasm of Xenopus oocytes. Binding of the major autoantigenic protein of the U3 snRNP, fibrillarin, and cap trimethylation can occur in the cytoplasm, but neither are required for import. The 3'-terminal 13 nucleotides are required for optimal import and cap trimethylation and participate in a phylogenetically conserved U3 structural element, a short 3'-terminal stem. An artificial construct containing the 3'-terminal 13 nucleotides, including the 3'-terminal stem, but only 56 nucleotides of the 217 nucleotides in U3, appears to be sufficient for import. The presence of the 3'-terminal stem in all snRNAs known to be imported suggests that it might be a universal element required for nuclear import.
Selective disruption of genes expressed in totipotent embryonal stem cells.
Authors: von Melchner H , DeGregori J V , Rayburn H , Reddy S , Friedel C , Ruley H E
Published: June 2007

Two retrovirus promoter trap vectors (U3His and U3Neo) have been used to disrupt genes expressed in totipotent murine embryonal stem (ES) cells. Selection in L-histidinol or G418 produced clones in which the coding sequences for histidinol-dehydrogenase or neomycin-phosphotransferase were fused to sequences in or near the 5' exons of expressed genes, including one in the developmentally regulated REX-1 gene. Five of seven histidinol-resistant clones and three of three G418-resistant clones generated germ-line chimeras. A total of four disrupted genes have been passed to the germ line, of which two resulted in embryonic lethalities when bred to homozygosity. The ability to screen large numbers of recombinant ES cell clones for significant mutations, both in vitro and in vivo, circumvents genetic limitations imposed by the size and long generation time of mice and will facilitate a functional analysis of the mouse genome.
A novel modulator domain of Ets transcription factors.
Authors: Wasylyk C , Kerckaert J P , Wasylyk B
Published: June 2007

The ets gene family is composed of several oncogenes and codes for transcription factors. The Ets proteins have a similar sequence called the ets domain and bind to the core motif A/CGGAA. We show here that several members of the ets family have different trans-activating properties. The ets domain of Ets-1 is required for DNA binding. Adjacent to this domain there is a novel element that inhibits DNA binding. It appears to alter the structure of the DNA-binding domain before it interacts with DNA. There is a similar sequence in Ets-2 that also inhibits DNA binding. This sequence is absent in alternative splice products of h-Ets-1. PU1, the most distantly related member of the ets gene family, lacks this novel element. It has a distinct DNA-binding specificity that is determined by DNA sequences outside the core motif. These results have important implications for both the oncogenic and normal functions of ets family members.
A ubiquitous factor is required for C/EBP-related proteins to form stable transcription complexes on an albumin promoter segment in vitro.
Authors: Milos P M , Zaret K S
Published: June 2007

The liver-enriched transcription factor CCAAT/enhancer binding protein (C/EBP) binds to numerous liver-specific promoters, yet the mechanism by which the protein stimulates transcription has not been described. The serum albumin promoter, which is liver specific, contains a strong C/EBP-binding site tightly juxtaposed to a binding site for the ubiquitous factor nuclear factor-Y (NF-Y). The binding of C/EBP impairs the binding of NF-Y; yet surprisingly, this arrangement leads to strong synergistic activation of a minimal promoter in liver nuclear extracts. Transcriptional synergism is manifested by NF-Y facilitating the ability of C/EBP to form preinitiation complexes that are stable through multiple rounds of transcription. Binding by itself, C/EBP stimulates transcription weakly without forming stable complexes, and moving the NF-Y binding site 10 bp away from the C/EBP site increases NF-Y binding in the presence of C/EBP but reduces the efficiency of stable complex formation and transcriptional synergism. These findings show that C/EBP requires precise positioning next to a ubiquitous factor for optimal formation of stable complexes and provides a model to understand the dramatic activation of the albumin gene in hepatic development.
The human heat shock protein hsp70 interacts with HSF, the transcription factor that regulates heat shock gene expression.
Authors: Abravaya K , Myers M P , Murphy S P , Morimoto R I
Published: June 2007

Transcriptional regulation of the human hsp70 gene in response to heat shock and other forms of physiological stress occurs through the activation of heat shock transcription factor (HSF). Exposure of cells to a heat shock temperature of 42 degrees C results in transient activation of HSF; its DNA-binding activity increases rapidly, plateaus, and attenuates, during which the intracellular levels of hsp70 increase. In an effort to understand whether HSF is regulated negatively by hsp70, we have examined whether HSF associates with hsp70. We show that activated HSF associates with hsp70 and that the interaction is detected as the levels of hsp70 increase in the cell. Addition of ATP and other hydrolyzable nucleotides results in the dissociation of hsp70 from HSF while nonhydrolyzable nucleotide analogs do not disrupt the complex. We demonstrate that exogenous recombinant wild-type hsp70 can associate with activated HSF, whereas no association is observed with an amino-terminal or a carboxy-terminal deletion mutant of hsp70. We also show that hsp70 blocks the in vitro activation of HSF from its cryptic non-DNA-binding state to a DNA-binding form; this inhibitory effect of hsp70 is abolished by ATP. We suggest that hsp70 may negatively regulate the activation of HSF.
DnaK and DnaJ heat shock proteins participate in protein export in Escherichia coli.
Authors: Wild J , Altman E , Yura T , Gross C A
Published: June 2007

In Escherichia coli secreted proteins must be maintained in an export-competent state before translocation across the cytoplasmic membrane. This function is carried out by a group of proteins called chaperones. SecB is the major chaperone that interacts with precursor proteins before their secretion. We report results indicating that the DnaK and DnaJ heat shock proteins are also involved in the export of several proteins, most likely by acting as their chaperones. Translocation of alkaline phosphatase, a SecB-independent protein, was inhibited in dnaK- and dnaJ- mutant strains, suggesting that export of this protein probably involves DnaK and DnaJ. In addition, DnaK and DnaJ play a critical role in strains lacking SecB. They are required both for viability and for the residual processing of the SecB-dependent proteins LamB and maltose-binding protein (MBP) seen in secB null strains. Furthermore, overproduction of DnaK and DnaJ permits strains lacking SecB to grow in rich medium and accelerates the processing of LamB and MBP. These results suggest that under conditions where SecB becomes limiting, DnaK and DnaJ probably substitute for SecB and facilitate protein export. This provides the cell with a mechanism to overcome a temporary imbalance in the secretion process caused by an abrupt expansion in the pool of precursor proteins.
Isolation of novel murine maternal mRNAs regulated by cytoplasmic polyadenylation.
Authors: Sallés F J , Darrow A L , O'Connell M L , Strickland S
Published: June 2007

The cytoplasmic polyadenylation element (CPE) is an AU-rich sequence in the 3'-untranslated region of many stored maternal mRNAs. The CPE directs the meiotic maturation-specific cytoplasmic polyadenylation and translational activation of these dormant mRNAs in Xenopus. The work presented here demonstrates that the CPE controls a similar regulation in mouse oocytes and utilizes the information to isolate novel maternal mRNAs by polymerase chain reaction (PCR). A degenerate CPE primer was used in an anchored PCR reaction with cDNAs from primary mouse oocytes. Clones were identified that contained the canonical polyadenylation signal AATAAA. A novel PCR test was then used to determine the polyadenylation state of the respective mRNAs before and after meiotic maturation. Two mRNAs, OM-1 and OM-2, are cytoplasmically polyadenylated upon maturation. Another mRNA is not polyadenylated during maturation, although it contains multiple CPE-like elements, indicating that this sequence element is not sufficient for adenylation during this time. Microinjection into primary oocytes of antisense oligodeoxynucleotides directed against OM-1 destroys the mRNA but does not appear to interfere with maturation in vitro. These experiments identify two novel maternal mRNAs and establish a simple strategy for isolating other maternal messages that control meiotic maturation, fertilization, and early mouse development.
Translational activation of the non-AUG-initiated c-myc 1 protein at high cell densities due to methionine deprivation.
Authors: Hann S R , Sloan-Brown K , Spotts G D
Published: June 2007

c-myc belongs to a small, yet growing, group of eukaryotic mRNAs that initiate translation inefficiently from a non-AUG codon upstream from a more efficient AUG codon. We have examined the translational regulation of non-AUG-initiated c-myc 1 and AUG-initiated c-myc 2 protein synthesis in avian and mouse cells during proliferation. As lymphoid, erythroid, and embryo fibroblast cells approached high densities in culture, there was a sustained 5- to 10-fold induction in the synthesis of c-myc 1 protein to levels greater than or equal to c-myc 2 protein synthesis. Treatment with conditioned/depleted media from high-density cells was able to reproduce this activation in low-density cells within 5 hr. Additional studies with the conditioned/depleted media revealed that amino acid availability, specifically methionine deprivation, was responsible for this unique translational control. Our results describe a specific and dramatic regulation of dual translational initiation. Furthermore, these results represent a novel translational activation of a specific gene in higher eukaryotes in response to nutrient deprivation.
Signal transduction in Saccharomyces cerevisiae requires tyrosine and threonine phosphorylation of FUS3 and KSS1.
Authors: Gartner A , Nasmyth K , Ammerer G
Published: June 2007

The FUS3 and KSS1 kinases are components of the pheromone-dependent signal transduction pathway in yeast. We show that FUS3 and KSS1 become rapidly phosphorylated after pheromone treatment. Similar to mammalian MAP kinases, this modification occurs at two amino acids of FUS3, threonine-180 and tyrosine-182. A mutation introduced at either position results in complete loss of function in vivo. Amino acid substitutions that destroy catalytic activity of the kinase do not prevent phosphorylation of the mutant products, a result that excludes an autocatalytic activation pathway. The modification of FUS3 is dependent on kinases encoded by the STE11 and STE7 genes. Furthermore, a hyperactive allele of STE11 causes increased phosphorylation of FUS3 in the absence of pheromone stimulation. Thus, either STE7 or STE11 could be the kinase responsible for the phosphorylation of FUS3.
Topoisomerases and yeast rRNA transcription: negative supercoiling stimulates initiation and topoisomerase activity is required for elongation.
Authors: Schultz M C , Brill S J , Ju Q , Sternglanz R , Reeder R H
Published: June 2007

Previous work has shown that rRNA synthesis is strongly inhibited in yeast top1-top2 double mutants. Here, we show that inactivation of yeast topoisomerases can have paradoxical effects on transcription by RNA polymerase I. For example, transcription of ribosomal minigenes on extrachromosomal plasmids is greatly stimulated in top1-top2 cells while accumulation of full-length endogenous rRNA is strongly inhibited. We present evidence for a mechanism that can partly account for these opposing effects on transcription. On the one hand, transcription initiation can be stimulated owing to an accumulation of negative superhelicity because polymerase I prefers to initiate on negatively supercoiled templates. Conversely, synthesis of full-length rRNA is inhibited owing to the fact that chain elongation requires a DNA relaxing activity.
The RNA polymerase II ternary complex cleaves the nascent transcript in a 3'----5' direction in the presence of elongation factor SII.
Authors: Izban M G , Luse D S
Published: June 2007

The process by which RNA polymerase II elongates RNA chains remains poorly understood. Elongation factor SII is known to be required to maximize readthrough at intrinsic termination sites in vitro. We found that SII has the additional and unanticipated property of facilitating transcript cleavage by the ternary complex. We first noticed that the addition of SII caused a shortening of transcripts generated by RNA polymerase II at intrinsic termination sites during transcription reactions in which a single NTP was limiting. Truncation of the nascent transcript was subsequently observed using a series of ternary complexes artificially paused after the synthesis of 15-, 18-, 20-, 21-, and 35-nucleotide transcripts. Transcripts as short as 9 or 10 nucleotides were generated in 5-min reactions. All of these shortened RNAs remained in active ternary complexes because they could be chased quantitatively. Continuation of the truncation reaction produced RNAs as short as 4 nucleotides; however, once cleavage had proceeded to within 8 or 9 bases of the 5' end, the resulting transcription complexes could not elongate the RNAs with NTP addition. Transcript cleavage requires a divalent cation, appears to proceed primarily in 2-nucleotide increments, and is inhibited by alpha-amanitin. The catalytic site of RNA polymerase II is repositioned after transcript cleavage such that polymerization resumes at the proper location on the template strand. The extent and kinetics of the transcript truncation reaction are affected by both the position at which RNA polymerase is halted and the sequence of the transcript.
The mechanism of somatic inhibition of Drosophila P-element pre-mRNA splicing: multiprotein complexes at an exon pseudo-5' splice site control U1 snRNP binding.
Authors: Siebel C W , Fresco L D , Rio D C
Published: June 2007

Somatic inhibition restricts splicing of the Drosophila P-element third intron (IVS3) to the germ line. We have exploited this simple system to provide a model for a mechanism of alternative pre-mRNA splicing. Biochemical complementation experiments revealed that Drosophila somatic extracts inhibited U1 snRNP binding to the 5' splice site. Using sensitive RNase protection and modification-interference assays, we found that U1 snRNP bound to a pseudo-5' splice site in the 5' exon and that multiprotein complexes bound to an adjacent site. Binding of these factors appeared to mediate the inhibitory effect, because mutations in the pseudo-5' splice sites blocked binding and activated splicing in vitro. Likewise, wild-type, but not mutant, 5' exon RNA titrated inhibitory factors away from the pre-mRNA and activated splicing. Thus, we have defined the pseudo-5' splice sites as crucial components of the regulatory element, correlated the inhibitory activity with specific RNA binding factors from Drosophila somatic cells, and provided a mechanistic description of somatic inhibition. Because the inhibitory activity involves general splicing functions such as protein recognition of 5' splice site sequences and changes in the distribution of bound U1 snRNP, our data may also provide insights into how splice sites are selected.