bluuebunny/crossref_metadata_2025 huggingface dataset:
Synergistic regulation of human beta-globin gene switching by locus control region elements HS3 and HS4.
Authors: Bungert J , Davé U , Lim K C , Lieuw K H , Shavit J A , Liu Q , Engel J D
Published: June 2007

Proper tissue- and developmental stage-specific transcriptional control over the five genes of the human beta-globin locus is elicited in part by the locus control region (LCR), but the molecular mechanisms that dictate this determined pattern of gene expression during human development are still controversial. By use of homologous recombination in yeast to generate mutations in the LCR within a yeast artificial chromosome (YAC) bearing the entire human beta-globin gene locus, followed by injection of each of the mutated YACs into murine ova, we addressed the function of LCR hypersensitive site (HS) elements 3 and 4 in human beta-globin gene switching. The experiments revealed a number of unexpected properties that are directly attributable to LCR function. First, deletion of either HS3 or HS4 core elements from an otherwise intact YAC results in catastrophic disruption of globin gene expression at all erythroid developmental stages, despite the presence of all other HS elements in the YAC transgenes. If HS3 is used to replace HS4, gene expression is normal at all developmental stages. Conversely, insertion of the HS4 element in place of HS3 results in significant expression changes at every developmental stage, indicating that individual LCR HS elements play distinct roles in stage-specific beta-type globin gene activation. Although the HS4 duplication leads to alteration in the levels of epsilon- and gamma-globin mRNAs during embryonic erythropoiesis, total beta-type globin mRNA synthesis is balanced, thereby leading to the conclusion that all of the human beta-locus genes are competitively regulated. In summary, the human beta-globin HS elements appear to form a single, synergistic functional entity called the LCR, and HS3 and HS4 appear to be individually indispensable to the integrity of this macromolecular complex.
The POU domain transcription factor Brn-2 is required for the determination of specific neuronal lineages in the hypothalamus of the mouse.
Authors: Nakai S , Kawano H , Yudate T , Nishi M , Kuno J , Nagata A , Jishage K , Hamada H , Fujii H , Kawamura K
Published: June 2007

We generated mice carrying a loss-of-function mutation in Brn-2, a gene encoding a nervous system specific POU transcription factor, by gene targeting in embryonic stem cells. In homozygous mutant embryos, migratory precursor cells for neurons of the paraventricular nuclei (PVN) and the supraoptic nuclei (SO) of the hypothalamus die at approximately E12.5. All homozygous mutants suffered mortality within 10 days after birth, possibly because of a complete deficiency of these neurons in the hypothalamus. Although neither developmental nor histological abnormalities were observed in heterozygous mice, the levels of expression of vasopressin and oxytocin in the hypothalamus of these animals were half these of wild-type mice. These results strongly suggest that Brn-2 plays an essential role in the determination and development of the PVN and SO neuronal lineages in the hypothalamus.
Development and survival of the endocrine hypothalamus and posterior pituitary gland requires the neuronal POU domain factor Brn-2.
Authors: Schonemann M D , Ryan A K , McEvilly R J , O'Connell S M , Arias C A , Kalla K A , Li P , Sawchenko P E , Rosenfeld M G
Published: June 2007

Neurons comprising the endocrine hypothalamus are disposed in several nuclei that develop in tandem with their ultimate target the pituitary gland, and arise from a primordium in which three related class III POU domain factors, Brn-2, Brn-4, and Brn-1, are initially coexpressed. Subsequently, these factors exhibit stratified patterns of ontogenic expression, correlating with the appearance of distinct neuropeptides that define three major endocrine hypothalamic cell types. Strikingly, deletion of the Brn-2 genomic locus results in loss of endocrine hypothalamic nuclei and the posterior pituitary gland. Lack of Brn-2 does not affect initial hypothalamic developmental events, but instead results in a failure of differentiation to mature neurosecretory neurons of the paraventricular and supraoptic nuclei, characterized by an inability to activate genes encoding regulatory neuropeptides or to make correct axonal projections, with subsequent loss of these neurons. Thus, both neuronal and endocrine components of the hypothalamic-pituitary axis are critically dependent on the action of specific POU domain factors at a penultimate step in the sequential events that underlie the appearance of mature cellular phenotypes.
Targeted disruption of mammalian hairy and Enhancer of split homolog-1 (HES-1) leads to up-regulation of neural helix-loop-helix factors, premature neurogenesis, and severe neural tube defects.
Authors: Ishibashi M , Ang S L , Shiota K , Nakanishi S , Kageyama R , Guillemot F
Published: June 2007

Mammalian hairy and Enhancer of split homolog-1 (HES-1) encodes a helix-loop-helix (HLH) factor that is thought to act as a negative regulator of neurogenesis. To directly investigate the functions of HES-1 in mammalian embryogenesis, we performed a targeted disruption of the HES-1 locus. Mice homozygous for the mutation exhibited severe neurulation defects and died during gestation or just after birth. In the developing brain of HES-1-null embryos, expression of the neural differentiation factor Mash-1 and other neural HLH factors was up-regulated and postmitotic neurons appeared prematurely. These results suggest that HES-1 normally controls the proper timing of neurogenesis and regulates neural tube morphogenesis.
Binding sites for transcription factor NTF-1/Elf-1 contribute to the ventral repression of decapentaplegic.
Authors: Huang J D , Dubnicoff T , Liaw G J , Bai Y , Valentine S A , Shirokawa J M , Lengyel J A , Courey A J
Published: June 2007

The Dorsal morphogen is a transcription factor that activates some genes and represses others to establish multiple domains of gene expression along the dorsal/ventral axis of the early Drosophila embryo. Repression by Dorsal appears to require accessory proteins that bind to corepression elements in Dorsal-dependent regulatory modules called ventral repression regions (VRRs). We have identified a corepression element in decapentaplegic (dpp), a zygotically active gene that is repressed by the Dorsal morphogen. This dpp repression element (DRE) is located within a previously identified VRR and close to essential Dorsal-binding sites. We have purified a factor from Drosophila embryo extracts that binds to the DRE but not to mutant forms of the DRE that fail to support efficient repression. This protein also binds to an apparently essential region in a VRR associated with the zerknüllt (zen) gene. One of the DREs in the dpp VRR overlaps the binding site for a potential activator protein suggesting that one mechanism of ventral repression may be the mutually exclusive binding of repressor and activator proteins. We have found the DRE-binding protein to be identical to NTF-1 (equivalent to Elf-1, the product of the grainyhead gene), a factor originally identified as an activator of the Ultrabithorax and Dopa decarboxylase promoters. NTF-1 mRNA is synthesized during oogenesis and deposited in the developing oocyte where it is available to contribute to ventral repression during early embryogenesis. Previous studies have shown that overexpression of NTF-1 in the postblastoderm embryo results in a phenotype that is consistent with a role for this factor in the repression of dpp later in embryogenesis.
Regulation of human RPS14 transcription by intronic antisense RNAs and ribosomal protein S14.
Authors: Tasheva E S , Roufa D J
Published: June 2007

RNase protection studies reveal two stable RNAs (250 and 280 nucleotides) transcribed from the antisense strand of the human ribosomal protein gene RPS14's first intron. These transcripts, designated alpha-250 and alpha-280, map to overlapping segments of the intron's 5' sequence. Neither RNA encodes a polypeptide sequence, and both are expressed in all human cells and tissues examined. Although alpha-280 is detected among both the cells' nuclear and cytoplasmic RNAs, the great majority of alpha-250 is found in the cytoplasmic subcellular compartment. As judged by its resistance to high concentrations of alpha-amanitin, cell-free transcription of alpha-250 and alpha-280 appears to involve RNA polymerase I. Tissue culture transfection and cell-free transcription experiments demonstrate that alpha-250 and alpha-280 stimulate S14 mRNA transcription, whereas free ribosomal protein S14 inhibits it. Electrophoretic mobility shift experiments indicate specific binary molecular interactions between r-protein S14, its message and the antisense RNAs. In light of these data, we propose a model for fine regulation of human RPS14 transcription that involves RPS14 intron 1 antisense RNAs as positive effectors and S14 protein as a negative effector.
The SABRE gene is required for normal cell expansion in Arabidopsis.
Authors: Aeschbacher R A , Hauser M T , Feldmann K A , Benfey P N
Published: June 2007

Regulated cell expansion is an important determinant of organ shape in higher plants. The sabre mutation results in abnormal cell expansion in Arabidopsis. There is a shift in the orientation of expansion evident primarily in root cortex cells. The SABRE gene has been cloned and found to encode a novel protein. Reduction of effective levels of the plant phytohormone ethylene through use of inhibitors and an insensitive mutant resulted in partial rescue of the sabre phenotype. This suggested that one of the roles of SABRE is to counter the action of ethylene in promoting radial expansion in plant cells.
G1 cyclin turnover and nutrient uptake are controlled by a common pathway in yeast.
Authors: Barral Y , Jentsch S , Mann C
Published: June 2007

Entry into a new cell cycle is triggered by environmental signals at a point called Start in G1 phase. A key regulator of this transition step in yeast is the CDC28 kinase together with its short-lived regulatory subunits called G1-cyclins or CLN proteins. To identify genes involved in G1-cyclin degradation, we employed a genetic screen by selecting for stable CLN1-beta-galactosidase fusion proteins. Surprisingly, one group of mutants was found to be allelic to GRR1, a gene previously described to be involved in glucose uptake, glucose repression, and divalent cation transport. In grr1 mutants, both CLN1 and CLN2 cyclins are significantly stabilized. A suppressor analysis indicated that G1-cyclin stabilization in grr1 was not a consequence of the nutrient uptake defect. This suggests that the GRR1 gene product is part of a common regulatory pathway linking two functions important for cell growth, nutrient uptake, and G1 cyclin-controlled cell division.
Identification and characterization of genes that are required for the accelerated degradation of mRNAs containing a premature translational termination codon.
Authors: Cui Y , Hagan K W , Zhang S , Peltz S W
Published: June 2007

In both prokaryotes and eukaryotes nonsense mutations in a gene can enhance the decay rate of the mRNA transcribed from the gene, a phenomenon described as nonsense-mediated mRNA decay. In yeast, the products of the UPF1 and UPF3 genes are required for this decay pathway, and in this report we focus on the identification and characterization of additional factors required for rapid decay of nonsense-containing mRNAs. We present evidence that the product of the UPF2 gene is a new factor involved in this decay pathway. Mutation of the UPF2 gene or deletion of it from the chromosome resulted in stabilization of nonsense-containing mRNAs, whereas the decay of wild-type transcripts was not affected. The UPF2 gene was isolated, and its transcript was characterized. Our results demonstrate that the UPF2 gene encodes a putative 126.7-kD protein with an acidic region at its carboxyl terminus (-D-E)n found in many nucleolar and transcriptional activator proteins. The UPF2 transcript is 3600 nucleotides in length and contains an intron near its 5' end. The UPF2 gene is dispensable for vegetative growth, but upf2 delta strains were found to be more sensitive to the translational elongation inhibitor cycloheximide than UPF2+. A genetic analysis of other alleles proposed to be involved in nonsense-mediated mRNA decay revealed that the UPF2 gene is allelic to the previously identified sua1 allele, a suppressor of an out-of-frame ATG insertion shown previously to reduce translational initiation from the normal ATG of the CYC1 gene. In addition, we demonstrate that another suppressor of this cyc1 mutation, sua6, is allelic to upf3, a previously identified lesion involved in nonsense-mediated mRNA decay.
Identification of a novel component of the nonsense-mediated mRNA decay pathway by use of an interacting protein screen.
Authors: He F , Jacobson A
Published: June 2007

Rapid turnover of nonsense-containing mRNAs in yeast in dependent on the product of the UPF1 gene (Upf1p). Mutations in UPF1 lead to the selective stabilization of mRNAs containing early nonsense mutations without affecting the decay rates of most other mRNAs. To identify other integral components of this decay pathway, we have employed a two-hybrid screen, seeking those cellular factors that specifically interact with Upf1p. Screening of yeast genomic libraries identified six genes encoding potential Upf1p-interacting proteins. These include four previously uncharacterized genes, NMD1-4 (nonsense-mediated mRNA decay), DBP2, a gene encoding a putative RNA helicase with homology to mammalian p68 RNA helicase, and SNP1, a gene encoding a U1 snRNP 70-kD protein homolog. In this paper we report the identification and characterization of NMD2, a yeast gene that encodes a specific Upf1p-interacting protein. Disruption of NMD2 yields a nonsense-mediated mRNA decay phenotype identical to that obtained in UPF1-deletion strains, indicating that the NMD2 gene product (Nmd2p) is a new factor in the nonsense-mediated mRNA decay pathway. Deletion analysis demonstrated that the acidic carboxyl terminus of Nmd2p constituted the Upf1p-interacting domain. High-level expression of a fragment of Nmd2p containing this domain had a dominant-negative effect on nonsense-mediated mRNA decay when the protein was localized the cytoplasm but not when it was localized to the nucleus, indicating that this decay pathway has a cytoplasmic component. The association of a dominant-negative phenotype with a gene fragment identified in a two-hybrid screen suggests a generalized approach to confirming the function of genes identified in such screens.
Inhibition of viral gene expression by the catalytic RNA subunit of RNase P from Escherichia coli.
Authors: Liu F , Altman S
Published: June 2007

The catalytic RNA subunit (M1 RNA) of RNase P from Escherichia coli has been converted to an endoribonuclease that specifically cleaves the mRNA that encodes thymidine kinase (TK) of herpes simplex virus 1 (HSV-1). Covalent attachment to the 3' end of M1 RNA of a sequence complementary to TK mRNA results in very efficient cleavage of the target RNA in vitro. This reaction can be stimulated by proteins extracted from both E. coli and HeLa cells. When mouse cells in culture that express the novel RNA construct are infected with HSV-1, the levels of both TK mRNA and protein are reduced by approximately 80% as compared with cells that either do not express the novel RNA construct or express constructs with certain deletions that are known to abolish the catalytic activity of M1 RNA.
enabled, a dosage-sensitive suppressor of mutations in the Drosophila Abl tyrosine kinase, encodes an Abl substrate with SH3 domain-binding properties.
Authors: Gertler F B , Comer A R , Juang J L , Ahern S M , Clark M J , Liebl E C , Hoffmann F M
Published: June 2007

Genetic screens for dominant second-site mutations that suppress the lethality of Abl mutations in Drosophila identified alleles of only one gene, enabled (ena). We report that the ena protein contains proline-rich motifs and binds to Abl and Src SH3 domains, ena is also a substrate for the Abl kinase; tyrosine phosphorylation of ena is increased when it is coexpressed in cells with human or Drosophila Abl and endogenous ena tyrosine phosphorylation is reduced in Abl mutant animals. Like Abl, ena is expressed at highest levels in the axons of the embryonic nervous system and ena mutant embryos have defects in axonal architecture. We conclude that a critical function of Drosophila Abl is to phosphorylate and negatively regulate ena protein during neural development.
The Drosophila tumor suppressor gene warts encodes a homolog of human myotonic dystrophy kinase and is required for the control of cell shape and proliferation.
Authors: Justice R W , Zilian O , Woods D F , Noll M , Bryant P J
Published: June 2007

Homozygous loss of the warts (wts) gene of Drosophila, caused by mitotic recombination in somatic cells, leads to the formation of cell clones that are fragmented, rounded, and greatly overgrown compared with normal controls. Therefore, the gene is required for the control of the amount and direction of cell proliferation as well as for normal morphogenesis. The absence of wts function also results in apical hypertrophy of imaginal disc epithelial cells. Secretion of cuticle over and between the domed apical surfaces of these cells leads to a honeycomb-like structure and gives the superficial wart-like phenotype of mitotic clones on the adult. One wts allele allows survival of homozygotes to the late larval stage, and these larvae show extensive imaginal disc overgrowth. Because of the excess growth and abnormalities of differentiation that follow homozygous loss, we consider wts to be a tumor suppressor gene. The wts gene is defined by the breakpoints of overlapping deficiencies in the right telomeric region of chromosome 3, region 100A, and by lethal P-element insertions and excisions. It encodes a protein kinase that is most similar to human myotonic dystrophy kinase, the Neurospora cot-1 protein kinase, two cell-cycle regulated kinases of yeast, and several putative kinases from plants. These proteins define a new subfamily of protein kinases that are closely related to but distinct from the cyclic AMP-dependent kinases. Although myotonic dystrophy is defined by a neuromuscular disorder, it is sometimes associated with multiple pilomatrixomas, which are otherwise rare epithelial tumors, and with other tumors including neurofibromas and parathyroid adenomas. Our results raise the possibility that homozygous loss of the myotonic dystrophy kinase may contribute to the development of these tumors.
Promoter-proximal pausing of RNA polymerase II defines a general rate-limiting step after transcription initiation.
Authors: Krumm A , Hickey L B , Groudine M
Published: June 2007

We have shown previously that the majority of RNA polymerase II complexes initiated at the c-myc gene are paused in the promoter-proximal region, similar to observations in the Drosophila hsp70 gene. Our analyses define the TATA box or initiator sequences in the c-myc gene as necessary components for the establishment of paused RNA polymerase II. Deletion of upstream sequences or even the TATA box does not influence significantly the degree of transcriptional initiation or pausing. Deletion of both the TATA box and sequences at the transcription initiation site, however, abolishes transcriptional pausing of transcription complexes but still allows synthesis of full-length RNA. Further analyses with synthetic promoter constructs reveal that the simple combination of upstream activator with TATA consensus sequences or initiator sequences act synergistically to recruit high levels of RNA polymerase II complexes. Only a minor fraction of these complexes escapes into regions further downstream. Several different trans-activation domains fused to GAL4-DNA-binding domains, including strong activators such as VP16, do not eliminate promoter-proximal pausing of RNA polymerase. Thus, we conclude that pausing of RNA polymerase II is a common phenomenon in eukaryotic transcription and does not require complex promoter structures. Further analyses reveal that enhancers have a modest influence on transcription initiation and on release of transcription complexes out of the pause site but may function primarily to increase the elongation competence of transcription complexes.
SMC2, a Saccharomyces cerevisiae gene essential for chromosome segregation and condensation, defines a subgroup within the SMC family.
Authors: Strunnikov A V , Hogan E , Koshland D
Published: June 2007

We characterized the SMC2 (structural maintenance of chromosomes) gene that encodes a new Saccharomyces cerevisiae member of the growing family of SMC proteins. This family of evolutionary conserved proteins was introduced with identification of SMC1, a gene essential for chromosome segregation in budding yeast. The analysis of the putative structure of the Smc2 protein (Smc2p) suggests that it defines a distinct subgroup within the SMC family. This subgroup includes the ScII, XCAPE, and cut14 proteins characterized concurrently. Smc2p is a nuclear, 135-kD protein that is essential for vegetative growth. The temperature-sensitive mutation, smc2-6, confers a defect in chromosome segregation and causes partial chromosome decondensation in cells arrested in mitosis. The Smc2p molecules are able to form complexes in vivo both with Smc1p and with themselves, suggesting that they can assemble into a multimeric structure. In this study we present the first evidence that two proteins belonging to two different subgroups within the SMC family carry nonredundant biological functions. Based on genetic, biochemical, and evolutionary data we propose that the SMC family is a group of prokaryotic and eukaryotic chromosomal proteins that are likely to be one of the key components in establishing the ordered structure of chromosomes.
Pbx proteins display hexapeptide-dependent cooperative DNA binding with a subset of Hox proteins.
Authors: Chang C P , Shen W F , Rozenfeld S , Lawrence H J , Largman C , Cleary M L
Published: June 2007

The human proto-oncogene PBX1 codes for a homolog of Drosophila extradenticle, a divergent homeo domain protein that modulates the developmental and DNA-binding specificity of select HOM proteins. We demonstrate that wild-type Pbx proteins and chimeric E2a-Pbx1 oncoproteins cooperatively bind a consensus DNA probe with HoxB4, B6, and B7 of the Antennapedia class of Hox/HOM proteins. Specificity of Hox-Pbx interactions was suggested by the inability of Pbx proteins to cooperatively bind the synthetic DNA target with HoxA10 or Drosophila even-skipped. Site-directed mutagenesis showed that the hexapeptide motif (IYPWMK) upstream of the Hox homeo domain was essential for HoxB6 and B7 to cooperatively bind DNA with Pbx proteins. Engraftment of the HoxB7 hexapeptide onto HoxA10 endowed it with robust cooperative properties, demonstrating a functional role for the highly conserved hexapeptide element as one of the molecular determinants delimiting Hox-Pbx cooperativity. The Pbx homeo domain was necessary but not sufficient for cooperativity, which required conserved amino acids carboxy-terminal of the homeo domain. These findings demonstrate that interactions between Hox and Pbx proteins modulate their DNA-binding properties, suggesting that Pbx and Hox proteins act in parallel as heterotypic complexes to regulate expression of specific subordinate genes.
Drosophila MEF2, a transcription factor that is essential for myogenesis.
Authors: Bour B A , O'Brien M A , Lockwood W L , Goldstein E S , Bodmer R , Taghert P H , Abmayr S M , Nguyen H T
Published: June 2007

mef2 encodes the only apparent Drosophila homolog of the vertebrate myocyte-specific enhancer factor 2 (MEF2). We show herein that the Drosophila MEF2 protein is expressed throughout the mesoderm following gastrulation. Later in embryogenesis, its expression is maintained in precursors and differentiated cells of the somatic and visceral musculature, as well as the heart. We have characterized genetic deficiencies and EMS-induced point mutations that result in complete loss of MEF2 protein in homozygous mutant embryos. These embryos exhibit a dramatic absence of myosin heavy chain (MHC)-expressing myoblasts and lack differentiated muscle fibers. Examination of earlier events of muscle development indicates that the specification and early differentiation of somatic muscle precursors are not affected because even-skipped-, nautilus-, and beta 3-tubulin-expressing myoblasts are present. However, these partially differentiated cells are unable to undergo further differentiation to form muscle fibers in the absence of mef2. The later aspects of differentiation of the visceral mesoderm and the heart are also disrupted in mef2 mutant embryos, although the specification and early development of these tissues appear unaffected. Midgut morphogenesis is disrupted in the mutant embryos, presumably as a consequence of abnormal development of the visceral mesoderm. In the heart, the cardial cells do not express MHC. These results indicate that MEF2 is required for later aspects of differentiation of the three major types of musculature, which include body wall muscles, gut musculature, and the heart, in the Drosophila embryo.
MEK-2, a Caenorhabditis elegans MAP kinase kinase, functions in Ras-mediated vulval induction and other developmental events.
Authors: Wu Y , Han M , Guan K L
Published: June 2007

Activated Ras initiates a cascade of sequential phosphorylation events, including the protein kinases Raf, MEK, and MAP kinase. The Let-60 Ras-mediated signal transduction pathway controls vulval induction in Caenorhabditis elegans. Both Lin-45 Raf and Sur-1 MAP kinase have been determined to be essential factors during vulval induction; however, the C. elegans mek gene has not been identified. In this paper, we have cloned a C. elegans mek gene, mek-2, and demonstrated that the MEK-2 protein possesses the biochemical properties of MAP kinase kinases: The C. elegans MEK-2 protein can phosphorylate and activate a human MAP kinase (ERK1), and MEK-2 itself can be phosphorylated and activated by immunoprecipitated mammalian Raf. The mek-2 gene plays a key role in the let-60 ras-mediated vulval induction pathway, as loss-of-function mutations in the gene (ku114 and h294) significantly reduce the signal transmitted through Ras. mek-2(ku114) completely suppressed the Multivulva (Muv) phenotype of a hyperactive let-60 ras mutation, and animals homozygous for mek-2(ku114) also displayed a partial larval lethal phenotype. Animals homozygous for mek-2(h294) exhibited a highly penetrant sterile and Vulvaless phenotype. Microinjection of a gain-of-function mek-2 mutation resulted in Muv and other mutant phenotypes, whereas microinjection of a dominant-negative mutation not only suppressed the Muv phenotype of an activated let-60 ras mutation but also caused an egg-laying defective phenotype in otherwise wild type animals. Our results demonstrate that mek-2 acts between lin-45 raf and sur-1/mpk-1 in a signal transduction pathway used in the control of vulval differentiation and other developmental events.
A novel pathway for vitamin A signaling mediated by RXR heterodimerization with NGFI-B and NURR1.
Authors: Perlmann T , Jansson L
Published: June 2007

In addition to its role as a 9-cis retinoic acid receptor, RXR has an important role in the regulation of multiple hormonal pathways through heterodimerization with nuclear receptors. Here, we show that two orphan receptors, NGFI-B and NURR1, which have been shown previously to interact with DNA as monomers, also can heterodimerize with RXR. These heterodimers bind selectively to a class of retinoic acid response elements composed of direct repeats spaced by 5 nucleotides. In this respect they are similar to heterodimers formed between RXR and the receptor for all-trans retinoic acid, RAR. However, whereas RXR is inhibited in the RXR-RAR heterodimer, NGFI-B/NURR1 promote efficient activation in response to RXR ligands and therefore shift RXR from a silent to an active heterodimerization partner. These data show that NGFI-B and NURR1 can increase the potential of RXR to modulate gene expression in a ligand-dependent manner by allowing a distinct class of direct repeats to serve as specific RXR response elements. Because expression of both NGFI-B and NURR1 is rapidly induced by various growth factors, these findings also suggest a novel mechanism for convergence between vitamin A or retinoid and growth factor signaling pathways.
A novel U2-U6 snRNA structure is necessary for mammalian mRNA splicing.
Authors: Sun J S , Manley J L
Published: June 2007

Splicing of mRNA precursors requires a complex and dynamic set of RNA-RNA base-pairing interactions in which the U2 and U6 snRNAs play central roles. Using a genetic suppression assay, we refine and extend a U2-U6 snRNA structure that may comprise the catalytic center of the spliceosome. We first show that a critical U2-U6 helix proven in yeast, helix Ia, is also essential for mammalian splicing. Mutations in the adjacent helix Ib, however, cannot be similarly suppressed, and relevant residues in both U2 and U6 are shown to participate in intramolecular, rather than intermolecular, base-pairing. We next demonstrate the requirement for a novel U2-U6 helix, helix III, which involves bases extending 3' from the branch site recognition sequence in U2 and 5' from an evolutionarily invariant sequence in U6 implicated previously in 5' splice site recognition. This configuration suggests that helix III may help juxtapose the pre-mRNA 5' splice site and branch site. We provide evidence for this by demonstrating that a branch site mutation can be suppressed by a mutation in the 5' splice site, provided that compensatory changes are made in the appropriate bases in U2 and U6. Our results provide new insights into how U2 and U6 snRNAs interact with each other and with the pre-mRNA to initiate the first catalytic step in splicing.
Assembly and function of a TCR alpha enhancer complex is dependent on LEF-1-induced DNA bending and multiple protein-protein interactions.
Authors: Giese K , Kingsley C , Kirshner J R , Grosschedl R
Published: June 2007

In this study we examine the molecular basis for the synergistic regulation of the minimal TCR alpha enhancer by multiple proteins. We find that reconstitution of TCR alpha enhancer function in nonlymphoid cells requires expression of the lymphoid-specific proteins LEF-1, Ets-1 and PEBP2 alpha (CBF alpha), and a specific arrangement of their binding sites in the enhancer. We show that Ets-1 cooperates with PEBP2 alpha to bind adjacent sites at one end of the enhancer, forming a ternary complex that is unstable by itself. Stable occupancy of the Ets-1- and PEBP2 alpha-binding sites in a DNase I protection assay was found to depend on both a specific helical phasing relationship with a nonadjacent ATF/CREB-binding site at the other end of the enhancer and on LEF-1. The HMG domain of LEF-1 was found previously to bend the DNA helix in the center of the TCR alpha enhancer. We now show that the HMG domain of the distantly related SRY protein, which also bends DNA, can partially replace LEF-1 in stimulating enhancer function in transfection assays. Taken together with the observation that Ets-1 and members of the ATF/CREB family have the potential to associate in vitro, these data suggest that LEF-1 can coordinate the assembly of a specific higher-order enhancer complex by facilitating interactions between proteins bound at nonadjacent sites.
Raf1 interaction with Cdc25 phosphatase ties mitogenic signal transduction to cell cycle activation.
Authors: Galaktionov K , Jessus C , Beach D
Published: June 2007

The Ras and Raf1 proto-oncogenes transduce extracellular signals that promote cell growth. Cdc25 phosphatases activate the cell division cycle by dephosphorylation of critical threonine and tyrosine residues within the cyclin-dependent kinases. We show here that Cdc25 phosphatase associates with raf1 in somatic mammalian cells and in meiotic frog oocytes. Furthermore, Cdc25 phosphatase can be activated in vitro in a Raf1-dependent manner. We suggest that activation of the cell cycle by the Ras/Raf1 pathways might be mediated in part by Cdc25.
The conserved Schizosaccharomyces pombe kinase plo1, required to form a bipolar spindle, the actin ring, and septum, can drive septum formation in G1 and G2 cells.
Authors: Ohkura H , Hagan I M , Glover D M
Published: June 2007

We have identified a Schizosaccharomyces pombe gene with homology to the budding yeast gene CDC5, the Drosophila gene polo, and the mammalian family of genes encoding polo-like kinases. Disruption of this gene, plo1+, indicates that it is essential. Loss of plo1+ function leads to a mitotic arrest in which condensed chromosomes are associated with a monopolar spindle or to the failure of septation following the completion of nuclear division. In the latter case, cells show a failure both in the formation of an F-actin ring and in the deposition of septal material, suggesting that plo1+ function is required high in the regulatory cascade that controls septation. The overexpression of plo1+ in wild-type cells also results in the formation of monopolar spindles but also induces the formation of multiple septa without nuclear division. Septation can also be induced in the absence of mitotic commitment and concomitant spindle formation by the overexpression of plo1+ in cdc25-22 or cdc2-33 cells arrested in G2; in G1 cells arrested at Start by the cdc10-V50 mutation, or in cells lacking the cyclin B homolog cdc13 that undergo repeated S phases in the absence of mitosis.
Distribution of GAGA protein on Drosophila genes in vivo.
Authors: O'Brien T , Wilkins R C , Giardina C , Lis J T
Published: June 2007

GAGA protein binds specific CT.GA-rich DNA sequences in vitro, and many of these sequences are required for transcription in vivo. GAGA protein has been implicated in the transcription of numerous Drosophila genes, including hsp70, hsp26, actin 5C, and Ubx. Here, we examine the in vivo distribution of GAGA protein on a number of Drosophila genes that do and do not have CT-rich sequences by use of a UV cross-linking technique. Prior to heat shock, GAGA protein is associated with the promoter regions of the uninduced hsp70 and hsp26 genes. Upon heat shock induction, GAGA protein is recruited to their transcription units with its distribution coincident with that of RNA polymerase II. The recruitment of GAGA protein to the hsp70 gene after an instantaneous heat shock occurs in a 5' to 3' manner with kinetics similar to RNA polymerase. GAGA protein has been shown to disrupt nucleosome both in vivo and in vitro. We propose that GAGA protein may function in vivo both by binding constitutively to its high-affinity binding sites and by spreading through the induced gene opening the chromatin structure allowing polymerase to elongate efficiently.
DNA polymerase fidelity and the polymerase chain reaction.
Authors: Eckert K A , Kunkel T A
Published: June 2007

High-fidelity DNA synthesis conditions are those that exploit the inherent ability of polymerases to discriminate against errors. This review has described several experimental approaches for controlling the fidelity of enzymatic DNA amplification. One of the most important parameters to consider is the choice of which polymerase to use in PCR. As demonstrated by the data in Tables 2 and 3, high-fidelity DNA amplification will be best achieved by using a polymerase with an active 3'-->5' proofreading exonuclease activity (Fig. 1E). For those enzymes that are proofreading-deficient, the in vitro reaction conditions can significantly influence the polymerase error rates. To maximize fidelity at the dNTP insertion step (Fig. 1A,B), any type of deoxynucleoside triphosphate pool imbalance should be avoided. Similarly, stabilization of errors by polymerase extension from mispaired or misaligned primer-termini (Fig. 1D) can be minimized by reactions using short synthesis times, low dNTP concentrations, and low enzyme concentrations. Additional improvements in fidelity can be made by further manipulating the reaction conditions. To perform high-fidelity PCR with Taq polymerase, reactions should contain a low MgCl2 concentration, not in large excess over the total concentration of dNTP substrates, and be buffered to approximately pH 6 (70 degrees C) using Bis-Tris Propane or PIPES (Table 2). These buffers have a pKa between pH 6 and pH 7 and a small temperature coefficient (delta pKa/degree C), allowing the pH to be maintained stably throughout the PCR cycle. For amplifications in which fidelity is the critical issue, one should avoid the concept that conditions generating more DNA product are the better conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
Optimization of the polymerase chain reaction with regard to fidelity: modified T7, Taq, and vent DNA polymerases.
Authors: Ling L L , Keohavong P , Dias C , Thilly W G
Published: June 2007

The fidelity of DNA polymerases used in the polymerase chain reaction (PCR) can be influenced by many factors in the reaction mixture. To maximize the fidelity of DNA polymerases in the PCR, pH, concentrations of deoxynucleoside triphosphates, and magnesium ion were varied. Denaturing gradient gel electrophoresis was used to separate the polymerase-induced mutants from wild-type DNA sequences. Thermolabile modified T7 DNA polymerase, thermostable Taq, and Vent DNA polymerases were studied. Fidelity of all three DNA polymerases was sensitive to concentrations of deoxynucleoside triphosphates, magnesium ion, and pH. Within conditions that permitted efficient amplification, optimization with regard to these three factors yielded an average error rate in error/base pair incorporated of 7.2 x 10(-5) for Taq, 4.5 x 10(-5) for Vent, and 4.4 x 10(-5) for modified T7 (Sequenase) DNA polymerases.
Capture PCR: efficient amplification of DNA fragments adjacent to a known sequence in human and YAC DNA.
Authors: Lagerström M , Parik J , Malmgren H , Stewart J , Pettersson U , Landegren U
Published: June 2007

We have devised a procedure, termed capture PCR (CPCR), that permits the rapid isolation of DNA segments situated adjacent to a characterized nucleotide sequence. In this procedure, a DNA sample is restriction-digested and a linker, comprising two base-paired oligonucleotides, is added to the ends by ligation. Multiple extension reactions are performed using a biotinylated primer derived from the known sequence, permitting the subsequent isolation of extension products on a streptavidin-coated support. The enriched fragments are amplified exponentially using another specific oligonucleotide, hybridizing 3' to the biotinylated primer in combination with one of the linker oligonucleotides, now functioning as a PCR primer. The convenience of CPCR is greatly enhanced by using a novel streptavidin-coated manifold, which is constructed so that it projects into each individual well of a microtiter plate. The procedure permits the simultaneous isolation of fragments from large numbers of DNA samples and minimizes the risk of contamination between reactions. We have applied this method to identify DNA sequences located downstream of known sequences in the human genome. The technique has also been used to identify end fragments of sequences cloned in a yeast artificial chromosome (YAC) vector. The reactions can be initiated directly from yeast colonies and provide access to DNA sequence information for these end fragments in a minimal number of steps. With the aid of the present technique, we have isolated over 100 end fragments from YACs derived from the human X chromosome. Isolated end sequences have been used to order YAC clones into a contig.
OSP: a computer program for choosing PCR and DNA sequencing primers.
Authors: Hillier L , Green P
Published: June 2007

OSP (Oligonucleotide Selection Program) selects oligonucleotide primers for DNA sequencing and the polymerase chain reaction (PCR). The user can specify (or use default) constraints for primer and amplified product lengths, %(G+C), (absolute or relative) melting temperatures, and primer 3' nucleotides. To help minimize nonspecific priming and primer secondary structure, OSP screens candidate primer sequences, using user-specifiable cutoffs, against potential base-pairing with a variety of sequences present in the reaction, including the primer itself, the other primer (for PCR), the amplified product, and any other sequences desired (e.g., repetitive element sequences in genomic templates, vector sequence in cloned templates, or other primer pair sequences in multiplexed PCR reactions). Base-pairing involving the primer 3' end is considered separately from base-pairing involving internal sequences. Primers meeting all constraints are ranked by a "combined score," a user-definable weighted sum of any of the above parameters. OSP is being routinely and extensively used to select sequencing primers for the Caenorhabditis elegans genome sequencing project and human genomic PCR primer pairs for the Washington University Genome Center mapping project, with success rates exceeding 96% and 81%, respectively. It is available for research purposes from the authors, at no cost, in both text output and interactive graphics (X windows) versions.
Generation of competitor DNA fragments for quantitative PCR.
Authors: Uberla K , Platzer C , Diamantstein T , Blankenstein T
Published: June 2007

A convenient and generally applicable method for the generation of competitor DNA fragments for quantitative PCR is described. Using mouse-specific primers, fragments are amplified from DNA of an evolutionarily distantly related species under low-stringency annealing conditions. Because these artificially created fragments contain the mouse primer specific ends, they can be used for the quantification of the mouse DNA amplified by these primers. Competitor DNA fragments that differ in size from the corresponding mouse DNA are selected to distinguish both fragments visually by gel electrophoresis. Competitor DNA fragments were generated for mouse beta-actin, interleukin-1, and tumor necrosis factor (TNF). Co-amplification of beta-actin cDNA for adjustment of equal amounts of input cDNA and subsequently TNF cDNA from lipopolysaccharide (LPS)-activated and nonactivated spleen cells with serial dilutions of the respective competitor DNA fragments allowed a semiquantitative comparison of the ratio of TNF mRNA present in both cDNA samples. Under certain conditions, the competitor DNA fragments can be used to determine the approximate molar concentration of a mRNA.
A PCR-based assay for reporter gene expression.
Authors: Sperisen P , Wang S M , Reichenbach P , Nabholz M
Published: June 2007

Transient transfection is a widely used tool for the identification of cis-acting regulatory elements. These elements are detected by their effect on the expression of a reporter gene, which is quantified by measuring the reporter gene product in the form of mRNA, protein (hGH), or enzymes (CAT, luciferase). Measurements of mRNA levels have several advantages over enzyme or protein assays. However, mRNA quantification by RNase protection or S1 mapping has considerably lower signal-to-background ratio than protein assays and is therefore less sensitive. In this paper we report the development of a system that takes advantage of the polymerase chain reaction (PCR) to quantify rabbit beta-globin reporter gene expression. Cells are co-transfected with constructs whose activity is to be tested and a reference plasmid with a small deletion in the second exon of the beta-globin gene. We show that the ratio of the two amplified cDNA signals is a highly reliable measure of test gene expression. The sensitivity of this assay is at least 1000-fold higher than RNase protection.
A method for specific amplification and PCR sequencing of individual members of multigene families: application to the study of steroid 21-hydroxylase deficiency.
Authors: Collier S , Tassabehji M , Strachan T
Published: June 2007

Mutations at the human HLA-linked CYP21B locus are responsible for 21-hydroxylase deficiency, a recessively inherited disorder of steroidogenesis. The scope for PCR-based analysis of the CYP21B gene has been restricted by the very high sequence homology between CYP21B and a closely related pseudogene, CYP21A. Here we describe a novel PCR sequencing strategy that allows the independent amplification of the entire CYP21B coding sequence and the subsequent enzyme-mediated conversion of the PCR product to a single-stranded form for dideoxy sequencing. We have used this approach to characterize the 21-hydroxylase deficiency allele associated with HLA-B55, the most frequent HLA marker associated with a CYP21B point mutation in the British population, and also an HLA-B35 associated allele of Asian origin. Allele-specific oligonucleotide (ASO) hybridization analyses have confirmed the selective amplification of CYP21B genes and the identity of the pathological mutations. The method can be adapted to permit selective amplification and PCR sequencing of individual closely related members of other multigene families and small-copy-number repetitive DNA families.
Detection of influenza A and B in respiratory secretions with the polymerase chain reaction.
Authors: Donofrio J C , Coonrod J D , Davidson J N , Betts R F
Published: June 2007

Influenza A and B are RNA-containing viruses that frequently infect humans. Currently, sensitive detection of these viruses requires fresh respiratory secretions and special facilities for culture. To facilitate diagnosis of influenza, the polymerase chain reaction (PCR) was used in the present studies to detect DNA produced by reverse transcription of influenzal RNA in vaccines, tissue culture fluids, and stored respiratory secretions. Primers were directed at targets on the highly conserved segment 7 (matrix gene) of influenza A (212-bp product) and B (365-bp product). The primers were completely type specific. Critical variables in the assay were the concentration of pleotropic salts used during preparation of samples, the use of carrier RNA and RNase inhibitors during sample preparation, and the use of optimum K+ and Mg2+ levels at each step. Studies of 33 patients with symptoms of viral respiratory infection whose nasal washes had been cultured and frozen for up to 1 year before assay showed that PCR provided type-specific detection of influenza with a sensitivity comparable to that of culture of the fresh secretions. The assay offers a powerful test for detection of devitalized influenza viruses and may be useful in both diagnostic work and epidemiological studies of influenza.
Mismatch amplification mutation assay (MAMA): application to the c-H-ras gene.
Authors: Cha R S , Zarbl H , Keohavong P , Thilly W G
Published: June 2007

We have found that under appropriate conditions, an allele-specific polymerase chain reaction (PCR) can achieve a sensitivity suitable for measuring specific, infrequent mutations in single cell systems or in animal tissues. Using the 12th codon GC-to-AT mutation in the rat c-Ha-ras gene as a model system, we have defined conditions that allow for measurement of mutations present at frequencies as low as one in 10(5) gene copies. Our approach involved the use of PCR primers that created a single mismatch with the mutated allele (GAA) but created a double mismatch with the wild-type allele (GGA). Five out of the six such double-mismatch primers we tested permitted amplification of the mutant allele (GAA) with a high degree of specificity. The specificity of the assay was further enhanced by using a two-step PCR cycle consisting of a denaturation step (1 min incubation at 94 degrees C) and an annealing/extension step (1 min incubation at 50 degrees C) in the presence of 10% (vol/vol) glycerol. Reconstruction experiments using genomic DNA demonstrate that this procedure cna measure the presence of 30 copies of the transforming ras allele present amongst 3 x 10(6) copies of the wild-type allele.