bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo transcriptional pausing and cap formation on three Drosophila heat shock genes.
Authors: Rasmussen E B , Lis J T
Published: May 2006

The regulation of many eukaryotic genes occurs at the level of transcriptional elongation. On the uninduced hsp70 gene of Drosophila melanogaster, for example, an RNA polymerase II complex has initiated transcription but has paused early in elongation. In this study, we examine pausing on hsp70 and two of the small heat shock genes (hsp27 and hsp26) at high resolution, using a technique that utilizes paramagnetic particle-mediated selection of terminated run-on transcripts. This technique provides precise information on the distribution of RNA polymerase within each transcription unit. It also details the progression of 5' cap formation on the elongating transcripts. For each gene, we find polymerases paused over a relatively narrow promoter-proximal region. The regions are generally around 20 nucleotides wide, with two preferred pausing positions spaced roughly 10 nucleotides apart or about one turn of the helix. The bulk of capping occurs as transcripts pass between 20 and 30 nucleotides in length. Interestingly, in the three genes examined here, elongational pausing and 5' cap formation appear largely coincident.
The primary structure of rat brain (cytoplasmic) dynein heavy chain, a cytoplasmic motor enzyme.
Authors: Zhang Z , Tanaka Y , Nonaka S , Aizawa H , Kawasaki H , Nakata T , Hirokawa N
Published: May 2006

Overlapping cDNA clones encoding the heavy chain of rat brain cytoplasmic dynein have been isolated. The isolated cDNA clones contain an open reading frame of 13,932 bp encoding 4644 aa (M(r), 532,213). The deduced protein sequence of the heavy chain of rat brain dynein shows significant similarity to sea urchin flagellar beta-dynein (27.0% identical) and to Dictyostelium cytoplasmic dynein (53.5% identical) throughout the entire sequence. The heavy chain of rat brain (cytoplasmic) dynein contains four putative nucleotide-binding consensus sequences [GX4GK(T/S)] in the central one-third region that are highly similar to those of sea urchin and Dictyostelium dyneins. The N-terminal one-third of the heavy chain of rat brain (cytoplasmic) dynein shows high similarity (43.8% identical) to that of Dictyostelium cytoplasmic dynein but poor similarity (19.4% identical) to that of sea urchin flagellar dynein. These results suggested that the C-terminal two-thirds of the dynein molecule is conserved and plays an essential role in microtubule-dependent motility activity, whereas the N-terminal regions are different between cytoplasmic and flagellar dyneins.
Inhibition of human immunodeficiency virus type 1 replication by regulated expression of a polymeric Tat activation response RNA decoy as a strategy for gene therapy in AIDS.
Authors: Lisziewicz J , Sun D , Smythe J , Lusso P , Lori F , Louie A , Markham P , Rossi J , Reitz M , Gallo R C
Published: May 2006

We are investigating a strategy for somatic gene therapy to treat human immunodeficiency virus type 1 (HIV-1) infection by intracellular expression of an RNA decoy and a ribozyme. The RNA decoy, consisting of polymeric Tat activation response elements (TARs), is designed to compete for Tat binding in an equilibrium with viral TAR RNA, thereby inhibiting viral replication. The expression of polymeric TAR is regulated by the HIV long terminal repeat (LTR) and transcriptional activation is dependent on the presence of HIV Tat. Our initial studies indicated that plasmids expressing up to 50 tandem copies of TAR RNA (50TAR) inhibited tat-mediated gene expression by > 90% in a transient transfection assay. A HIV LTR-driven 50TAR construct was subcloned into a replication-defective retroviral vector to ensure high-efficiency gene transfer into T lymphocytes. In addition, a gag RNA-specific ribozyme gene was introduced into the 50TAR containing retroviral vector to enhance the inhibitory effect of the construct (designated TAR-Rib). A human T-cell line (Molt3) was infected (transduced) with the TAR-Rib recombinant retrovirus and challenged with either HIV-1 or simian immunodeficiency virus (SIV). HIV-1 replication was inhibited by 99% in the TAR-Rib-transduced T cells and was maintained over a 14-month period, suggesting that this antiviral strategy represses the formation of escape mutants. Interestingly, the TAR-Rib also inhibited SIV replication in transduced T cells, which suggests that polymeric TAR is a general inhibitor of primate lentiviruses; therefore, the macaque model could be used for further in vivo testing of this antiviral gene therapy strategy.
Crystal structure of yeast TATA-binding protein and model for interaction with DNA.
Authors: Chasman D I , Flaherty K M , Sharp P A , Kornberg R D
Published: May 2006

The C-terminal 179-aa region of yeast (Saccharomyces cerevisiae) TATA-binding protein (TBP), phylogenetically conserved and sufficient for many functions, formed crystals diffracting to 1.7-A resolution. The structure of the protein, determined by molecular replacement with coordinates from Arabidopsis TBP and refined to 2.6 A, differed from that in Arabidopsis slightly by an angle of about 12 degrees between two structurally nearly identical subdomains, indicative of a degree of conformational flexibility. A model for TBP-DNA interaction is proposed with the following important features: the long dimension of the protein follows the trajectory of the minor groove; two rows of basic residues conserved between the subdomains lie along the edges of the protein in proximity to the DNA phosphates; a band of hydrophobic residues runs down the middle of the groove; and amino acid residues whose mutation alters specificity for the second base of the TATA sequence are juxtaposed to that base.
Physicochemical characterization of a ouabain isomer isolated from bovine hypothalamus.
Authors: Tymiak A A , Norman J A , Bolgar M , DiDonato G C , Lee H , Parker W L , Lo L C , Berova N , Nakanishi K , Haber E
Published: May 2006

Recent reports have shown the presence of a ouabain-like inhibitor of Na+/K(+)-ATPase in humans. We have purified a bovine hypothalamic Na+/K(+)-ATPase inhibitory factor (HIF) by using affinity chromatography combined with HPLC. This inhibitor has a molecular weight of 584 as determined by ion-spray mass spectrometry, making it isobaric with ouabain. Glycosidase treatment or acid hydrolysis of HIF released only L-rhamnose, the hexose isomer found in ouabain, as detected by chiral GC/MS. Additionally, enzymatically generated desrhamnosyl HIF was found to have a molecular weight of 438, as does ouabagenin, the aglycone of ouabain. HIF and its aglycone were indistinguishable from ouabain and ouabagenin, respectively, by reversed-phase HPLC retention times. However, derivatization with naphthoylimidazole followed by HPLC revealed different retention times for naphthoylation products of HIF and ouabain. Subsequent CD spectroscopy on isolated naphthoylation products of HIF and ouabain confirmed that they were different. This study provides chromatographic and spectroscopic evidence that ouabain and HIF are isomeric cardenolides. The structural difference is presumed to account for the significant differences in biological properties observed for HIF and ouabain.
Characterization of a hepatoma mRNA transcribed from a third promoter of a 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-encoding gene and controlled by ets oncogene-related products.
Authors: Dupriez V J , Darville M I , Antoine I V , Gegonne A , Ghysdael J , Rousseau G G
Published: May 2006

6-Phosphofructo-2-kinase (EC 2.7.1.105)/fructose-2,6-bis-phosphatase (EC 3.1.3.46) catalyzes the synthesis and degradation of fructose 2,6-bisphosphate, a ubiquitous stimulator of glycolysis. The liver (L-type) and muscle (M-type) mRNAs for this bifunctional enzyme arise from distinct promoters of the same gene. We have now characterized in rat hepatoma FTO2B cells another mRNA, which is transcribed from a third promoter of that gene. This F-type mRNA is present in fetal rat liver and muscle, in rat placenta, and in several established rat cell lines. The F promoter contains no TATA box but contains several binding sites for Sp1 and for members of the ets oncogene family. Transfection of FTO2B cells with constructs containing the intact or mutagenized F promoter showed that its activity depends mainly on one of these sites. This site bound a heteromeric FTO2B cell protein indistinguishable from the ets-related GA binding protein alpha/ankyrin-repeats GA binding protein beta transcription factor.
Zn2+ potentiates excitatory action of ATP on mammalian neurons.
Authors: Li C , Peoples R W , Li Z , Weight F F
Published: May 2006

Despite the increasing recognition that ATP is an important extracellular excitatory mediator in the nervous system, the regulation of ATP receptors is poorly understood. Because the extracellular Zn2+ concentration is regulated in a variety of biological tissues, we studied modulation of the ATP-gated cation channel by Zn2+ in mammalian neurons using the whole-cell patch-clamp technique. In approximately 73% of cells tested, the amplitude of ATP-activated membrane ion current increased up to 5-fold in the presence of micromolar concentrations of Zn2+. The characteristics of this action suggest that Zn2+ increases the apparent affinity of the receptor for ATP. In addition, Zn2+ increased membrane depolarization and action potential firing elicited by ATP. These observations suggest that Zn2+ may play a physiological role in regulating the excitatory action of ATP on mammalian neurons.
Phospholipase A2 activity can protect renal tubules from oxygen deprivation injury.
Authors: Zager R A , Schimpf B A , Gmur D J , Burke T J
Published: May 2006

During hypoxic or ischemic renal tubular injury, phospholipase A2 (PLA2) induces membrane deacylation, causing fatty acid accumulation and phospholipid breakdown. Because these changes can compromise cellular integrity, PLA2 activity has been widely proposed as a critical mediator of hypoxic renal tubular injury and, hence, of ischemic acute renal failure. To explore this hypothesis, isolated rat proximal tubules were subjected to continuous oxygenation or to hypoxic injury with or without exogenous PLA2 addition (porcine or bovine pancreatic PLA2; bee or snake venom PLA2). Cell death was quantified by lactic dehydrogenase (LDH) release. Pancreatic PLA2 (0.4 unit/ml) caused no LDH release under oxygenated conditions, and it dramatically attenuated hypoxic cell death (e.g., no PLA2, 55 +/- 3% LDH release; porcine pancreatic PLA2, 22 +/- 1% LDH release; P < 0.001). Bee and snake venom PLA2 (0.4 unit/ml) were directly toxic to tubules under oxygenated conditions, and this injury was additive with that induced by hypoxia. However, when these venoms were serially diluted (removing their overt toxicity), they, too, mitigated hypoxic cell death (LDH release with PLA2, 33 +/- 2%; without PLA2, 60 +/- 1% LDH release; P < 0.001). PLA2-mediated cytoprotection was Ca2+ dependent (negated by Ca2+ chelation), and it was expressed despite worsening hypoxia-associated membrane deacylation/fatty acid accumulation (12 times) and ATP depletion. These results indicate that PLA2 activity can exert both beneficial and deleterious effects on O2-deprived renal tubules, the net result of which can be a salvaging of cells from hypoxic cell death.
Transgenic mouse models of lipoprotein metabolism and atherosclerosis.
Authors: Breslow J L
Published: May 2006

Lipoprotein transport genes have either been added to the germ line of mice by transgenic techniques or knocked out by homologous recombination in embryonic stem cells. The resultant over- or underexpression of these genes has resulted in new insights about how these genes function in the body and their role in lipoprotein metabolism. Either singly or in combination, these genetic modifications can be used to engineer the mouse to make it a better model for human lipoprotein disorders and atherosclerosis.
Fighting for a harem of queens: physiology of reproduction in Cardiocondyla male ants.
Authors: Heinze J , Hölldobler B
Published: May 2006

Several species of the ant genus Cardiocondyla produce dimorphic males, which exhibit sharply different mating strategies. Winged males typically disperse to mate outside the nest, whereas wingless, ergatoid males stay in the nest and aggressively employ their mandibles against competing ergatoid males to monopolize the virgin queens eclosing in the nest. Such aggressive mating strategy would only be adaptive if the males had unlimited sperm supply. Histological studies showed that, contrary to the rule in the Hymenoptera order, the ergatoid Cardiocondyla males are indeed able to produce sperm during their entire adult life. Winged males, on the other hand, have only a limited sperm supply since spermatogenesis ceases in the late pupal stage.
Cellular interactions implicated in the mechanism of photoreceptor degeneration in transgenic mice expressing a mutant rhodopsin gene.
Authors: Huang P C , Gaitan A E , Hao Y , Petters R M , Wong F
Published: May 2006

Photoreceptors of transgenic mice expressing a mutant rhodopsin gene (Pro347-->Ser) slowly degenerate. The mechanism of degeneration was studied by aggregation of embryos of normal and transgenic mice to form chimeras. In these chimeras, mosaicism was observed in the coat color, retinal pigment epithelium, and retina. In the retina, the genotype of adjacent patches of normal and transgenic photoreceptors was determined by in situ hybridization with a transgene-specific RNA probe. Photoreceptors in the chimeric retina degenerated uniformly, independent of the genotype and similar to the photoreceptors in transgenic mice. However, the chimeric retinas showed varying proportions of normal and transgenic cells. The chimeric retina with a nearly even proportion of normal and transgenic photoreceptors displayed uniform but slower degeneration than that observed in a transgenic mouse of the same age. Our results demonstrate non-autonomy of gene action for the mutated rhodopsin gene and imply that cellular interactions between photoreceptors in the retina probably play a role in degeneration.
Polyoma virus early-late switch: regulation of late RNA accumulation by DNA replication.
Authors: Liu Z , Carmichael G G
Published: May 2006

Early in infection of permissive mouse cells, messages from the early region of the polyoma virus genome accumulate preferentially over those from the late region. After initiation of DNA replication, the balance between early and late gene expression is reversed in favor of the late products. In previous work from our laboratory, we showed that viral early proteins do not activate the polyoma late promoter in the absence of DNA replication. Here we show that activation of the late genes in replication-incompetent viral genomes can occur if actively replicating genomes are present in the same cell. A low level of DNA replication, however, is insufficient to induce the early-late switch. Furthermore, replication-competent genomes that fail to accumulate late RNA molecules are defective in the transactivation of replication-incompetent genomes. We suggest that titration of an unknown diffusible factor(s) after DNA replication relieves the block to late RNA accumulation seen in the early phase, with most of this titration being attributable to late-strand RNA molecules themselves.
Cytolytic activity of intestinal intraepithelial lymphocytes in germ-free mice is strain dependent and determined by T cells expressing gamma delta T-cell antigen receptors.
Authors: Kawaguchi M , Nanno M , Umesaki Y , Matsumoto S , Okada Y , Cai Z , Shimamura T , Matsuoka Y , Ohwaki M , Ishikawa H
Published: May 2006

We have compared the cytolytic activities and the cellular compositions of the intestinal intraepithelial lymphocyte (i-IEL) populations in three different combinations of conventional (CV) and germ-free (GF) mice. Cytolytic activity of i-IELs expressing gamma delta T-cell antigen receptors (TCRs) is strain dependent in CV mice (high vs. low), and this strain-dependent variability is unaltered in the GF condition. Although absolute numbers of gamma delta i-IELs are slightly decreased, the composition of CD8 alpha alpha+ and CD4-CD8- subsets and the usage of TCR gamma- and delta-chain variable gene segments by gamma delta i-IELs remain the same in GF mice. By contrast, cytolytic activity of alpha beta TCR-expressing i-IELs is uniformly high in CV mice but attenuated sharply in the GF condition. A conspicuous decrease in the total numbers of alpha beta i-IELs is also noted, and CD8 alpha beta+ and CD4+CD8+ subsets are reduced, whereas the CD8 alpha alpha+ subset is expanded in GF mice. These results indicate that microbial deprivation preferentially influences the alpha beta i-IEL population to decrease and become noncytolytic but has little effect on the pool size or characteristics of gamma delta i-IELs. Consequently, cytolytic activity of freshly isolated i-IELs from GF mice is determined by T cells expressing gamma delta TCRs and is found to be strain dependent.
Isolation of virulence genes directing surface glycosyl-phosphatidylinositol synthesis by functional complementation of Leishmania.
Authors: Ryan K A , Garraway L A , Descoteaux A , Turco S J , Beverley S M
Published: May 2006

Trypanosomatid parasites of the genus Leishmania cause a spectrum of widespread tropical diseases. In the vertebrate host they reside within the macrophage phagolysosome; however, the mechanisms employed in this remarkable survival strategy are not well understood. Recent advances in the molecular genetics of these parasites prompted us to develop methods of functional genetic complementation in Leishmania and apply them to the isolation of genes involved in the biosynthesis of the virulence determinant lipophosphoglycan, an abundant glycosyl-phosphatidylinositol-anchored polysaccharide. LPG1, the gene product identified by complementation of the R2D2 mutant, appears to be a glycosyltransferase responsible for the addition of galactofuranosyl residues to the nascent lipophosphoglycan chain. As galactofuranose is not found in mammalian cells, inhibition of the addition of this sugar could be exploited for chemotherapy. Overall, the success of the functional complementation approach opens the way to the identification of a variety of genes involved in pathogenesis and parasitism.
Characterization of distinct functional domains of transforming growth factor beta.
Authors: Burmester J K , Qian S W , Roberts A B , Huang A , Amatayakul-Chantler S , Suardet L , Odartchenko N , Madri J A , Sporn M B
Published: May 2006

Three distinct isoforms of transforming growth factor beta (TGF-beta) are expressed in mammalian cells. Although many cells respond equivalently to all three isoforms, certain cells respond selectively. Using chimeric proteins in which selected regions of the different isoforms were interchanged, we have identified two distinct functional domains of TGF-beta involved in determining the biological potencies and functions of the molecule. The first domain is important for determining whether TGF-beta can be sequestered by alpha 2-macroglobulin. By replacing aa 45 and 47 of TGF-beta 2 with the corresponding amino acids of TGF-beta 1, sequestration of the TGF-beta molecule by alpha 2-macroglobulin was markedly reduced. The second domain is functionally different from the alpha 2-macroglobulin sequestration site and is important for determining the potency of TGF-beta to inhibit growth of the LS513 human colorectal cancer cell line. Neither the TGF-beta 2/beta 1-(40-47) replacement construct nor a chimera containing aa 1-39 of TGF-beta 2, aa 40-82 of TGF-beta 1, and aa 83-112 of TGF-beta 2 was equivalent to TGF-beta 1 in inhibiting growth of LS513 cells. This fact suggests that additional amino acids outside of the aa 40-82 region are required to specify TGF-beta 1 activity with these cells.
T- and B-cell functions and epitope expression in nonhuman primates immunized with simian immunodeficiency virus antigen by the rectal route.
Authors: Lehner T , Brookes R , Panagiotidi C , Tao L , Klavinskis L S , Walker J , Walker P , Ward R , Hussain L , Gearing J H
Published: May 2006

Transmission of human immunodeficiency virus (HIV) in North America and Europe occurs most commonly through the rectal mucosa during homosexual intercourse. The simian immunodeficiency virus (SIV) macaque model has been used to investigate rectal immunization. The vaccine used was a recombinant SIV gag p27 expressed as hybrid Ty virus-like particles (Ty-VLP). Sequential ororectal (OR) mucosal immunization was compared with i.m. immunization. Whereas both routes of immunization induced serum IgA and IgG p27 antibodies, only OR immunization induced rectal secretory IgA antibodies. Specific CD4+ T-cell proliferative responses to stimulation with p27 were found after i.m. immunization only in the blood and spleen, but after OR immunization they were found in the internal iliac and inferior mesenteric lymph nodes in addition to the blood and spleen. T-cell epitope mapping of the proliferative responses of short-term cell lines (STCLs) grown from peripheral blood or lymphoid cells revealed a major epitope within the polypeptide 121-150 after either route of immunization. Two minor T-cell epitopes were found within peptide 41-80 in STCLs from splenic and circulating cells. B-cell epitope mapping of serum or biliary IgA and IgG antibodies revealed two overlapping or adjacent immunodominant epitopes to the T-cell epitopes within the polypeptides 121-170 and 51-90. The results suggest that rectal augmented by oral immunization with a recombinant particulate antigen in nonhuman primates elicits secretory IgA and to a lesser extent IgG responses in the draining lymph nodes and the rectal mucosa, whereas systemic immunization targets predominantly splenic and circulating T- and B-cell responses. These findings may have important implications in the strategy of designing vaccines in prevention of homosexual transmission of HIV infection.
GTP-dependent association of Raf-1 with Ha-Ras: identification of Raf as a target downstream of Ras in mammalian cells.
Authors: Koide H , Satoh T , Nakafuku M , Kaziro Y
Published: May 2006

Ras is involved in signal transduction of various factors for growth, differentiation, and oncogenesis. Recent studies have revealed several proteins that function upstream and downstream of the Ras signaling pathway. However, its immediate downstream target molecular has not yet been identified. In an effort to identify the Ras-associated downstream proteins, we added recombinant Ha-Ras in a GTP-bound form to cell-free lysates and used several antibodies against Ras to immunoprecipitate Ras complexes. We found that a serine/threonine kinase, Raf-1, was coimmunoprecipitated with Ha-Ras by two anti-Ras antibodies (LA069 and Y13-238), whereas a neutralizing antibody against Ras (Y13-259) could not precipitate Raf-1. The coimmunoprecipitation was observed with a complex of Ras and guanosine 5'-[gamma- thio]triphosphate but not with a complex of Ras and guanosine 5'-[beta-thio]diphosphate. The GTP-dependent association of Ha-Ras with Raf-1 was observed with lysates of various types of cultured cells, including NIH 3T3, pheochromocytoma (PC) 12, Ba/F3, and Jurkat T cells, and also with crude extracts from rat brain. Furthermore, Raf-1 was precipitated with a transforming Ha-Ras mutant ([Val12]Ras) and wild-type Ha-Ras but not with an effector-region mutant ([Leu35,ARg37]Ras) that lacks transforming activity. These results indicate that Ras.GTP physically associates with Raf either directly or through other component(s) and strongly suggest that Raf functions in close downstream proximity to Ras in mammalian cells.
Release from quiescence of CD34+ CD38- human umbilical cord blood cells reveals their potentiality to engraft adults.
Authors: Cardoso A A , Li M L , Batard P , Hatzfeld A , Brown E L , Levesque J P , Sookdeo H , Panterne B , Sansilvestri P , Clark S C
Published: May 2006

Using optimal culture conditions in which the transforming growth factor beta 1 (TGF-beta 1) inhibitory loop has been interrupted by antisense TGF-beta 1 oligonucleotides or anti-TGF-beta serum, we have compared the proliferative capacities and the abilities of the CD34+ CD38- cell populations from bone marrow and umbilical cord blood to generate early progenitors in long-term cultures. The CD34+ CD38- fraction of umbilical cord blood accounts for 4% of the CD34+ fraction compared to only 1% in bone marrow, indicating that umbilical cord blood may be relatively enriched in stem cells. We estimate that the CD34+ CD38- cells from a typical umbilical cord blood sample produce equivalent numbers of colony-forming units (CFU)-granulocyte/erythrocyte/macrophage/megakaryocyte, twice as many CFU-granulocyte/macrophage (GM) and 3 times as many burst-forming units-erythroid as the same population from an average bone marrow sample used in adult transplantation. In addition, the colonies resulting from the umbilical cord blood samples were significantly larger than those from bone marrow, indicating a greater growth potential. However, the content of later progenitors, which may be important for short-term reconstitution, was less in umbilical cord blood-derived than in bone marrow-derived cell preparations, as estimated by a 4-fold lower production of CFU-GM in long-term cultures of CD34+ CD38+ cells. This deficit is partially compensated by the higher growth capacity of the resulting CFU-GM. These studies suggest that umbilical cord blood is a suitable source of cells for adult transplantation.
Growth of the cyanobacterium Anabaena on molecular nitrogen: NifJ is required when iron is limited.
Authors: Bauer C C , Scappino L , Haselkorn R
Published: May 2006

The nifJ gene of Klebsiella pneumoniae encodes an oxidoreductase required for the transfer of electrons from pyruvate to flavodoxin, which reduces nitrogenase. The nifJ gene of Anabaena 7120, isolated from a cosmid bank, was found to contain an open reading frame encoding a 1197-aa protein. The deduced amino acid sequence shows 50% identity to the Klebsiella homolog. The nifJ gene in Anabaena 7120 was inactivated by chromosomal interruption. The resulting mutant was unable to grow on medium depleted of both iron and combined nitrogen but grew normally, fixing nitrogen, when iron was present. NifJ transcripts of 2.7 and 4.3 kb are induced by iron depletion irrespective of nitrogen status. One particular stretch of the Anabaena 7120 nifJ gene encodes 12 aa with no complementary matches in the Klebsiella protein. This insert contains five tandem repeats of the heptamer CCCCAGT. These heptamers, as well as heptamers and octamers of other related sequences, have been located in a number of cyanobacterial genomes but are usually not found within the coding region of a gene. The site of the Anabaena 7120 heptamers in the nifJ genes of other filamentous cyanobacteria contains a surprising diversity of repeated sequences, both octamers and heptamers. The corresponding protein inserts range in length from 1 to 21 aa, relative to Klebsiella NifJ.
Translation of reovirus RNA species m1 can initiate at either of the first two in-frame initiation codons.
Authors: Roner M R , Roner L A , Joklik W K
Published: May 2006

The m1 species of reovirus RNA, which encodes the minor protein component mu 2, possesses two initiation codons, one "strong" according to Kozak rules and preceded by 13 residues (IC1), the other "weak" and located 49 codons downstream of the first (IC2). In reovirus-infected cells only IC2 is used, but initiation from IC1 can be activated, and efficiency of initiation from either initiation codon modulated over a wide range, by coupling unrelated sequences to either or both ends of m1 RNA. For example, when the M1 genome segment is cloned into the thymidine kinase gene of vaccinia virus in such a way that various "irrelevant" stretches of nucleotides comprising restriction endonuclease cleavage sites or promoter remnants are coupled to the 5' end of m1 RNA, translation of the resultant transcripts is also initiated at IC2, with frequencies controlled by the nature of the attached sequences. However, in rabbit reticulocyte lysates these same transcripts are translated from IC1 as well as from IC2, and transcripts in which m1 RNA is preceded by long sequences of encephalomyocarditis virus RNA (from the T7 polymerase-controlled pTM1 vector) are translated exclusively from IC1. By contrast, m1 RNA itself is translated only from IC2. It appears that the most important factor that controls the extent to which translation is initiated from IC1 and IC2 is their "availability," which is likely to be a function of the extent to which the regions on either side of them interact with each other (and also, to a lesser extent, with the 3' untranslated region) either directly or via interaction with host cell proteins. The effects described here are of considerable potential significance when genetic material is rearranged as a result of translocations, insertions, deletions, and amplifications--that is, when sequences that are normally separated are brought into apposition.
Processing of a fusion protein by endoprotease in COS-1 cells for secretion of mature peptide by using a chimeric expression vector.
Authors: Liu Y C , Kawagishi M , Mikayama T , Inagaki Y , Takeuchi T , Ohashi H
Published: May 2006

The subtilisin-related proprotein convertase furin is expressed in various mammalian tissues. Expecting that COS-1 cells have a furin-like endoprotease, we constructed a fusion expression vector for production of a recombinant foreign protein having no signal peptide or a protein in truncated form into secreted mature protein. A cDNA fragment encoding N-terminal procalcitonin (pro-CT) of human calcitonin precursor was inserted into the mammalian expression vector pME18S. We used PCR techniques to generate four kinds of cDNAs encoding the C terminus of the pro-CT with Arg residues at P4 (Arg-Xaa-Lys-Arg), P6 (Arg-Xaa-Xaa-Xaa-Lys-Arg), or both (Arg-Xaa-Arg-Xaa-Lys-Arg), in addition to the Lys-Arg motif at the cleavage site, in order to determine the conditions for efficient processing in nonendocrine cells, such as COS-1 cells. The cDNA coding for the Fc fragment of human immunoglobulin G1 was fused in-frame to the cDNA encoding pro-CT at its C terminus. Upon transfection of the chimeric plasmids into COS-1 cells, almost all of the fusion protein with the Arg residues at both P4 and P6 were processed into secreted Fc product, even without cotransfection of furin. These results indicate that COS-1 cells have a furin-like endoprotease and suggest that pro-CT, with the Arg residues at both P4 and P6, can be used as a carrier peptide for expression of a foreign protein having no signal peptide or a protein in truncated form in COS-1 cells.
A chromatin folding model that incorporates linker variability generates fibers resembling the native structures.
Authors: Woodcock C L , Grigoryev S A , Horowitz R A , Whitaker N
Published: May 2006

The "30-nm" chromatin fibers, as observed in eukaryotic nuclei, are considered a discrete level in a hierarchy of DNA folding. At present, there is considerable debate as to how the nucleosomes and linker DNA are organized within chromatin fibers, and a number of models have been proposed, many of which are based on helical symmetry and imply specific contacts between nucleosomes. However, when observed in nuclei or after isolation, chromatin fibers show considerable structural irregularity. In the present study, chromatin folding is considered solely in terms of the known properties of the nucleosome-linker unit, taking into account the relative rotation between consecutive nucleosomes that results from the helical twist of DNA. Model building based on this premise, and with a constant length of linker DNA between consecutive nucleosomes, results in a family of fiber- and ribbon-like structures. When the linker length between nucleosomes is allowed to vary, as occurs in nature, fibers showing the types of irregularity observed in nuclei and in isolated chromatin are created. The potential application of the model in determining the three-dimensional organization of chromatin in which nucleosome positions are known is discussed.
The primary signal in the biological perception of temperature: Pd-catalyzed hydrogenation of membrane lipids stimulated the expression of the desA gene in Synechocystis PCC6803.
Authors: Vigh L , Los D A , Horváth I , Murata N
Published: May 2006

One of the well-characterized phenomena associated with the acclimation of organisms to changes in ambient temperature is the regulation of the molecular motion or "fluidity" of membrane lipids via changes in the extent of unsaturation of the fatty acids of membrane lipids. The enzymes responsible for this process when the temperature is decreased are the desaturases, the activities of which are enhanced at low temperature. To examine whether the change in the fluidity of membrane lipids is the first event that signals a change in temperature, we studied the effect of the Pd-catalyzed hydrogenation of membrane lipids on the expression of the desA gene, which is responsible for the desaturation of fatty acids of membrane lipids in the cyanobacterium Synechocystis PCC6803. The Pd-catalyzed hydrogenation of plasma membrane lipids stimulated the expression of the desA gene. We also found that, for unexplained reasons, the hydrogenation was much more specific to a minor phospholipid, phosphatidylglycerol, than to members of other lipid classes. These results suggest that the organism perceives a decrease in the fluidity of plasma membrane lipids when it is exposed to a decrease in temperature.
X-ray crystallographic identification of a protein-binding site for both all-trans- and 9-cis-retinoic acid.
Authors: Newcomer M E , Pappas R S , Ong D E
Published: May 2006

The elucidation of how a protein-binding site might specifically recognize both the all-trans and 9-cis isomers of retinoic acid is of particular interest because of the recently discovered binding specificities of the nuclear receptors for retinoic acid. Two families of nuclear receptors for retinoic acid have been described, which are designated RAR (for retinoic acid receptor) and RXR (for retinoid-X receptor). The RXR family of receptors is specific for 9-cis-retinoic acid, whereas the RAR-type receptor is activated by either 9-cis- or all-trans-retinoic acid. During the x-ray structure determination of a secreted epididymal retinoic acid-binding protein, with and without retinoic acid, we observed an electron density for the bound all-trans-retinoic acid that indicates the protein-bound all-trans form of the vitamin/hormone adopts a horseshoe-like conformation that resembles the structure of the 9-cis isomer of the ligand. We detail here the experiments that indicate the electron density is indeed due to all-trans-retinoic acid and that protein can also bind the 9-cis isomer. This observation and the fact that the same protein also binds the synthetic retinoid (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1- propenyl]-benzoic acid (TTNPB), a retinoic acid analog that activates RAR but does not activate RXR, suggest that the mechanism by which this protein recognizes both 9-cis- and all-trans-retinoic acids may be analogous to the mechanism used by RAR. Three crystallographic structures of retinol-binding proteins have been described. In each of these structures the retinol binds with the isoprene tail fully extended. This report represents an x-ray crystallographic description of a protein-bound retinoid conformer that adopts a nonextended conformation, and we believe this observation is relevant to the ligand specificities described for the retinoic acid receptors.
Suppression of tumorigenicity in simian virus 40-transformed 3T3 cells transfected with alpha-actinin cDNA.
Authors: Glück U , Kwiatkowski D J , Ben-Ze'ev A
Published: May 2006

Human cytoskeletal alpha-actinin cDNA was transfected into highly malignant simian virus 40-transformed BALB/c 3T3 (SVT2) cells that express 6-fold lower levels of alpha-actinin than nontransformed BALB/c 3T3 cells. SVT2 clones expressing various levels of alpha-actinin were isolated and their structure and tumorigenic properties were determined. Transfected SVT2 clones expressing alpha-actinin at levels found in nontumorigenic 3T3 cells displayed a flatter phenotype, a decreased ability to grow in suspension culture in soft agar, and a marked reduction in their ability to form tumors in syngeneic BALB/c mice and in athymic nude mice. Clones overexpressing alpha-actinin at the highest level (about 2-fold higher than 3T3 cells) were completely suppressed in their ability to form tumors in syngeneic BALB/c mice. The results suggest that alpha-actinin, an actin-crosslinking protein that is also localized in cell junctions, may have an effective suppressive ability on the transformed phenotype.
Protein folding--what's the question?
Authors: Lattman E E , Rose G D
Published: May 2006

The folding reactions of many small, globular proteins exhibit two-state kinetics, in which the folded and unfolded states interconvert readily without observable intermediates. Typically, the free energy difference, delta G, between the native and denatured states of such a protein is quite small, lying in the range of approximately -5 to -15 kcal/mol. We point out that, under these circumstances, a population of native-like molecules will persist, even in the presence of mutations sufficiently destabilizing to change the sign of delta G. Therefore, it is not energy per se that determines conformation. A corollary to this argument is that specificity--not stability--would be the more informative focus in future folding studies.
Phospholipid transmembrane domains and lateral diffusion in fibroblasts.
Authors: el Hage Chahine J M , Cribier S , Devaux P F
Published: May 2006

The lateral diffusion of fluorescent phospholipids in cultured Chinese hamster lung fibroblasts was examined by modulated fringe pattern photobleaching. When cells were labeled and maintained at 7 degrees C, the fluorescence remained localized at the plasma membrane. N-[6-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl-amino)caproyl] sphingosylphosphocholine (C6-NBD-SphPCho) and 1-acyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)caproyl] phosphatidylcholine (C6-NBD-PtdCho) both diffused with the same apparent lateral diffusion coefficient (D1 approximately 0.3 x 10(-9) cm2/s). By contrast, the phosphatidylserine derivative (1-acyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)caproyl] phosphatidylserine (C6-NBD-Ptd-Ser)) gave rise to two diffusional components: a slow component, D1, analogous to that measured with the choline-containing lipids, and a fast component (D2 approximately 2 x 10(-9) cm2/s). The fast component only exists in ATP-containing cells. It was shown to be associated with C6-NBD-PtdSer translocated to the inner leaflet. This indicates that the two leaflets form very different membranous domains. At higher temperature, the same difference in mobility was observed between the choline-containing lipids and the aminolipid. However, with C6-NBD-SphPCho, a fraction of very slowly diffusing or quasi-immobilized probes gradually appeared with time. This could be attributed to sphingomyelin located in small organelles after internalization. From the amplitude of this component registered at different intervals, we calculated that approximately 50% of the plasma membrane sphingomyelin is recycled in less than 30 min in Chinese hamster fibroblasts by an ATP- and microtubule-dependent process.
Mouse placental macrophages have a decreased ability to present antigen.
Authors: Chang M D , Pollard J W , Khalili H , Goyert S M , Diamond B
Published: May 2006

Large numbers of macrophages can be found in an animal's uteroplacental unit. This high concentration of macrophages suggests they must play an important role during placental development. To gain a better understanding of the functional capacity of placental macrophages, we have obtained a highly enriched placental macrophage culture and have derived several cell lines from this population. Both placental macrophages and cell lines show colony-stimulating factor 1-dependent growth, express Fc receptors, and can perform Fc-receptor-mediated phagocytosis. In addition, they express macrophage markers Mac-1, F4/80, and CD14. Although placental macrophages express major histocompatibility complex class II molecules constitutively, they display a decreased ability to present protein antigens to T cells. Since primary fetal liver macrophages of the same gestational stage also show a decreased ability to present antigens, this phenomenon may reflect a developmental stage of macrophages.
In vivo analysis of Chlamydomonas chloroplast petD gene expression using stable transformation of beta-glucuronidase translational fusions.
Authors: Sakamoto W , Kindle K L , Stern D B
Published: May 2006

We have used the Escherichia coli beta-glucuronidase (uidA) gene as a reporter gene to localize the promoter and analyze the function of the 5' untranslated region (UTR) of the Chlamydomonas chloroplast petD gene. Using particle bombardment, petD-uidA transcriptional and translational fusion genes were introduced into the chloroplast genome in the large inverted repeat flanking the atpB gene. In transformants carrying a petD-uidA transcriptional fusion, uidA mRNA accumulated but was not translated. However, in a translational fusion that included the entire petD 5' UTR, uidA mRNA accumulated and a high level of beta-glucuronidase activity was detected. When approximately 70% of the petD 5' UTR was deleted from the translational fusion, uidA mRNA accumulation and beta-glucuronidase activity decreased 4- to 6-fold and 8-fold, respectively. Run-on transcription assays demonstrated that all strains transcribe the uidA gene at equivalent rates. Our results show that sequences essential for translation reside in the petD 5' UTR and also that sequences within the 5' UTR directly or indirectly affect mRNA stability. The expression of beta-glucuronidase under the control of chloroplast transcriptional and translational signals will facilitate further studies of chloroplast gene regulatory mechanisms.
Double keystone bird in a keystone species complex.
Authors: Daily G C , Ehrlich P R , Haddad N M
Published: May 2006

Species in a Colorado subalpine ecosystem show subtle interdependences. Red-naped sapsuckers play two distinct keystone roles. They excavate nest cavities in fungus-infected aspens that are required as nest sites by two species of swallows, and they drill sap wells into willows that provide abundant nourishment for themselves, hummingbirds, orange-crowned warblers, chipmunks, and an array of other sap robbers. The swallows thus depend on, and the sap robbers benefit from, a keystone species complex comprised of sapsuckers, willows, aspens, and a heartwood fungus. Disappearance of any element of the complex could cause an unanticipated unraveling of the community.
Rhizobium NodB protein involved in nodulation signal synthesis is a chitooligosaccharide deacetylase.
Authors: John M , Röhrig H , Schmidt J , Wieneke U , Schell J
Published: May 2006

The common nodulation genes nodABC are conserved in all rhizobia and are involved in synthesis of a lipooligosaccharide signal molecule. This bacterial signal consists of a chitooligosaccharide backbone, which carries at the nonreducing end a fatty acyl chain. The modified chitooligosaccharide molecule triggers development of nodules on the roots of the leguminous host plant. To elucidate the specific role of the NodB protein in nodulation factor synthesis, we have purified recombinant NodB and determined its biochemical role by direct assays. Our data show that the NodB protein of Rhizobium meliloti deacetylates the nonreducing N-acetylglucosamine residue of chitooligosaccharides. The monosaccharide N-acetylglucosamine is not deacetylated by NodB. In the pathway of Nod factor synthesis, deacetylation at the nonreducing end of the oligosaccharide backbone may be a necessary requirement for attachment of the fatty acyl chain.
A histidine protein kinase is involved in polar organelle development in Caulobacter crescentus.
Authors: Wang S P , Sharma P L , Schoenlein P V , Ely B
Published: May 2006

Mutations having pleiotropic effects on polar organelle development (pod) in Caulobacter crescentus have been identified and shown to occur in at least 13 genes scattered throughout the genome. Mutations at each locus affect a unique combination of polar traits, suggesting that complex interactions occur among these genes. The DNA sequence of one of these genes, pleC, indicates that it is homologous to members of the family of histidine protein kinase genes. Membes of this family include the senor components of the bacterial two-component regulatory systems. Furthermore, in vitro experiments demonstrated that the PleC protein was capable of autophosphorylation. These results suggest that the PleC protein (and perhaps the proteins encoded by the other pod genes as well) regulates the expression of genes involved in polar organelle development through the phosphorylation of key regulatory proteins. The use of a phosphorelay system cued to internal changes in the cell would provide a mechanism for coordinating major changes in gene expression with the completion of specific cell cycle events.
Three small nucleolar RNAs of unique nucleotide sequences.
Authors: Ruff E A , Rimoldi O J , Raghu B , Eliceiri G L
Published: May 2006

Three small RNA species were detected in human cells, and their cDNAs were synthesized and cloned. These RNAs are nucleolar, are 207, 154, and 135 nucleotides long, and are named E1, E2, and E3, respectively, and their unique nucleotide sequences suggest that they may belong to an additional family of small nucleolar RNAs. The 5' ends of these three RNAs do not appear to have a trimethylguanosine cap or another type of cap. Apparent homologs of these three RNAs were detected in mouse, rabbit, and frog cells, suggesting their universal importance. They are housekeeping RNA species, since they are present in all rabbit tissues analyzed.
Mouse mammary tumor virus infection accelerates mammary carcinogenesis in Wnt-1 transgenic mice by insertional activation of int-2/Fgf-3 and hst/Fgf-4.
Authors: Shackleford G M , MacArthur C A , Kwan H C , Varmus H E
Published: May 2006

Transgenic mice carrying the Wnt-1 protooncogene modified for expression in mammary epithelial cells exhibit hyperplastic mammary glands and stochastically develop mammary carcinomas, suggesting that additional events are necessary for tumorigenesis. To induce such events and to identify the genes involved, we have infected Wnt-1 transgenic mice with mouse mammary tumor virus (MMTV), intending to insertionally activate, and thereby molecularly tag, cooperating protooncogenes. Infection of breeding female Wnt-1 transgenics decreased the average age at which tumors appeared from approximately 4 months to approximately 2.5 months and increased the average number of primary tumors per mouse from 1-2 to > 5. A smaller effect was observed in virgin females, and infection of transgenic males showed no significant effect on tumor latency. More than half of the tumors from the infected breeding group contained one or more newly acquired MMTV proviruses in a pattern suggesting that most cells in tumors arose from a single infected cell. Analyses of provirus-containing tumors for induced or altered expression of int-2/Fgf-3, hst/Fgf-4, int-3, and Wnt-3 showed activation of int-2 in 39% of tumors, hst in 3%, and both int-2 and hst in 3%. DNA analyses with probes for protooncogenes and MMTV confirmed that the activations resulted from proviral insertions. There was no evidence for proviral insertions at the int-3, Wnt-3, or Wnt-1 loci. These findings provide further evidence that fibroblast growth factors Int-2 and Hst can cooperate with Wnt-1, another secreted factor, in mammary tumorigenesis, and they illustrate the capacity of this system to identify cooperating oncogenes.