bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of the carboxyl terminus of vertebrate poly(A) polymerase in U1A autoregulation and in the coupling of splicing and polyadenylation.
Authors: Gunderson S I , Vagner S , Polycarpou-Schwarz M , Mattaj I W
Published: June 2007

Interactions required for inhibition of poly(A) polymerase (PAP) by the U1 snRNP-specific U1A protein, a reaction whose function is to autoregulate U1A protein production, are examined. PAP inhibition requires a substrate RNA to which at least two molecules of U1A protein can bind tightly, but we demonstrate that the secondary structure of the RNA is not highly constrained. A mutational analysis reveals that the carboxy-terminal 20 amino acids of PAP are essential for its inhibition by the U1A-RNA complex. Remarkably, transfer of these amino acids to yeast PAP, which is otherwise not affected by U1A protein, is sufficient to confer U1A-mediated inhibition onto the yeast enzyme. A glutathione S-transferase fusion protein containing only these 20 PAP residues can interact in vitro with an RNA-U1A protein complex containing two U1A molecules, but not with one containing a single U1A protein, explaining the requirement for two U1A-binding sites on the autoregulatory RNA element. A mutational analysis of the U1A protein demonstrates that amino acids 103-119 are required for PAP inhibition. A monomeric synthetic peptide consisting of the conserved U1A amino acids from this region has no detectable effect on PAP activity. However, the same U1A peptide, when conjugated to BSA, inhibits vertebrate PAP. In addition to this activity, the U1A peptide-BSA conjugate specifically uncouples splicing and 3'-end formation in vitro without affecting uncoupled splicing or 3'-end cleavage efficiencies. This suggests that the carboxy-terminal region of PAP with which it interacts is involved not only in U1A autoregulation but also in the coupling of splicing and 3'-end formation.
The ROOT HAIR DEFECTIVE3 gene encodes an evolutionarily conserved protein with GTP-binding motifs and is required for regulated cell enlargement in Arabidopsis.
Authors: Wang H , Lockwood S K , Hoeltzel M F , Schiefelbein J W
Published: June 2007

In plants, morphogenesis is largely determined by the orientation and extent of cell enlargement. To define the molecular mechanisms regulating plant cell enlargement, we have conducted a molecular genetic analysis of the ROOT HAIR DEFECTIVE3 (RHD3) gene of Arabidopsis thaliana. Mutations affecting the RHD3 gene were found to alter cell size, but not cell number, in tissues throughout the plant. Genetic and physiological analyses suggest that the RHD3 gene is not required for proper cell type specification, and it is likely to act downstream of the hormones auxin and ethylene. The RHD3 gene was cloned by a T-DNA tagging method and confirmed by the molecular complementation of the rhd3 mutant phenotype and by the analyses of six rhd3 mutant alleles. Consistent with the global effects of the rhd3 mutations, the RHD3 gene is expressed in all major plant organs. The deduced RHD3 product is a novel 89-kD polypeptide with putative GTP-binding motifs near the amino terminus. RHD3-like genes were identified from a protozoan (Entamoeba histolytica), a fungus (Saccharomyces cerevisiae), and another plant species (Oryza sativa), with the sequence identity including the putative GTP-binding motifs. These results imply that the RHD3 protein is a member of a new class of GTP-binding proteins that is widespread in eukaryotes and required for regulated cell enlargement.
The winged helix transcription factor MFH1 is required for proliferation and patterning of paraxial mesoderm in the mouse embryo.
Authors: Winnier G E , Hargett L , Hogan B L
Published: June 2007

The gene mfh1, encoding a winged helix/forkhead domain transcription factor, is expressed in a dynamic pattern in paraxial and presomitic mesoderm and developing somites during mouse embryogenesis. Expression later becomes restricted to condensing mesenchyme of the vertebrae, head, limbs, and kidney. A targeted disruption of the gene was generated by homologous recombination in embryonic stem cells. Most homozygous mfh1 null embryos die prenatally but some survive to birth, with multiple craniofacial and vertebral column defects. Using molecular markers, we show that the initial formation and patterning of somites occurs normally in mutants. Differentiation of sclerotome-derived cells also appears unaffected, although a reduction of the level of some markers [e.g., mtwist, mf1, scleraxis, and alpha1(II) collagen] is seen in the anterior of homozygous mutants. The most significant difference, however, is a marked reduction in the proliferation of sclerotome-derived cells, as judged by BrdU incorporation. This proliferation defect was also seen in micromass cultures of somite-derived cells treated with transforming growth factor beta1 and fibroblast growth factors. Our findings establish a requirement for a winged helix/forkhead domain transcription factor in the development of the paraxial mesoderm. A model is proposed for the role of mfh1 in regulating the proliferation and differentiation of cell lineages giving rise to the axial skeleton and skull.
Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis.
Authors: Molkentin J D , Lin Q , Duncan S A , Olson E N
Published: June 2007

The zinc finger transcription factor GATA4 has been implicated in heart development based on its early expression in precardiogenic splanchnic mesoderm and its ability to activate the expression of a number of cardiac-specific genes. To determine the role of GATA4 in embryogenesis, we generated mice homozygous for a GATA4 null allele. Homozygous GATA4 null mice arrested in development between E7.0 and E9.5 because of severe developmental abnormalities. Mutant embryos most notably lacked a primitive heart tube and foregut and developed partially outside the yolk sac. In the mutants, the two bilaterally symmetric promyocardial primordia failed to migrate ventrally but instead remained lateral and generated two independent heart tubes that contained differentiated cardiomyocytes. We show that these deformities resulted from a general loss in lateral to ventral folding throughout the embryo. GATA4 is most highly expressed within the precardiogenic splanchnic mesoderm at the posterior lip of the anterior intestinal portal, corresponding to the region of the embryo that undergoes ventral fusion. We propose that GATA4 is required for the migration or folding morphogenesis of the precardiogenic splanchnic mesodermal cells at the level of the AIP.
Inhibition of cyclin D1 phosphorylation on threonine-286 prevents its rapid degradation via the ubiquitin-proteasome pathway.
Authors: Diehl J A , Zindy F , Sherr C J
Published: June 2007

The expression of D-type G1 cyclins and their assembly with their catalytic partners, the cyclin-dependent kinases 4 and 6 (CDK4 and CDK6), into active holoenzyme complexes are regulated by growth factor-induced signals. In turn, the ability of cyclin D-dependent kinases to trigger phosphorylation of the retinoblastoma (Rb) protein in the mid- to late G1 phase of the cell cycle makes the inactivation of Rb's growth suppressive function a mitogen-dependent step. The ability of D-type cyclins to act as growth factor sensors depends not only on their rapid induction by mitogens but also on their inherent instability, which ensures their precipitous degradation in cells deprived of growth factors. However, the mechanisms governing the turnover of D-type cyclins have not yet been elucidated. We now show that cyclin D1 turnover is governed by ubiquitination and proteasomal degradation, which are positively regulated by cyclin D1 phosphorylation on threonine-286. Although "free" or CDK4-bound cyclin D1 molecules are intrinsically unstable (t1/2 < 30 min), a cyclin D1 mutant (T286A) containing an alanine for threonine-286 substitution fails to undergo efficient polyubiquitination in an in vitro system or in vivo, and it is markedly stabilized (t1/2 approximately 3.5 hr) when inducibly expressed in either quiescent or proliferating mouse fibroblasts. Phosphorylation of cyclin D1 on threonine-286 also occurs in insect Sf9 cells, and although the process is enhanced significantly by the binding of cyclin D1 to CDK4, it does not depend on CDK4 catalytic activity. This implies that another kinase can phosphorylate cyclin D1 to accelerate its destruction and points to yet another means by which cyclin D-dependent kinase activity may be exogenously regulated.
Direct regulation of rhodopsin 1 by Pax-6/eyeless in Drosophila: evidence for a conserved function in photoreceptors.
Authors: Sheng G , Thouvenot E , Schmucker D , Wilson D S , Desplan C
Published: June 2007

Pax-6 is a transcription factor containing both a homeodomain (HD) and a Paired domain (PD). It functions as an essential regulator of eye development in both Drosophila and vertebrates, suggesting an evolutionarily conserved origin for different types of metazoan eyes. Classical morphological and phylogenetic studies, however, have concluded that metazoan eyes have evolved many times independently. These apparently contradictory findings may be reconciled if the evolutionarily ancient role of Pax-6 was to regulate structural genes (e.g., rhodopsin) in primitive photoreceptors, and only later did it expand its function to regulate the morphogenesis of divergent and complex eye structures. In support of this, we present evidence that eyeless (ey), which encodes the Drosophila homolog of Pax-6, directly regulates rhodopsin 1 (rh1) expression in the photoreceptor cells. We detect ey expression in both larval and adult terminally differentiated photoreceptor cells. We show that the HD of Ey binds to a palindromic HD binding site P3/RCS1 in the rh1 promoter, which is essential for rh1 expression. We further demonstrate that, in vivo, P3/RCS1 can be replaced by binding sites specific for the PD of Ey. P3/RCS1 is conserved in the promoters of all Drosophila rhodopsin genes as well as in many opsin genes in vertebrates. Mutimerized P3 sites in front of a basal promoter are able to drive the expression of a reporter gene in all photoreceptors. These results suggest that Pax-6/Ey directly regulates rhodopsin 1 gene expression by binding to the conserved P3/RCS1 element in the promoter.
Distribution of pre-mRNA splicing factors at sites of RNA polymerase II transcription.
Authors: Neugebauer K M , Roth M B
Published: June 2007

If pre-mRNA splicing begins during RNA synthesis, then transcriptionally active genes may be expected to contain high concentrations of pre-mRNA splicing factors. However, previous studies have localized splicing factors to a network of "speckles," which is distinct from individual sites of gene transcription where pre-mRNA is spliced. Speckles have been detected with antibodies specific for splicing snRNPs and members of the SR family of splicing factors. Here we report that dilution of these probes results in the visualization of hundreds of sites throughout the HeLa cell nucleus, the size and distribution of which are consistent with transcription units viewed with light microscopy. Importantly, these sites of highest SR protein concentration frequently coincide in three-dimensional space with active sites of RNA polymerase II transcription. A newly developed reagent specific for a single member of the SR family, SRp20, detects a subset (approximately 20%) of these sites, suggesting the gene-specific accumulation of these splicing regulators, which have distinct functions in pre-mRNA splicing. These observations question the view that the nucleus and its functions are highly compartmentalized; instead, they support a model in which the localization of these and possibly other gene regulators is determined primarily by their function.
The Rhizobium meliloti PII protein, which controls bacterial nitrogen metabolism, affects alfalfa nodule development.
Authors: Arcondéguy T , Huez I , Tillard P , Gangneux C , de Billy F , Gojon A , Truchet G , Kahn D
Published: June 2007

Symbiotic nitrogen fixation involves the development of specialized organs called nodules within which plant photosynthates are exchanged for combined nitrogen of bacterial origin. To determine the importance of bacterial nitrogen metabolism in symbiosis, we have characterized a key regulator of this metabolism in Rhizobium meliloti, the uridylylatable P(II) protein encoded by glnB. We have constructed both a glnB null mutant and a point mutant making nonuridylylatable P(II). In free-living conditions, P(II) is required for expression of the ntrC-dependent gene glnII and for adenylylation of glutamine synthetase I. P(II) is also required for efficient infection of alfalfa but not for expression of nitrogenase. However alfalfa plants inoculated with either glnB mutant are nitrogen-starved in the absence of added combined nitrogen. We hypothesize that P(II) controls expression or activity of a bacteroid ammonium transporter required for a functional nitrogen-fixing symbiosis. Therefore, the P(II) protein affects both Rhizobium nitrogen metabolism and alfalfa nodule development.
Isolation of chicken vitellogenin I and III cDNAs and the developmental regulation of five estrogen-responsive genes in the embryonic liver.
Authors: Evans M I , Silva R , Burch J B
Published: June 2007

The isolation of cDNA clones that code for portions of the two minor chicken vitellogenin (VTG) genes (VTGI and VTGIII) is reported. These clones represent unique sequences that are expressed exclusively in the livers of estrogenized birds. In the liver of the egg-laying hen, the levels of RNAs encoding VTGI, VTGII, and VTGIII are approximately 11,000, 30,000, and 3,000 molecules per cell, respectively. We have used the newly isolated clones, as well as the yolk protein cDNAs previously available [VTGII, apolipoprotein II (apoVLDLII), and apolipoprotein B], as probes to examine several aspects of the regulation of these genes by estradiol. First, we demonstrate that the capacity of each gene to respond to estradiol is acquired between 8 and 13 days in ovo. The response of four of these genes to estradiol is diminished during late fetal development, but the responsiveness is recovered within a week after hatching. Second, we demonstrate that these genes display distinct kinetic response profiles following the addition of estradiol. Third, as has been described previously for the VTGII and apoVLDLII genes, we demonstrate that a single injection of estradiol effects a long-term reprogramming event (hepatic memory) that allows a faster onset of the rapid accumulation of both VTGI and VTGIII RNAs following a subsequent rechallenge by estradiol. Collectively, these three sets of data suggest molecular parameters that may contribute to both the coordinate and noncoordinate regulation of this set of genes by estradiol.
Pattern of transcription of the homeo gene Hox-3.1 in the mouse embryo.
Authors: Le Mouellic H , Condamine H , Brûlet P
Published: June 2007

A cDNA from the Hox-3.1 locus, isolated from a 10.5-day postcoitum (p.c.) mouse embryo cDNA library, and the putative encoded protein are described. The spatial distribution of Hox-3.1 gene transcripts from late gastrulation to embryonic day 14.5 p.c. was monitored by in situ hybridization, using a cDNA probe. When first detectable in 8.5-day p.c. embryos, the transcripts are distributed in all the tissues of the posterior end. At later stages, the distribution becomes progressively spatially restricted and tissue specific. By 12.5 days p.c., transcription is localized most intensely in the neural tube region lying above the heart. The early transcription pattern thus appears to be compatible with a regionalizing role for the Hox-3.1 gene.
Cell-cycle regulation of a human histone H2b gene is mediated by the H2b subtype-specific consensus element.
Authors: LaBella F , Sive H L , Roeder R G , Heintz N
Published: June 2007

Mammalian histone gene transcription is increased approximately fivefold during the transition from the G1 phase to the S phase of the cell cycle. In this study, we present a detailed in vivo analysis of the human histone H2b promoter, which establishes that transcriptional regulation of this gene is mediated by a subtype-specific consensus element containing the core octanucleotide ATTTGCAT. Our results demonstrate that the activity of this sequence is specific for S phase. Comparative analysis of different replication variant mammalian histone gene promoters and our knowledge of the transcription factors interacting with the human histone H2b and H4 promoters allow us to conclude that coordinate regulation of histone gene transcription in higher eukaryotes is mediated by distinct factors. We propose a simple model for transcriptional regulation of mammalian histone gene expression, which incorporates both the distinct features of the individual histone gene promoters and the apparent functional equivalence of the specific sequence elements regulating transcription of each histone gene subtype.
Functional mapping of the human papillomavirus type 11 transcriptional enhancer and its interaction with the trans-acting E2 proteins.
Authors: Hirochika H , Hirochika R , Broker T R , Chow L T
Published: June 2007

The transcriptional enhancer sequences of the papillomaviruses are regulated by trans-acting factors encoded by the viral E2 open reading frame. We have performed detailed functional and physical analyses of the enhancer of the human papillomavirus type 11 (HPV-11). Using the chloramphenicol acetyltransferase (CAT) assay in transiently transfected monkey CV-1 cells, the enhancer region has been localized to a 270-bp tract immediately preceding the E6 open reading frame, and it consists of two functional components. The first is a constitutive enhancer containing sequences homologous to the GT-, Sph-, and P-motifs found in the SV40 and polyomavirus enhancers; others resemble the recognition sequence for CTF (NF-1), a factor which stimulates transcription of certain eukaryotic genes and replication of adenovirus DNA. The second component is an inducible enhancer with a consensus sequence ACCN6GGT responsive to the E2 protein encoded by papillomaviruses. Tandem copies of portions of the constitutive enhancer function as an E2-independent enhancer, whereas multiple copies of HPV-11 DNA restriction fragments or synthetic oligonucleotides containing the E2-responsive sequence (E2-RS) act as an enhancer in the presence of the E2 protein encoded by HPV-1, HPV-11, or bovine papillomavirus type 1 (BPV-1). The inducible activity is lost when mutations are introduced into the E2-RS or when a mutant palindromic sequence is substituted. We have also expressed the E2 proteins of HPV-1, HPV-11, and BPV-1 in Escherichia coli and studied their physical interactions with the E2-responsive sequence in vitro. Filter-binding analyses with crude Escherichia coli lysates show that the E2 proteins bind to the E2-RS, but not to mutated motifs, with an affinity proportional to the copy number. These E2 proteins have been purified to near-homogeneity by sequence-specific DNA affinity chromatography using the synthetic E2-RS as a ligand. The purified proteins protect a DNA segment containing the E2-RS and several flanking nucleotides in pancreatic DNase I footprinting analyses. Based on these results, we conclude that E2 proteins activate the enhancer by binding directly to the E2-RS and interacting with other transcriptional factors and that the sequence ACCN6GGT is both necessary and sufficient for the E2 protein binding in vitro and for activation of RNA transcription in vivo.
The Drosophila su(Hw) gene, which controls the phenotypic effect of the gypsy transposable element, encodes a putative DNA-binding protein.
Authors: Parkhurst S M , Harrison D A , Remington M P , Spana C , Kelley R L , Coyne R S , Corces V G
Published: June 2007

Homozygous mutations at the suppressor of Hairy-wing [su(Hw)] locus reverse the phenotype of gypsy-induced alleles in a number of genes located throughout the Drosophila genome. To understand the molecular basis of this phenomenon, the su(Hw) locus was isolated by chromosomal walking from a cloned homeo-box-containing sequence. The exact location of the gene was determined by Southern analysis of the DNA alterations associated with several su(Hw) alleles. A 9.5-kb KpnI-SalI fragment, where all the DNA changes associated with su(Hw) mutations were mapped, was able to rescue the su(Hw) mutant phenotype after P-element-mediated germ-line transformation. This DNA fragment encodes a 3.3-kb RNA that is expressed in all stages of Drosophila development; the size or abundance of this RNA is affected in several su(Hw) alleles tested. This transcript encodes a protein that contains a highly acidic region and 12 repeats of the 'Zn finger' domain characteristic of some DNA-binding and transcription-activating proteins, supporting the hypothesis that the su(Hw) locus might encode a transcription factor that plays a role in the expression of the gypsy element.
Octamer-binding proteins from B or HeLa cells stimulate transcription of the immunoglobulin heavy-chain promoter in vitro.
Authors: LeBowitz J H , Kobayashi T , Staudt L , Baltimore D , Sharp P A
Published: June 2007

The B-cell-type specificity of the immunoglobulin (Ig) heavy-chain and light-chain promoters is mediated by an octanucleotide (OCTA) element, ATGCAAAT, that is also a functional component of other RNA polymerase II promoters, such as snRNA and histone H2B promoters. Two nuclear proteins that bind specifically and with high affinity to the OCTA element have been identified. NF-A1 is present in a variety of cell types, whereas the presence of NF-A2 is essentially confined to B cells, leading to the hypothesis that NF-A2 activates cell-type-specific transcription of the Ig promoter and NF-A1 mediates the other responses of the OCTA element. Extracts of the B-cell line, BJA-B, contain high levels of NF-A2 and specifically transcribe Ig promoters. In contrast, extracts from HeLa cells transcribed the Ig promoter poorly. Surprisingly, addition of either affinity-enriched NF-A2 or NF-A1 to either a HeLa extract or a partially purified reaction system specifically stimulates the Ig promoter. This suggests that the constitutive OCTA-binding factor NF-A1 can activate transcription of the Ig promoter and that B-cell-specific transcription of this promoter, at least in vitro, is partially due to a quantitative difference in the amount of OCTA-binding protein. Because NF-A1 can stimulate Ig transcription, the inability of this factor to activate in vivo the Ig promoter to the same degree as the snRNA promoters probably reflects a difference in the context of the OCTA element in these two types of promoters.
Molecular mechanisms in the developmental regulation of the maize Suppressor-mutator transposable element.
Authors: Banks J A , Masson P , Fedoroff N
Published: June 2007

The maize Suppressor-mutator (Spm) element can exist in one of three heritable forms: (1) a stably active form, (2) a stably inactive form, termed cryptic, and (3) a labile form, here termed programmable, in which the element exhibits one of a variety of heritable developmental programs of expression. Active elements are transcribed and are hypomethylated at sites upstream of the transcription start site, whereas inactive elements are transcriptionally silent and largely methylated at the upstream sites. Active (both stable and programmable), inactive programmable, and cryptic elements are unmethylated, partially methylated, and fully methylated, respectively, at sites within an 0.35-kb 80% G + C region just downstream from the transcription start site. An active Spm element in a genome with a cryptic element promotes its partial demethylation but not its transcriptional activation. In contrast, a trans-acting Spm promotes extensive demethylation and transcriptional activation of an inactive programmable element, as well as its heritable reactivation. These observations define the molecular components of the Spm element's developmental regulatory mechanism. We discuss their general relevance to the developmental regulation of gene expression.
Characterization of a murine homeo box gene, Hox-2.6, related to the Drosophila Deformed gene.
Authors: Graham A , Papalopulu N , Lorimer J , McVey J H , Tuddenham E G , Krumlauf R
Published: June 2007

The Hox-2 locus on chromosome 11 represents one of the major clusters of homeo-box-containing genes in the mouse. We have identified two new members (Hox-2.6 and Hox-2.7), which form part of this cluster of seven linked genes, and it appears that the Hox-2 locus is related by duplication and divergence to at least one other mouse homeo box cluster, Hox-1. The Hox-2.6 gene encodes a predicted protein of 250 amino acids, which displays extensive similarity in multiple regions to certain mouse, human, Xenopus, and zebra fish homeo domain proteins. The Drosophila Deformed (Dfd) gene also shares these same regions of similarity, and based on this sequence conservation, we suggest that Hox-2.6 forms part of a vertebrate 'Dfd-like' family. Hox-2.6 is expressed in fetal and adult tissues and is modulated during the differentiation of F9 teratocarcinoma stem cells. In situ hybridization analysis of mouse embryos shows that the Hox-2.6 is expressed in ectodermal derivatives: spinal cord, hindbrain, dorsal root ganglia, and the Xth cranial ganglia. In the central nervous system, expression is observed in the most posterior parts of the spinal cord, with the anterior limit residing in a region of the hindbrain and no expression in the mid- or forebrain. In mesodermal structures, Hox-2.6 is expressed in the kidney, the mesenchyme of the stomach and lung, and the longitudinal muscle layer of the gut. Expression has not been observed in derivatives of embryonic endoderm. The patterns of Hox-2.6 expression in both mesoderm and ectoderm are spatially restricted and may reflect a role for the gene in the response to or establishment of positional cues in the embryo.
Constitutive and metal-inducible protein:DNA interactions at the mouse metallothionein I promoter examined by in vivo and in vitro footprinting.
Authors: Mueller P R , Salser S J , Wold B
Published: June 2007

A method of high resolution in vivo footprinting has been developed and used to survey the mouse metallothionein I (MT-I) promoter for protein : DNA interactions associated with basal-level transcription and with high-level metal-induced transcription. This promoter and its associated regulatory region is structurally complex. It contains multiple potential binding sites for metal regulatory factors and for other transcription factors, including SP1 and MLTF. In several cases potential recognition sites overlap, and the experiments reported here provide a view of which sites are utilized in vivo. These data also show how the pattern of protein : DNA contacts changes when cells are shifted from basal-level expression to metal-induced expression. The noninduced footprint pattern consists of interactions at basal elements that are thought to be responsible for the moderate transcription of this gene in the absence of added metals. These interactions remain unchanged upon metal induction. When MT-I expression is increased by exposing cells to zinc or cadmium, a new footprint pattern is observed. It includes the basal interactions and a new set of metal-dependent footprints that are positioned over all five genetically defined metal responsive elements (MREs), MRE-A--MRE-E. In addition, these data identify a sixth probable MRE, MRE-F, which displays a dimethylsulfate (DMS) footprint similar to that at other MREs.
A chemical modification/interference study of yeast pre-mRNA spliceosome assembly and splicing.
Authors: Rymond B C , Rosbash M
Published: June 2007

A chemical modification/interference assay was used to determine the yeast pre-mRNA sequence requirements for in vitro spliceosome assembly and splicing. Modifications of any of the nucleotides within the 5' splice site and branch point (TACTAAC box) consensus sequences as well as less conserved intron and exon positions were found to inhibit assembly and/or splicing. The interference pattern of the 5' splice site and TACTAAC box lesions increased as spliceosome assembly proceeded (complex III----complex I----complex II) and as splicing proceeded, suggesting that these sequence elements play multiple roles in the assembly of yeast spliceosomes and in the removal of intervening sequences. Furthermore, modification (or mutation) of a TACTAAC-like sequence upstream of the branch point was found to inhibit the rate of spliceosome assembly, implying a possible role for degenerate branch point sequences in modulating the efficiency of spliceosome assembly.
Expression of an activated ras gene causes developmental abnormalities in transgenic Drosophila melanogaster.
Authors: Bishop J G , Corces V G
Published: June 2007

We describe the developmental effects of the expression of a normal and mutated Drosophila ras gene ras2 in transgenic Drosophila. A Gly14----Val14 mutant (Gly14 in ras2 is equivalent to Gly12 in mammalian ras proteins) was constructed in ras2 by site-directed mutagenesis. Inducible constructs of ras2 and ras2Val14 were made by coupling the hsp70 promoter to each of the genes. In addition, the endogenous ras2 promoter was placed upstream of the mutated ras2Val14. These constructs were introduced into the germ line of Drosophila to yield transgenic lines by P-element-mediated transformation. A wide variety of developmental disorders were displayed in flies carrying the mutated ras2 gene driven by either the endogenous or the hsp70 promoter. The basal level of transcription of mutated ras from the uninduced hsp70 promoter was sufficient to produce disturbances in the development of several tissue types. In contrast, phenotypic disturbances were not seen with the normal ras2 gene driven by the hsp70 promoter even when induced by heat shock to very high levels of transcription over normal ras2 levels. A subset of the tissues expressing ras2 during development was particularly sensitive to the expression of ras2Val14. The compound eye was found to develop a dorsal-to-ventral 'scar' correlated with the wave of differentiation occurring in the eye imaginal disc at the time of a single brief induction of the hsp70 promoted ras2Val14 construct.
Translational inactivation of ribosomal protein mRNAs during Xenopus oocyte maturation.
Authors: Hyman L E , Wormington W M
Published: June 2007

Ribosomal protein synthesis ceases upon maturation of Xenopus oocytes. We find that this cessation results from the dissociation of ribosomal protein mRNAs from polysomes and is accompanied by the deadenylation of these transcripts. A synthetic mRNA encoding ribosomal protein L1, microinjected into stage VI oocytes, is deadenylated and released from polysomes upon maturation. Our results indicate that sequences located within 387 bp of the 3' terminus of L1 mRNA direct both the deadenylation and polysomal release of this ribosomal protein mRNA. The proper translational regulation of an exogenous ribosomal protein mRNA in microinjected oocytes provides a basis for determining the sequence specificity for the differential utilization of maternal mRNAs during oocyte maturation.
Molecular cloning and transcript analysis of fem-3, a sex-determination gene in Caenorhabditis elegans.
Authors: Rosenquist T A , Kimble J
Published: June 2007

The fem-3 gene is required for specification of the male fate in the nematode Caenorhabditis elegans: XO males need fem-3 for male differentiation in both soma and germ line; XX hermaphrodites need it for the production of sperm. We have cloned fem-3 by transposon tagging. Among eight spontaneous fem-3 mutations generated in a strain in which the transposable element Tc1 is mobile, six contain Tc1 insertions in a 2-kb region of the genome. From this region, we have identified three fem-3 transcripts. Two, 1.7 kb and 1.62 kb, are presented in embryos, and two, 1.62 kb and 1.55 kb, are present in L4s and adults. The fem-3 transcripts are not XO specific; however, in XX adult hermaphrodites, they appear to be limited to the germ line--a tissue involved in male development (both for spermatogenesis and for the maternal contribution of fem-3 to the embryo). The amount of fem-3 RNA in XO embryos is approximately sixfold greater than in XX embryos. The significance of this difference in specifying male development in XO but not in XX embryos is discussed.
Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression.
Authors: Triezenberg S J , Kingsbury R C , McKnight S L
Published: June 2007

Mature virions of herpes simplex virus type 1 contain an activating factor that primes transcription from the five virally encoded immediate early (IE) genes. This activator is specified by a 65-kD polypeptide termed VP16. The action of VP16 is mediated through cis-regulatory elements located in regions adjacent to each IE gene. Although VP16 is normally introduced into cells by infecting virions, its trans-activating function can also be observed by cotransfecting cells with a plasmid that encodes VP16 along with a reporter gene driven by IE cis-regulatory sequences. We have used such an assay to examine the function of mutant forms of VP16. Our results provide tentative identification of two domains of VP16 that are crucial to its role in the induction of IE gene expression. One domain is located within the carboxy-terminal 78 amino acids of VP16 and is characterized by its acidity. Another domain, located in a more amino-terminal region of the protein, appears to tailor the specificity of VP16 for IE genes. According to the results presented in this and the accompanying paper, we predict that VP16 achieves IE gene specificity via protein: protein, rather than protein: DNA, interaction.
The half-life of immunoglobulin mRNA increases during B-cell differentiation: a possible role for targeting to membrane-bound polysomes.
Authors: Mason J O , Williams G T , Neuberger M S
Published: June 2007

The increase in abundance of immunoglobulin heavy-chain (IgH) mRNA that accompanies development of a B cell into a plasma cell is mainly due to post-transcriptional events. By constructing mu genes, whose expression is under inducible control, we determined the half-life of pulsed mu transcripts to be approximately 20 hr in plasmacytoma hosts and approximately 3 hr in B-cell lymphomas. Interestingly, a mu gene with a mutated signal sequence that is found on free (rather than membrane-bound) polysomes decays in the plasmacytoma host with a shortened half-life of 3 hr. Thus, a change in the turnover rate of IgH mRNA plays an important role in the cell-type specificity of immunoglobulin gene expression; this change may be a consequence of the fact that IgH mRNA is located in the membrane-bound polysome fraction.
Expression, modification, and localization of the fushi tarazu protein in Drosophila embryos.
Authors: Krause H M , Klemenz R , Gehring W J
Published: June 2007

The fushi tarazu (ftz) protein of Drosophila is required during embryogenesis for the process of body segmentation. To study the biochemical properties of the ftz protein, ftz cDNA was expressed in Escherichia coli and the protein was purified to homogeneity. Polyclonal antibodies raised against the purified protein were used to localize and quantitate the protein during embryogenesis. Three temporally and spatially distinct phases of expression were observed, which include a previously undetected period later in embryogenesis. During this last phase, the protein is localized predominantly in the developing hindgut. Analysis of embryonic ftz protein on Western blots permitted us to approximate the number of protein molecules per nucleus. During the blastoderm phase of development, when ftz protein is most abundant, we estimate that there are approximately 20,000 molecules of protein per ftz-expressing nucleus. The embryonic ftz protein migrates more slowly on SDS-polyacrylamide gels than protein made either in E. coli or in a reticulocyte lysate system in vitro, indicating that it is modified in the embryo. To facilitate characterization of ftz protein made in embryos, an ftz overexpression system functional in Drosophila was developed. When fused to an hsp70 heat shock promoter and introduced into the germ line by P-element-mediated transformation, ftz could be overexpressed at all stages of development by heat shock. This protein is localized in the nucleus comigrates on SDS-polyacrylamide gels with endogenous ftz protein. Two-dimensional gel electrophoresis followed by Western blotting resolves the overexpressed protein into a series of isoforms that differ in charge and electrophoretic mobility. Post-translational modification may influence the biochemical properties and functions of the ftz protein during embryogenesis.
Region-specific alleles of the Drosophila segmentation gene hairy.
Authors: Howard K , Ingham P , Rushlow C
Published: June 2007

An essential step in the Drosophila segmentation mechanism is the expression of the pair-rule gene hairy in a periodic pattern in the blastoderm. In this paper we describe four regulatory hairy mutations. Using in situ hybridization, we show that these mutations cause alterations in the normal pattern of hairy expression. The new patterns are partial versions of the wild-type pattern and indicate that there are regulatory sequences associated with hairy that respond to distinct cues in different parts of the blastoderm. This suggests that a major function of hairy is to decode a prepattern consisting of complex cues (probably generated by the coordinate and gap classes of segmentation genes) into a simple periodic pattern. We have located the mutations on the DNA map of the hairy gene. They identify a 5' region of approximately less than 20 kb necessary for this decoding function.
Structure and expression of a Drosophila male accessory gland gene whose product resembles a peptide pheromone precursor.
Authors: Monsma S A , Wolfner M F
Published: June 2007

The accessory gland of male insects is a genital tissue that secretes many components of the ejaculatory fluid, some of which affect the female's receptivity to courtship and her rate of oviposition. We have examined the structure and expression of two tightly linked genes that are expressed exclusively in the male accessory glands of adult Drosophila melanogaster. The two genes are transcribed from the same strand of DNA, and are separated by 20 bases. Both genes are regulated by the sex determination hierarchy and are expressed in the absence of germ cells. Immunological analysis reveals the protein products of at least one of these genes in the secretion of the accessory gland. The proteins are transferred to the female fly during copulation and are rapidly altered in the female genital tract. The predicted sequence of one protein has features of a peptide hormone precursor, and a region in which 11 of 17 amino acids are identical to egg-laying hormone (ELH) of the California sea hare, Aplysia californica.
Sp1 transcription factor binds DNA and activates transcription even when the binding site is CpG methylated.
Authors: Höller M , Westin G , Jiricny J , Schaffner W
Published: June 2007

In vertebrates, a negative correlation between gene activity and CpG methylation of DNA, notably in the promoter region, is well established. Therefore, it is conceivable that differential binding of transcription factors to methylated versus unmethylated binding sites is crucial for gene activity. Since the consensus binding site of transcription factor Sp1 contains a central CpG, we have investigated the binding of Sp1 factor to unmethylated and synthetically CpG-methylated DNA. A strong Sp1 binding site was methylated on both strands at two CpG positions, located in the center and at the periphery of the recognition sequence. Our studies show that neither binding in vitro, nor transcription in vivo and in vitro are affected by methylation of the Sp1 binding site. We discuss the possibility that binding of Sp1 factor, which is often associated with promoters of housekeeping genes, prevents CpG methylation.
Molecular analysis of the armadillo locus: uniformly distributed transcripts and a protein with novel internal repeats are associated with a Drosophila segment polarity gene.
Authors: Riggleman B , Wieschaus E , Schedl P
Published: June 2007

During Drosophila embryogenesis, the segment polarity genes are required for the formation of specific pattern domains within each segment. Mutations in the armadillo (arm) gene primarily affect the posterior part of the segment and lead to the production of anterior structures within this region. To examine the molecular basis for these effects, we have cloned the arm region and identified the gene by germ-line transformation. The arm gene produces two types of very abundant 3.2-kb transcripts that differ only in their first exons. These RNAs appear to be formed by independent transcriptional initiation but have similar patterns of expression throughout development. Both arm transcripts are present in virtually all of the cell types contained in embryos, third-instar larvae, and adult ovaries, suggesting that arm may be required in all cells. In addition, the arm transcripts are uniformly distributed in embryonic segments, so the regional pattern defects associated with its embryonic phenotype may result from interactions between arm and other localized factors. Both arm RNAs encode the same 91-kD polypeptide. This protein has no probable secretory or membrane-spanning regions and contains a series of novel internal repeats that are conserved in sequence, length, and spacing. Considering these results and previous genetic observations, we discuss potential roles for the arm gene in pattern formation processes.
Regulation of transcription factor AP-2 by the morphogen retinoic acid and by second messengers.
Authors: Lüscher B , Mitchell P J , Williams T , Tjian R
Published: June 2007

The expression of the transcription factor AP-2 recently has been shown to be enhanced during retinoic acid (RA)-induced differentiation of NT2 cells, a human teratocarcinoma cell line. Here we show that this induction of AP-2 mRNA is at the level of transcription and is transient, reaching a peak 48-72 hr after the addition of RA and declining thereafter, even in the continuous presence of RA. Increased levels of AP-2 mRNA are reflected in a similar elevation of AP-2 protein and accompanied by an increase in the AP-2-binding site-dependent transcriptional activity of a reporter gene. AP-2 also has been proposed to confer TPA and cAMP inducibility on promoters/enhancers containing AP-2-binding sites. We investigated the effect of these agents on the expression of AP-2 protein and mRNA. Our experiments demonstrate that expression of the AP-2 gene in HeLa cells is not elevated significantly by TPA or by a calcium ionophore and is not enhanced at all by agents that increase intracellular cAMP concentration. In fact, AP-2 mRNA is repressed by both TPA and the calcium ionophore A23187 through a delayed response. These data suggest that the AP-2-binding site-mediated cAMP and TPA responses are not regulated at the level of AP-2 expression but, rather, achieved either by post-translational changes in AP-2 or in conjunction with another protein.
A compensatory base change in human U2 snRNA can suppress a branch site mutation.
Authors: Zhuang Y , Weiner A M
Published: June 2007

We have developed an assay to test whether U2 snRNA can base-pair with the branch site during mammalian mRNA splicing. The beta 110 point mutation (GG----AG) within the first intron of human beta-globin generates a new 3' splice site that is preferentially used. We show here that use of the normal 3' splice site can be restored either by improving the match of a cryptic branch site to the branch site consensus or by introducing mutant U2 snRNAs with greater complementarity to the cryptic branch site. These data indicate that human U2 snRNA can form base pairs with the mRNA precursor; however, base pairing appears to be optional because some mammalian branch sites do not match the consensus.
Mutations in the glucocorticoid receptor zinc finger region that distinguish interdigitated DNA binding and transcriptional enhancement activities.
Authors: Schena M , Freedman L P , Yamamoto K R
Published: June 2007

Mammalian glucocorticoid receptors bind specifically to glucocorticoid response element (GRE) DNA sequences and enhance transcription from GRE-linked promoters in mammalian cells and in yeast. We randomly mutagenized a segment of the receptor encompassing sequences responsible for DNA-binding and transcriptional regulation and screened in yeast for receptor defects. The mutations all mapped to a 66-amino-acid subregion that includes two zinc fingers; in general parallel phenotypes were observed in yeast and animal cells. Mutants defective for DNA binding also failed either to enhance or to repress transcription. However, several mutations in the second finger selectively impaired enhancement; we suggest that such 'positive control' mutants may alter protein-protein contacts required for transcriptional activation.
Phosphorylation and dephosphorylation of a bacterial transcriptional activator by a transmembrane receptor.
Authors: Igo M M , Ninfa A J , Stock J B , Silhavy T J
Published: June 2007

Signal transduction in the bacterial Omp, Che, and Ntr systems involves the phosphorylation and dephosphorylation of response regulators (OmpR, CheY and CheB, NRI) that share a homologous domain. We show that in the Omp system, the transmembrane sensor EnvZ, catalyzes both the phosphorylation of OmpR and the dephosphorylation of OmpR-P. The phosphorylation reaction proceeds by a mechanism shared with the Ntr and Che kinases, NRII, and CheA. EnvZ can phosphorylate NRI and can stimulate transcription from the glnAp2 promoter, and similarly, CheA can phosphorylate OmpR and can stimulate transcription from the ompF promoter. OmpR-P formed by either CheA or EnvZ is much more stable than CheY-P and NRI-P, but is rapidly hydrolyzed to OmpR and Pi by EnvZ in the presence of ATP, ADP, or nonhydrolyzable analogs of ATP. Because EnvZ is normally a transmembrane receptor with a periplasmic sensory domain, our results suggest that the role of EnvZ may be to control the intracellular concentration of OmpR-P in response to environmental signals.
Temporal and spatial control of the mother-cell regulatory gene spoIIID of Bacillus subtilis.
Authors: Kunkel B , Kroos L , Poth H , Youngman P , Losick R
Published: June 2007

Gene expression during endospore formation in Bacillus subtilis is compartmentalized between the mother-cell and forespore chambers of the sporangium, which follow separate pathways of cellular differentiation. The earliest acting regulatory gene so far identified in the mother-cell line of gene expression is spoIIID, whose product is required for the transcription of the composite gene (sigK) encoding the mother-cell RNA polymerase sigma-factor sigma K and for the chromosomal rearrangement that gives rise to the composite gene. Here we report the nucleotide sequence of spoIIID and studies on the temporal, spatial, and genetic control of its expression during sporulation. We show that the deduced spoIIID gene product, a 93-residue-long polypeptide, is a previously identified transcription factor that is known to activate the promoter for the sigK gene in vitro. Expression of spoIIID is largely confined to the mother-cell chamber of the sporangium and is turned on at, or shortly before, the time (hour 3 of sporulation) that the mother-cell chromosome is rearranged and transcription of the sigK gene commences. This gene expression depends strongly on the sporulation sigma-factor sigma E and partially on the spoIIID gene product, itself. We conclude that the timing and compartmentalization of the rearrangement and transcription of the sigK gene and, hence, of subsequent gene activation in the mother cell, are, in part, direct consequences of the temporal and spatial control of spoIIID gene expression.