bluuebunny/crossref_metadata_2025 huggingface dataset:
Pituitary cell phenotypes involve cell-specific Pit-1 mRNA translation and synergistic interactions with other classes of transcription factors.
Authors: Simmons D M , Voss J W , Ingraham H A , Holloway J M , Broide R S , Rosenfeld M G , Swanson L W
Published: June 2007

Development of the anterior pituitary gland involves proliferation and differentiation of ectodermal cells in Rathke's pouch to generate five distinct cell types that are defined by the trophic hormones they produce. A detailed ontogenetic analysis of specific gene expression has revealed novel aspects of organogenesis in this model system. The expression of transcripts encoding the alpha-subunit common to three pituitary glycoprotein hormones in the single layer of somatic ectoderm on embryonic day 11 established that primordial pituitary cell commitment occurs prior to formation of a definitive Rathke's pouch. Activation of Pit-1 gene expression occurs as an organ-specific event, with Pit-1 transcripts initially detected in anterior pituitary cells on embryonic day 15. Levels of Pit-1 protein closely parallel those of Pit-1 transcripts without a significant lag. Unexpectedly, Pit-1 transcripts remain highly expressed in all five cell types of the mature pituitary gland, but the Pit-1 protein is detected in only three cell types--lactotrophs, somatotrophs, and thyrotrophs and not in gonadotrophs or corticotrophs. The presence of Pit-1 protein in thyrotrophs suggests that combinatorial actions of specific activating and restricting factors act to confine prolactin and growth hormone gene expression to lactotrophs and somatotrophs, respectively. A linkage between the initial appearance of Pit-1 protein and the surprising coactivation of prolactin and growth hormone gene expression is consistent with the model that Pit-1 is responsible for the initial transcriptional activation of both genes. The estrogen receptor, which has been reported to be activated in a stereotypic fashion subsequent to the appearance of Pit-1, appears to be capable, in part, of mediating the progressive increase in prolactin gene expression characteristic of the mature lactotroph phenotype. This is a consequence of synergistic transcriptional effects with Pit-1, on the basis of binding of the estrogen receptor to a response element in the prolactin gene distal enhancer. These data imply that both transcriptional and post-transcriptional regulation of Pit-1 gene expression and combinatorial actions with other classes of transcription factors activated in distinct temporal patterns, are required for the mature physiological patterns of gene expression that define distinct cell types within the anterior pituitary gland.
Sequences 5' to the polyadenylation signal mediate differential poly(A) site use in hepatitis B viruses.
Authors: Russnak R , Ganem D
Published: June 2007

Most genetic elements that employ reverse transcription generate a terminally redundant genomic RNA that serves as the template for this reaction. Because the identical polyadenylation signal is present in each terminally redundant segment, synthesis of this RNA requires that this signal be ignored on the first pass of the transcription machinery, then recognized and used on the second pass. We have studied the mechanism of this differential poly(A) site use in one family of retroid elements, the hepatitis B viruses (hepadnaviruses). Our results indicate that two features are involved: the presence of a variant poly(A) signal (TATAAA) and the participation of multiple sequences 5' to this signal that act to increase the efficiency of its use. Deletion of these upstream elements abolishes proper poly(A) site use, despite the presence of the poly(A) signal and downstream GT- and T-rich motifs known to be required for polyadenylation. Sequences from the corresponding regions of retroviral genomes can restore proper processing to these hepadnaviral deletion mutants. Thus, functionally analogous upstream elements exist in other classes of retroid elements, including those employing the canonical AATAAA hexanucleotide signal.
Mechanism of 3' splice site selection by the catalytic core of the sunY intron of bacteriophage T4: the role of a novel base-pairing interaction in group I introns.
Authors: Michel F , Netter P , Xu M Q , Shub D A
Published: June 2007

The catalytic core of the sunY intron of bacteriophage T4 is separated from its 3' exon by 837 nucleotides, most of which are part of an open reading frame (ORF). Here, we report that transcripts truncated within the sunY ORF self-splice in vitro to a variety of sites in the segment immediately 3' of the core. Recognition of these proximal splice sites is shown to depend on (1) the presence on the intron side of a terminal G, which must not be part of a secondary structure; and (2) the ability of the penultimate intron nucleotide to base-pair with a 3' splice site-binding sequence (3'SSBS) located within the core. The counterpart of the 3'SSBS can be identified in most group I introns. The possible significance of such alternative splicing events for in vivo expression of intron-encoded proteins is discussed.
Escherichia coli RNase E has a role in the decay of bacteriophage T4 mRNA.
Authors: Mudd E A , Carpousis A J , Krisch H M
Published: June 2007

Bacteriophage T4 mRNAs are markedly stabilized, both chemically and functionally, in an Escherichia coli strain deficient in the RNA-processing endonuclease RNase E. The functional stability of total T4 messages increased 6-fold; we were unable to detect a T4 message whose functional stability was not increased. There was a 4-fold increase in the chemical stability of total T4 RNA. The degree of chemical stabilization of six specific T4 mRNAs examined varied from a maximum of 28-fold to a minimum of 1.5-fold. In the RNase E-deficient strain, several minutes delay and a slower rate of progeny production led to a reduction in final phage yield of approximately 50%. Although the effect of the rne temperature-sensitive mutation could be indirect, the simplest interpretation of our results is that RNase E acts directly in the degradation of many T4 mRNAs.
beta-globin dominant control region interacts differently with distal and proximal promoter elements.
Authors: Antoniou M , Grosveld F
Published: June 2007

We have studied the interaction between the dominant control region (DCR) and the promoter of the human beta-globin gene. Expression analysis in MEL cells has revealed that the DCR contains a number of elements capable of replacing the upstream (-250 to -100) erythroid-specific region of the promoter. The DCR strongly stimulates expression from a promoter possessing only a TATA box. However, this basic level of transcription is not induced upon erythroid differentiation of the cells. Mutational analysis of the minimal (-100, noninducible) promoter shows that only the combination of the DCR and the CAC/CCAAT elements provides erythroid-specific transcription. These regions act synergistically to produce full regulated expression during erythroid differentiation.
Structure-function analyses of the HTLV-I Rex and HIV-1 Rev RNA response elements: insights into the mechanism of Rex and Rev action.
Authors: Ahmed Y F , Hanly S M , Malim M H , Cullen B R , Greene W C
Published: June 2007

The ability of the Rex protein of the type I human T-cell leukemia virus (HTLV-I) to regulate expression of the retroviral gag and env structural genes post-transcriptionally is critically dependent on the presence of a Rex response element (RexRE). This cis-regulatory sequence is located within the retroviral 3' long terminal repeat and coincides with a predicted, highly stable RNA stem-loop structure. Rex action requires both the overall secondary structure intrinsic to the RexRE and specific sequences from one small subregion of this large structure. This small subregion likely forms a protein-binding site for Rex or a cellular RNA-binding factor. Whereas Rex can functionally replace the Rev protein of the type 1 human immunodeficiency virus (HIV-1) through its interaction with the analogous Rev response element (RevRE), distinct subregions of this HIV-1 RNA element mediate the responses to Rex and Rev. Strikingly, Rex acts as a dominant repressor of Rev action, following the deletion of the Rex responsive subregion of the RevRE. Similarly, Rev inhibits Rex function in a dominant manner when the Rev responsive subregion of the RevRE is deleted. Together, these findings suggest that Rex and Rev not only interact with their respective RNA response elements but also may either form mixed inactive multimers or interact with a common cellular factor(s). If binding of a common host protein is involved, this factor likely plays a central role either in spliceosome assembly or nuclear RNA transport.
Pan: a transcriptional regulator that binds chymotrypsin, insulin, and AP-4 enhancer motifs.
Authors: Nelson C , Shen L P , Meister A , Fodor E , Rutter W J
Published: June 2007

Two cDNAs were cloned that encode proteins (Pan-1 and Pan-2, with predicted molecular masses of 67.4 and 67.7 kD, respectively) that bind to a conserved transcriptional regulatory element present in pancreatic exocrine genes. The cDNAs are virtually identical in the region that encodes the amino-terminal 525 amino acids; they differ only by a 3-nucleotide insertion that results in the addition of a single amino acid. The cDNAs, however, code for related, but divergent, carboxy-terminal regions. Expression of the cloned cDNAs in HeLa cells activates expression of a cotransfected chimeric gene containing multimers of the pancreatic conserved element linked to a minimal promoter. Pan-1 and Pan-2 bind to the consensus sequence CAC/GCTGT/C, the CACCTGTC motif, which is present in transcriptional control elements of several genes. Both Pan-1 and Pan-2 bind to related motifs present in the rat insulin enhancer as well as a DNA segment containing the SV40 AP-4 element. The Pan-1 and Pan-2 cDNAs display extensive sequence identity with partial cDNA clones encoding E12 and E47, which bind to the immunoglobulin kappa E2 cis-active element, and may be derived from the same gene. These factors belong to a set of genetically distinct molecules, including AP-4 and MLTF, that bind to the CACCTGTC motif or related sequences. The Pan/E12,E47 proteins also show structural similarity with the Drosophila daughterless protein, MyoD, Myogenin, and Myf-5.
Cell motility is required for the transmission of C-factor, an intercellular signal that coordinates fruiting body morphogenesis of Myxococcus xanthus.
Authors: Kim S K , Kaiser D
Published: June 2007

There are striking similarities between the developmental phenotypes of two different mutant classes of Myxococcus xanthus. The first class, mglA mutants, are nonmotile under all conditions tested. The second class, csgA mutants, are motile but belong to a class of signal-defective developmental mutants that cannot develop alone but will develop when mixed with intact wild-type cells. Nevertheless, both csgA and mglA mutants fail to aggregate properly or to sporulate when induced to form fruiting bodies. An mglA mutation and a csgA mutation affect expression of a panel of lacZ fusions to developmental genes in the same way, indicating that nonmotile cells and csgA cells arrest development at a similar stage. One explanation for the similarity of developmental phenotypes between these mutants is that motility is required for the csgA-mediated cell interaction. In support of this hypothesis, we report that C-factor, a protein purified from nascent wild-type fruiting bodies based on its ability to rescue csgA mutant fruiting body development, also rescues sporulation and expression of beta-galactosidase from developmentally controlled lacZ fusions in mglA strains, apparently without restoring their motility. Wild-type levels of active C-factor can be purified from mglA cells, yet intact mglA cells do not rescue csgA cells upon cell-cell mixing. Intact wild-type cells are unable to restore the sporulation and beta-galactosidase expression of mglA mutants. These results support the hypothesis that donor and responder cell motility is required for C-factor transmission between cells during development.(ABSTRACT TRUNCATED AT 250 WORDS)
Assembly of alternative multiprotein complexes directs rRNA promoter selectivity.
Authors: Bell S P , Jantzen H M , Tjian R
Published: June 2007

How can trans-activators with the same DNA binding specificity direct different transcriptional programs? The rRNA transcriptional apparatus offers a useful model system to address this question and to dissect the mechanisms that generate alternative transcription complexes. Here, we compare the mouse and human transcription factors that govern species-specific RNA polymerase I promoter recognition. We find that both human and mouse rRNA transcription is mediated by a specific multiprotein complex. One component of this complex is the DNA-binding transcription factor, UBF. Paradoxically, human and mouse UBF display identical DNA binding specificities even though transcription of rRNA is species specific. Promoter selectivity is conferred by a second essential factor, SL1, which, for humans, does not bind DNA independently but, instead, cooperates with UBF in the formation of high-affinity DNA-binding complexes. In contrast, mouse SL1 can selectively interact with DNA in the absence of UBF. Reconstituted transcription experiments establish that UBF and RNA polymerase I from the two species are functionally interchangeable, whereas mouse and human SL1 exhibit distinct DNA binding and transcription activities. Together, these results suggest a critical role for a specific multiprotein assembly in RNA polymerase I promoter recognition and reveal distinct mechanisms through which such complexes can generate functional diversity.
Cell-cycle-regulated phosphorylation of DNA replication factor A from human and yeast cells.
Authors: Din S , Brill S J , Fairman M P , Stillman B
Published: June 2007

Replication factor A (RF-A) is a multisubunit, cellular protein that functions with SV40 T antigen during the initiation stage of DNA replication at the SV40 origin. It also cooperates with other replication factors to stimulate the activity of both polymerases alpha and delta during chain elongation. RF-A from both human and yeast cells is phosphorylated in a cell-cycle-dependent manner; the protein is phosphorylated at the G1- to S-phase transition, and dephosphorylation occurs at mitosis, thereby resetting this cycle. This observation provides a direct link between a protein required for DNA replication and cell-cycle-regulated protein phosphorylation.
Mutations in the Drosophila gene extradenticle affect the way specific homeo domain proteins regulate segmental identity.
Authors: Peifer M , Wieschaus E
Published: June 2007

We characterized a gene, extradenticle, which seems to interact with a specific subset of Drosophila homeo domain proteins, possibly affecting their target specificity. This interpretation is based on an examination of the zygotic and maternal effect phenotypes of extradenticle mutations. In embryos with reduced levels of extradenticle gene product, anterior and posterior segmental transformations occur. Segmental identity in Drosophila is mediated by the products of the Antennapedia and bithorax complexes. These homeo domain proteins are thought to regulate different target genes specifically in each segment, resulting in different morphologies. extradenticle alters segmental identity without affecting the pattern of expression of homeotic genes. Genetic tests demonstrate that in extradenticle mutants, the homeotic proteins are functional and act in their normal segmental domains, yet segmental identities are altered. Even when homeotic proteins are ectopically expressed under the control of a heterologous promoter, extradenticle mutations affect their consequences. Thus, in the absence of sufficient extradenticle product, altered segmental morphology results from alteration of the functional consequences of specific homeo domain proteins, possibly through alterations in their target gene specificity. extradenticle is also expressed maternally. Complete removal of extradenticle, maternally and zygotically, leads to specific alterations in segmentation, many of which result from failure to maintain the expression of the homeo domain protein engrailed.
A cloned human cDNA determines expression of a mouse stage-specific embryonic antigen and the Lewis blood group alpha(1,3/1,4)fucosyltransferase.
Authors: Kukowska-Latallo J F , Larsen R D , Nair R P , Lowe J B
Published: June 2007

The stage-specific embryonic antigen SSEA-1 is a cell-surface oligosaccharide molecule expressed with temporal precision during the murine preimplantation period and implicated in adhesive events involving the process of compaction. We used a mammalian transient expression system to isolate a cloned human cDNA that determines expression of the SSEA-1 molecule. The cDNA sequence predicts a type II transmembrane protein with a domain structure similar to mammalian glycosyltransferases, but without primary sequence similarity to these enzymes. The carboxy-terminal domain of this protein was shown to be catalytically active as a fucosyltransferase when expressed in COS-1 cells as a portion of a secreted protein A fusion peptide. The enzyme is an exceptional glycosyltransferase in that it can use both type I and type II oligosaccharides as acceptor substrates to generate subterminal Fuc alpha(1,4)- and Fuc alpha(1,3)-linkages, respectively, in a manner analogous to the human Lewis blood group fucosyltransferase. Southern blot analysis shows that the cDNA corresponds to sequences syntenic to the Lewis locus on chromosome 19. These results indicate that this cDNA is the product of the human Lewis blood group locus, provide genetic confirmation of the hypothesis that this enzyme can catalyze two distinct transglycosylation reactions, and outline an approach to the isolation of other sequences that determine expression of developmentally regulated oligosaccharide antigens.
Patterns of expression of cut, a protein required for external sensory organ development in wild-type and cut mutant Drosophila embryos.
Authors: Blochlinger K , Bodmer R , Jan L Y , Jan Y N
Published: June 2007

The loss of cut activity in Drosophila results in the transformation of the neurons and support cells of external sensory (es) organs into those of chordotonal (ch) organs. The cut locus encodes a homeo domain-containing protein, which is expressed in the cells of es, but not in ch, organs. We show by Western analyses the presence of two embryonic protein species whose approximate relative molecular masses of 280 and 320 kD are compatible with that predicted from the primary sequence. We also describe the development of the Cut protein expression pattern and show that Cut is expressed in sensory precursor cells that divide to give rise to es organs. Finally, we analyze the changes in the Cut expression pattern of several mutant alleles of the complex cut locus and show that the mutations affecting es organ development are associated with either altered protein distribution in the PNS or incorrect subcellular Cut protein localization.
Ig/EBP-1: a ubiquitously expressed immunoglobulin enhancer binding protein that is similar to C/EBP and heterodimerizes with C/EBP.
Authors: Roman C , Platero J S , Shuman J , Calame K
Published: June 2007

We report the isolation and characterization of cDNA clones that encode a protein with the same DNA binding specificity as the immunoglobulin heavy chain enhancer binding protein E (muEBP-E). We call the gene encoding this protein Ig/EBP-1. A fusion protein encoded by the cDNA binds specifically to muEBP-E-binding sites (E sites) in both the IgH enhancer and the VH1 promoter. Sequence analysis reveals that Ig/EBP-1 is a member of the "basic-zipper" family of DNA-binding proteins that are characterized by basic regions and heptad repeats of leucine residues. Among known family members, Ig/EBP-1 demonstrates highest homology to C/EBP throughout the DNA-binding domain and leucine repeat region. Ig/EBP-1 and C/EBP have highly overlapping binding specificities; both cloned proteins bind to the IgH enhancer and the VH1 promoter E sites, and Ig/EBP-1 binds to previously characterized C/EBP binding sites in the Rous sarcoma virus (RSV) LTR and the murine albumin promoter. Consistent with their homology in the leucine repeat region, Ig/EBP-1 and C/EBP form heterodimers; Ig/EBP-1 is the first member of this family that has been found to heterodimerize with the well-characterized C/EBP. Ig/EBP-1 mRNA is present in all tissues and cell lines examined, although its levels vary almost 20-fold from different sources, with highest levels in early B cells. In tissues where Ig/EBP-1 and C/EBP are both present, heterodimers may be functionally important. The presence of Ig/EBP-1 in fibroblasts and other tissues where C/EBP is not expressed suggests that Ig/EBP-1 may be functionally important for the activity of the RSV enhancer in these cell types. Finally, elevated expression of Ig/EBP-1 in early B cells may explain in part the enhancer-independent activity of VH promoters early in B-cell development.
In vivo monitoring of a cAMP-stimulated DNA-binding activity.
Authors: Weih F , Stewart A F , Boshart M , Nitsch D , Schütz G
Published: June 2007

The transcriptional activity of the tyrosine aminotransferase (TAT) gene is influenced by two major signal transduction pathways, by glucocorticoids and by glucagon acting via cAMP. We analyzed the effect of cAMP on protein-DNA interactions in vivo and on the transcription rate of the TAT gene. We demonstrate that a cAMP-responsive element (CRE) is located in a tissue-specific DNase I-hypersensitive region, 3.6 kb upstream of the start site of transcription. By using the genomic footprinting technique, we show that this sequence is occupied by protein in uninduced cells and that the in vivo footprint is transiently increased upon cAMP induction. Protein binding at the TAT-CRE correlates with the rate of transcription of the TAT gene. Cycloheximide treatment reveals that the genomic footprint is subject to rapid turnover; however, subsequent cAMP induction in the continued presence of cycloheximide restores the footprint partially. We conclude that as a part of the signal transduction pathway, a cAMP-dependent, post-translational modification increases the DNA-binding activity of a protein to the TAT-CRE and thereby stimulates the transcription rate of the TAT gene.
Floral homeotic mutations produced by transposon-mutagenesis in Antirrhinum majus.
Authors: Carpenter R , Coen E S
Published: June 2007

To isolate and study genes controlling floral development, we have carried out a large-scale transposon-mutagenesis experiment in Antirrhinum majus. Ten independent floral homeotic mutations were obtained that could be divided into three classes, depending on whether they affect (1) the identity of organs within the same whorl; (2) the identity and sometimes also the number of whorls; and (3) the fate of the axillary meristem that normally gives rise to the flower. The classes of floral phenotypes suggest a model for the genetic control of primordium fate in which class 2 genes are proposed to act in overlapping pairs of adjacent whorls so that their combinations at different positions along the radius of the flower can specify the fate and number of whorls. These could interact with class 1 genes, which vary in their action along the vertical axis of the flower to generate bilateral symmetry. Both of these classes may be ultimately regulated by class 3 genes required for flower initiation. The similarity between some of the homeotic phenotypes with those of other species suggests that the mechanisms controlling whorl identity and number have been highly conserved in plant evolution. Many of the mutations obtained show somatic and germinal instability characteristic of transposon insertions, allowing the cell-autonomy of floral homeotic genes to be tested for the first time. In addition, we show that the deficiens (def) gene (class 2) acts throughout organ development, but its action may be different at various developmental stages, accounting for the intermediate phenotypes conferred by certain def alleles. Expression of def early in development is not necessary for its later expression, indicating that other genes act throughout the development of specific organs to maintain def expression. Direct evidence that the mutations obtained were caused by transposons came from molecular analysis of leaf or flower pigmentation mutants, indicating that isolation of the homeotic genes should now be possible.
DNA bending is a determinant of binding specificity for a Drosophila zinc finger protein.
Authors: Spana C , Corces V G
Published: June 2007

The suppressor of Hairy-wing [su(Hw)] locus encodes a zinc finger-containing protein that binds to specific sequences of the Drosophila gypsy element, mediating the mutagenic effects of this retrotransposon. We carried out a detailed analysis of the su(Hw)-gypsy interaction using various biochemical assays. DNase I footprinting delimits a 37-bp region in the coding strand of gypsy that is protected from digestion by the binding of the su(Hw) protein. Specific DNA contacts involved in the interaction were determined by methylation protection analysis and by missing nucleoside experiments using hydroxyl radical. Results from these experiments indicate that the su(Hw) protein binds to sequences homologous to the octamer motif, which is recognized by homeo box-containing proteins in mammalian organisms. Furthermore, two DNA bends present on both sides of the binding site in the absence of protein favor the strength of this interaction.
abdA expression in Drosophila embryos.
Authors: Karch F , Bender W , Weiffenbach B
Published: June 2007

The abdominal A (abdA) gene is one of three transcription units in the Bithorax Complex of Drosophila encoding a homeo box protein; it is flanked by Ultrabithorax (Ubx) and Abdominal B (AbdB). The abdA gene is required for segmental identity of the second through eighth abdominal segments. The transcription unit of abdA is approximately 20 kb long and encodes a protein of 330 amino acids. The abdA homeo box is almost identical to the homeo box of Ubx but is quite different from the AbdB homeo box. A polyclonal antibody to abdA protein stains embryonic nuclei in segments A1-A7 (parasegments 7-13). The iab-2, 3, and 4 mutant classes define positive cis-regulatory elements that induce expression of abdA in segments A2-A4 (parasegments 7-9), respectively. Once a pattern of abdA expression is turned on in a given parasegment, it remains on in the more posterior parasegments, so that the complex pattern of expression is built up in the successive parasegments. The abdA product appears to repress expression of Ubx whenever they appear in the same cell, but abdA is repressed by AbdB only in the eighth and ninth abdominal segments.
Reversible inhibition of a thyroid-specific trans-acting factor by Ras.
Authors: Avvedimento V E , Musti A M , Ueffing M , Obici S , Gallo A , Sanchez M , DeBrasi D , Gottesman M E
Published: June 2007

Exposure of rat thyroid cells for 1 week to a temperature-sensitive variant of Kirsten murine sarcoma virus (KiMSV) Ras inactivated the thyroglobulin promoter (pTg). Cellular dedifferentiation was paralleled by the loss of the thyroid-specific trans-acting factor, TgTF1, which binds to pTg. When Ras was denatured by shifting cells to 39 degrees C, TgTF1 binding and pTg function recovered rapidly without the synthesis of new protein. TgTF1 could be reactivated in vitro by treating nuclear extracts with protein kinase A. After 4 weeks of exposure to the oncogene, denaturation of Ras no longer restored TgTF1 binding or reactivated pTg. Incubation of nuclear extracts with protein kinase A likewise did not reactivate TgTF1. Cells chronically exposed to Ras did, however, yield differentiated clones after treatment with 5-azacytidine. We suggest that Ras induces dedifferentiation in two sequential steps: (1) Ras reduces PKA activity; TgTF1 (or an auxiliary protein) becomes dephosphorylated, and binding to pTg is abolished. (2) The effects of Ras become imprinted by methylation, possibly of the TgTF1 gene.
Rapid in vivo footprinting technique identifies proteins bound to the TTR gene in the mouse liver.
Authors: Mirkovitch J , Darnell J E
Published: June 2007

In vivo examination of the occupancy of DNA elements that can regulate transcription is critical to reveal which proteins actually take part in establishing and maintaining gene expression. We describe a new genomic sequencing method involving the rapid purification of relevant DNA segments from the bulk of the genomic DNA using a biotinylated riboprobe. The purified sequences are revealed by a single primer extension using Taq DNA polymerase. We used this technique to study the promoter and the enhancer of mouse transthyretin (TTR), a gene highly expressed in the liver. Footprints showed high liver-specific occupancy of some, but not all, of the DNA sites that had been identified as important for expression by transfection studies in hepatoma cells. In addition, several previously undetected sites were observed that bound proteins specifically in liver. These results suggest that not all demonstrable binding sites are involved in ongoing transcription and that in vivo studies may reveal additional and probably more relevant sites.
Chromosome tangling and breakage at anaphase result from mutations in lodestar, a Drosophila gene encoding a putative nucleoside triphosphate-binding protein.
Authors: Girdham C H , Glover D M
Published: June 2007

We describe a Drosophila maternal-effect gene, lodestar, mutations in which cause chromatin bridges at anaphase. lodestar maps to cytological position 84D13-14, and we identified the lodestar gene in germ-line transformation experiments by the ability of a genomic fragment to restore fertility to females homozygous for lodestar mutations. lodestar encodes a potential nucleoside triphosphate binding protein, which is a novel member of the D-E-A-H box family of proteins. Antibodies raised against the lodestar gene product detect a protein that undergoes cell cycle-dependent changes in distribution in the embryo. The protein is cytoplasmic at interphase, and rapidly enters the nucleus early in prophase. It is restricted to the region enclosed by the spindle envelope during metaphase and anaphase; but by telophase, the lodestar protein is contained entirely within the reforming nucleus.
Temporal regulation of gene expression in the blastoderm Drosophila embryo.
Authors: Yasuda G K , Baker J , Schubiger G
Published: June 2007

The Drosophila embryo undergoes a developmental transition during cycle 14 when it initiates asynchronous mitotic cycles and markedly increases its rate of zygotic transcription. The nucleo-cytoplasmic ratio has been proposed to be the single factor that temporally regulates this developmental transition. We altered the ratio in the embryo and analyzed the consequences on the cell cycle program and on the transcripts of specific genes. These genes were chosen because their transcripts normally undergo changes in pattern during cycle 14. We found evidence that the nucleo-cytoplasmic ratio is read and interpreted locally to regulate the cell cycle program. Based on the response of the transcripts to changes in the ratio, we found evidence that at least two classes of temporal regulatory mechanisms control these transcripts. We therefore propose two corresponding classes of transcripts: (1) nucleo-cytoplasmic ratio dependent; and (2) nucleo-cytoplasmic ratio independent or time correlated. The temporal regulation of the ratio-independent transcripts may be dependent on developmental time. We conclude that multiple modes of temporal regulation underlie the events of the developmental transition in Drosophila embryogenesis.
Negative regulatory sequences in the lin-14 3'-untranslated region are necessary to generate a temporal switch during Caenorhabditis elegans development.
Authors: Wightman B , Bürglin T R , Gatto J , Arasu P , Ruvkun G
Published: June 2007

The heterchronic gene lin-14 controls the temporal sequence of developmental events in the Caenorhabditis elegans postembryonic cell lineage. It encodes a nuclear protein that normally is present in most somatic cells of late embryos and L1 larvae but is absent at later stages. Two lin-14 gain-of-function mutations delete 3'-untranslated sequences causing an inappropriately high level of the lin-14 nuclear protein late in development. These mutations identify a negative regulatory element that controls the formation of the lin-14 protein temporal gradient. The 21-kb lin-14 gene is differentially spliced to generate three lin-14 transcripts that encode protein products with variable amino-terminal regions and a constant carboxy-terminal region. The sequence of the gene revealed no protein sequence similarity to any proteins in various data bases.
Processing of TCP pilin by TcpJ typifies a common step intrinsic to a newly recognized pathway of extracellular protein secretion by gram-negative bacteria.
Authors: Kaufman M R , Seyer J M , Taylor R K
Published: June 2007

Biogenesis of the Vibrio cholerae toxin-coregulated pilus (TCP) requires the activities of at least seven accessory proteins. We demonstrate that a portion of this pathway involves a novel processing step in which a hydrophilic leader peptide is proteolytically removed from TcpA by the gene product characterized in this report, TcpJ, to yield the mature, export-competent form of the pilin. Cleavage of the pilin leader peptide is independent of known signal peptidases as demonstrated by pilin-processing profiles in Escherichia coli strains conditionally defective for production of leader peptidase or grown in the presence of the antibiotic globomycin. Additionally, pilin cleavage did not rely on the SecA protein, as evidenced by TcpA processing in azide-treated cells. These results suggest that TcpJ is representative of a new class of proteins involved in SecA-independent proteolytic cleavage of a set of atypical leader peptides during extracellular export.
Transcription from a TATA-less promoter requires a multisubunit TFIID complex.
Authors: Pugh B F , Tjian R
Published: June 2007

In eukaryotes, the TATA box-binding protein (TBP) is responsible for nucleating assembly of the transcription initiation machinery. Here, we report that a TFIID complex containing TBP is essential for transcription even at a promoter that lacks a TATA box. Immunopurification of TFIID reveals that the active species in reconstituting TATA-less transcription is a multisubunit complex consisting of TBP and many TBP-associated factors (TAFs).
xUBF contains a novel dimerization domain essential for RNA polymerase I transcription.
Authors: McStay B , Frazier M W , Reeder R H
Published: June 2007

Xenopus laevis upstream binding factor (xUBF) is an RNA polymerase I transcription factor that is required for formation of the stable initiation complex. The 701-amino-acid protein contains three regions of homology to the chromosomal protein HMG1 (the HMG boxes), which act in comparative independence to cause DNA binding. DNA binding is augmented by a 102-residue amino-terminal domain that causes xUBF to form dimers. The dimerization domain is bipartite in structure, consisting of two regions with the potential to form amphipathic helices, separated by a gap of at least 22 amino acids. The carboxyl half of xUBF is relatively dispensable for transcription (including an 87-residue acidic tail). However, either altering the number of HMG boxes or interfering with dimerization eliminates transcription. The gap region of the dimerization domain is dispensable for dimerization but is absolutely required for transcription. This suggests that the gap region has a critical function in transcription distinct from any effect on dimerization or DNA binding.
N-myristoylation is required for function of the pheromone-responsive G alpha protein of yeast: conditional activation of the pheromone response by a temperature-sensitive N-myristoyl transferase.
Authors: Stone D E , Cole G M , de Barros Lopes M , Goebl M , Reed S I
Published: June 2007

In a screen designed to identify novel mutations in the mating response pathway of Saccharomyces cerevisiae, we isolated conditional alleles of NMT1, the gene encoding N-myristoyl transferase. Genetic data indicate that Nmt1 deficiency results in the activation of the pheromone response at the level of Gpa1, the alpha subunit of the pheromone-responsive G protein. We show that Gpa1 is myristoylated by Nmt1, and without this normally stable modification, Gpa1 is unable to inhibit pheromone signaling. This loss of Gpa1 function is probably not the result of improper subcellular localization. Unlike the mammalian G alpha i proteins alpha i and alpha o, nonmyristoylated Gpa1 is able to associate with membranes. In addition to Gpa1, our data indicate that Nmt1 myristoylates other proteins essential to vegetative growth.
Schizosaccharomyces pombe ste11+ encodes a transcription factor with an HMG motif that is a critical regulator of sexual development.
Authors: Sugimoto A , Iino Y , Maeda T , Watanabe Y , Yamamoto M
Published: June 2007

Schizosaccharomyces pombe ste11 encodes a member of the family of HMG-box proteins. Its transcript is induced in response to nitrogen starvation and a concomitant decrease of the intracellular cAMP level. Expression of ste11 is essential for induction of sexual development, and its ectopic expression stimulates uncontrolled mating and sporulation. Ste11 protein regulates positively transcription of the following genes required for sexual development: the mating type genes, matP and matM, and the mei2 gene, which is essential for commitment to meiosis. Ste11 protein synthesized in vitro binds specifically to a DNA fragment carrying a 10-base motif TTCTTTGTTY that is an essential cis-acting element for the induction of mei2 and is commonly seen in the upstream regions of the genes inducible by nitrogen starvation. These observations strongly suggest that Ste11 serves as a key transcription factor for sexual development.
tat regulates binding of the human immunodeficiency virus trans-activating region RNA loop-binding protein TRP-185.
Authors: Wu F , Garcia J , Sigman D , Gaynor R
Published: June 2007

The TAR element extending from -17 to +80 in the human immunodeficiency virus long terminal repeat (HIV LTR) is required for activation of gene expression by the tat trans-activator protein. TAR RNA forms a stable stem-loop structure, and mutagenesis studies indicate that the stem structure, the primary sequence of the loop, and the bulge element are the major determinants for tat activation. RNA gel retardation analysis demonstrates that both tat and cellular proteins bind to TAR RNA, but the mechanism by which these proteins increase HIV gene expression is unknown. We have fractionated HeLa cell nuclear extracts in an attempt to identify cellular proteins that bind to TAR RNA and are involved in regulating HIV gene expression. RNA gel retardation and UV cross-linking reveal that a cellular protein of 185 kD, which we designate TAR RNA-binding protein 185 (TRP-185), binds with both high affinity and marked specificity to TAR RNA. RNA gel retardation and competition analyses indicate that TRP-185 binding is strongly dependent on the TAR RNA loop sequences. The binding of TRP-185 is modulated by both a set of cellular cofactors and the tat protein. Highly purified preparations of TRP-185 are capable of activating in vitro transcription of wild-type, but not mutated, HIV LTR chloramphenicol acetyltransferase (CAT) constructs. These results characterize a positively acting cellular RNA-binding factor, TRP-185, which is involved in the regulation of HIV gene expression.
Analysis of arginine-rich peptides from the HIV Tat protein reveals unusual features of RNA-protein recognition.
Authors: Calnan B J , Biancalana S , Hudson D , Frankel A D
Published: June 2007

Arginine-rich sequences are found in many RNA-binding proteins and have been proposed to mediate specific RNA recognition. Fragments of the HIV-1 Tat protein that contain the arginine-rich region of Tat bind specifically to a 3-nucleotide bulge in TAR RNA. To determine the amino acid requirements for specific RNA recognition, we synthesized a series of mutant Tat peptides spanning this domain (YGRKKRRQRRRP) and measured their affinity and specificity for TAR RNA. Several corresponding mutations were introduced into the full-length Tat protein, and trans-activation activity was measured. Systematic substitution of arginine residues with alanines or lysines suggested that overall charge density is important but did not point to any specific residues as being essential for binding. A glutamine-to-alanine substitution had no effect on binding. Remarkably, peptides with scrambled or reversed sequences showed the same affinity and specificity for TAR RNA as the wild-type peptide. Trans-activation activity of the mutant Tat proteins correlated with RNA binding. Arginine-rich peptides from SIV Tat and from HIV-1 Rev, which can functionally substitute for the basic region of HIV-1 Tat, also bound specifically to TAR. Circular dichroism spectra suggest that the arginine-rich region of Tat is unstructured in the absence of RNA, becomes partially or fully structured upon binding, and induces a conformational change in the RNA. These results suggest that arginine-rich RNA-binding domains have considerable sequence flexibility, reminiscent of acidic domains found in transcriptional activators, and that RNA structure may provide much of the specificity for the interaction.
The protein-coding region of c-myc mRNA contains a sequence that specifies rapid mRNA turnover and induction by protein synthesis inhibitors.
Authors: Wisdom R , Lee W
Published: June 2007

The stability of certain mRNAs is known to be affected by translation. Some mRNAs appear to be protected from rapid degradation by translation, whereas degradation is coupled to translation for other mRNAs. The molecular determinants of this selective effect of translation are unknown. One example of this effect is the induction of early-response gene mRNAs in the presence of translation inhibitors. To define the molecular basis of induction of early-response gene mRNA expression by inhibitors of protein synthesis, we have performed a mutational analysis of one member of the early response gene family, the c-myc gene. We find that induction by cycloheximide is due to stabilization of c-myc transcripts. The requirements for increased expression of c-myc mRNA by cycloheximide are the presence of the sequence encoding c-myc amino acids 335-439 on a mRNA that can be translated; all other portions of the c-myc gene are dispensable, and this sequence can confer induction of mRNA expression by protein synthesis inhibitors on a heterologous gene. By direct measurement of mRNA turnover in the absence of transcription-blocking drugs, we show that this sequence can function as a selective mRNA destabilizing element, that turnover mediated by this element is translation dependent, and turnover mediated by this element is inhibited by actinomycin D. Our results support the hypothesis that degradation of c-myc mRNA is coupled to translation, that the sequences specifying this form of degradation are contained in the protein-coding sequence, and that translation inhibitors induce expression of c-myc mRNA by blocking turnover mediated by this element.
Expression of two members of the Wnt family during mouse development--restricted temporal and spatial patterns in the developing neural tube.
Authors: Roelink H , Nusse R
Published: June 2007

The Wnt gene family encodes a group of cysteine-rich proteins implicated in intercellular signaling during several stages of vertebrate development. This family includes Wnt-1 and Wnt-3, both discovered as activated oncogenes in mouse mammary tumors. Here we describe the molecular cloning of an additional member of the Wnt family, called Wnt-3A, and the spatial and temporal expression pattern of this gene as well as that of its close relative Wnt-3. The putative amino acid sequences of both proteins are almost 90% identical, but in situ hybridization to mouse embryo sections showed highly restricted patterns of expression of Wnt-3 and Wnt-3A, largely in separate areas in the developing nervous system. In the spinal cord Wnt-3 was expressed at low levels in the alar laminae and in the ventral horns, whereas Wnt-3A expression was confined to the roof plate. In the developing brain Wnt-3 was expressed broadly across the dorsal portion of the neural tube with a rostral boundary of expression at the diencephalon. In contrast, Wnt-3A was expressed in a narrow region very close to the midline; expression extended into the bifurcating telencephalon, in a highly localized fashion. Both Wnt-3 and Wnt-3A were expressed in the ectoderm, and Wnt-3A was also expressed in the periumbilical mesenchyme. Characteristic expression patterns of these two closely related genes suggest that Wnt-3 and Wnt-3A play distinct roles in cell-cell signaling during morphogenesis of the developing neural tube.
Does steric interference between splice sites block the splicing of a short c-src neuron-specific exon in non-neuronal cells?
Authors: Black D L
Published: June 2007

The neuron-specific splicing of the mouse c-src N1 exon was analyzed. Model src genes, transiently expressed in HeLa and LA-N-5 neuroblastoma cells, were assayed for the insertion of the 18-nucleotide neuron-specific N1 exon into their product mRNA. The normal clone fails to use this exon in HeLa cells but inserts the exon into 50% of the mature mRNA in LA-N-5 cells. When the exon and flanking intron sequences are placed between two adenovirus exons, the N1 exon is still only inserted in the neural cells. Thus, the neural specificity is a property of the exon itself and its immediate flanking sequences. Simply extending the length of the N1 exon to 109 nucleotides allows its efficient use in HeLa cells, implying that the exon is normally skipped because it is too short to allow spliceosomes to assemble at both ends simultaneously. This model predicts that exclusion of the exon should be sensitive to proteins or mutations that alter the relative strength of the flanking splice sites. Mutations that change these splice sites support this hypothesis.
Hepatocyte nuclear factor 3 alpha belongs to a gene family in mammals that is homologous to the Drosophila homeotic gene fork head.
Authors: Lai E , Prezioso V R , Tao W F , Chen W S , Darnell J E
Published: June 2007

By analysis of cDNA clones that cross-hybridized with a portion of the cDNA encoding the recently described rat protein hepatocyte nuclear factor 3 alpha (HNF-3 alpha, previously called HNF-3A), we now describe two additional members, HNF-3 beta and HNF-3 gamma, of this gene family. A 110-amino-acid region in the DNA-binding domain of this family is not only very highly conserved in rodents (HNF-3 alpha, -3 beta, and -3 gamma are identical in 93 of 110 amino acids in this region) but also in Drosophila where the homeotic gene fork head has 88 of the 93 residues that are identical in the three rat genes. The HNF-3 family in rodents is expressed in cells that derive from the lining of the primitive gut; some of the embryonic Drosophila cells in which fork head is expressed also give rise to gut and salivary glands. Thus, it appears that this gene family, the DNA-binding portion of which is unlike that of any previously recognized DNA-binding proteins, may contribute to differentiation of cells in internal organs in both vertebrates and invertebrates.