bluuebunny/crossref_metadata_2025 huggingface dataset:
Transformation of Tetrahymena thermophila by microinjection of a foreign gene.
Authors: Kahn R W , Andersen B H , Brunk C F
Published: May 2006

Tetrahymena thermophila has been transformed to paromomycin-resistant phenotypes by microinjection of an aminoglycoside 3'-phosphotransferase (neo) gene under the control of the T. thermophila histone H4-I promoter. This chimeric neo gene, by itself or on a vector containing a rRNA-encoding DNA (rDNA) origin of replication, transforms T. thermophila. In cells transformed with the rDNA origin vector, the neo gene is usually found integrated into the endogenous rDNA molecules and is present in high copy number. In transformants obtained by microinjecting only the linear chimeric gene, the neo gene is found to have replaced the histone H4-I gene or is found integrated into the 5' flanking region of the H4-I gene. The relative transcript levels of the neo gene in T. thermophila transformed by the linear chimeric gene are much higher than in cells transformed with the vector. The neo gene provides an effective selectable marker for transformation of T. thermophila.
Ethylene induces de novo synthesis of chlorophyllase, a chlorophyll degrading enzyme, in Citrus fruit peel.
Authors: Trebitsh T , Goldschmidt E E , Riov J
Published: May 2006

Chlorophyllase (Chlase; EC 3.1.1.14) was extracted from plastid fractions of ethylene-treated orange fruit peel and purified 400-fold to homogeneity by gel filtration, hydrophobic chromatography, and preparative SDS/PAGE of nonheated protein. SDS/PAGE of nonheated purified enzyme indicated that Chlase activity is associated with a single protein band migrating at an apparent molecular mass of 25 kDa whereas the heated purified enzyme had a molecular mass of 35 kDa. The N-terminal sequence of the purified protein was determined. The purified enzyme was used as an immunogen for raising antibodies in rabbits. The antiserum was highly specific and on Western blots recognized both the heated and the nonheated form of Chlase. The antibodies also recognized the solubilized enzyme, as shown by an immunoprecipitation assay and by antigen-antibody capture assays in microtiter plates. Treatment with ethylene, which enhances degreening, increased Chlase activity 12-fold. Immunoblot analyses of crude extracts from ethylene-treated fruit detected a strong signal of the Chlase protein, while only a trace level of the enzyme protein could be detected in air. Gibberellin A3 and N6-benzyladenine partly counteracted the ethylene-induced increase in Chlase activity as well as the immunodetected upsurge of the Chlase protein. Ethylene appears to enhance the degreening of citrus fruit through de novo synthesis of the Chlase protein, which in turn is inhibited by the senescence-delaying regulators, gibberellin A3 and N6-benzyladenine. The Chlase enzyme protein may, therefore, serve as a model system for studying the hormonal molecular regulation of fruit ripening and senescence.
Herpes simplex virus infected cell polypeptide 4 preferentially represses Sp1-activated over basal transcription from its own promoter.
Authors: Gu B , Rivera-Gonzalez R , Smith C A , DeLuca N A
Published: May 2006

Herpes simplex virus type 1 infected cell polypeptide 4 (HSV-1 ICP4) is a multifunctional phosphoprotein that is essential for viral infection. It is both a repressor and an activator of viral gene expression depending upon the promoter. ICP4 represses transcription from its own promoter. In the present study, we used general transcription factors from HeLa cell nuclear extracts, recombinant TATA binding protein (TBP) and TFIIB, and the transcriptional activator Sp1 to reconstitute in vitro transcription for the ICP4 promoter and to examine the effects of purified ICP4 on transcription. ICP4 was able to effectively repress Sp1-induced transcription from ICP4 promoter templates that contain one or multiple Sp1 binding sites. The observed inhibition required the ICP4 binding site that spans the transcription initiation site. ICP4 did not inhibit basal transcription as inferred by its inability to inhibit transcription when (i) Sp1 was not included in transcription reactions, (ii) the templates contained no Sp1 binding sites, and (iii) TBP was used in place of TFIID in the reactions. The in vitro observations were consistent with the behavior of the same constructs expressed in cells from the herpes simplex virus type 1 genome. DNase I footprinting experiments revealed that ICP4 could co-occupy the ICP4 promoter region with TBP-TFIIB, indicating that ICP4 does not necessarily exclude these factors from binding to the TATA region. The data suggest that the repressive effects of ICP4 observed in this study result from ICP4 interfering with the interactions contributing to Sp1-induced transcription.
Loss of retinoic acid receptor gamma function in F9 cells by gene disruption results in aberrant Hoxa-1 expression and differentiation upon retinoic acid treatment.
Authors: Boylan J F , Lohnes D , Taneja R , Chambon P , Gudas L J
Published: May 2006

Retinoic acid (RA) signal transduction is believed to be mediated through several high-affinity nuclear receptors [RA receptors (RARs) and retinoid X receptors], which are members of the steroid/thyroid/vitamin D superfamily and function as transcription factors. Why multiple RARs exist and what gene targets are regulated by each of the three receptors remain compelling questions in developmental biology. Through targeted disruption of both RAR gamma alleles, we have identified several differentiation-specific genes that are regulated either directly or indirectly by RAR gamma in F9 embryonal carcinoma cells. These include genes encoding Hoxa-1 (Hox-1.6) and the extracellular matrix proteins laminin B1 and collagen type IV (alpha 1), all of which are RA inducible in wild-type F9 embryonal carcinoma cells but are not significantly induced in the RAR gamma-/- lines. In contrast, transcripts encoding Hoxb-1 (Hox-2.9) and cellular RA binding protein II (CRABPII) are activated by RA for a longer period of time in the RAR gamma-/- lines compared to the wild-type F9 line. Not all RA-responsive genes are aberrantly expressed; Rex-1, RAR beta, and SPARC transcripts are regulated in the RAR gamma-/- lines as they are in F9 wild-type cells. Our results support the idea that each RAR may regulate different subsets of RA-responsive genes, which may explain, in part, the complex regulation of developmental processes by retinoids.
A 30-kDa alternative translation product of the CCAAT/enhancer binding protein alpha message: transcriptional activator lacking antimitotic activity.
Authors: Lin F T , MacDougald O A , Diehl A M , Lane M D
Published: May 2006

Full-length (42 kDa) CCAAT/enhancer binding protein alpha (C/EBP alpha) (p42) has been implicated in the transcriptional activation of adipocyte genes including the 422(aP2) and C/EBP alpha genes during differentiation of 3T3-L1 preadipocytes. We have identified a 30-kDa isoform (p30) of C/EBP alpha that is expressed by 3T3-L1 adipocytes, mouse adipose tissue, and rat liver. In vitro translation of wild-type C/EBP alpha mRNA or transient transfection with a wild-type C/EBP alpha vector gave rise to similar levels of p42 and p30. Mutational analysis revealed that p30 is an alternative translation product initiated at the third in-frame methionine codon of the C/EBP alpha message. p30C/EBP alpha binds to the C/EBP sites within and activates reporter gene expression driven by the 422(aP2) and C/EBP alpha gene promoters. Although transfection of 3T3-L1 preadipocytes with a strong p30C/EBP alpha expression vector is insufficient to induce differentiation, this vector advances the differentiation program. Unlike p42C/EBP alpha, which inhibits cell proliferation, p30C/EBP alpha is not antimitotic. Thus, the N-terminal 12-kDa segment of full-length C/EBP alpha contains an amino acid sequence necessary for antimitotic activity. During differentiation of 3T3-L1 preadipocytes and during hepatocyte development, the cellular p42C/EBP alpha/p30C/EBP alpha ratio changes, raising the possibility of a regulatory role.
[d-Ala2]deltorphin I-like immunoreactivity in the adult rat brain: immunohistochemical localization.
Authors: Tooyama I , Abe H , Renda T , Erspamer V , Kimura H
Published: May 2006

Using a specific antiserum recently raised against [D-Ala2]deltorphin I (DADTI: Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2), a highly selective ligand for delta-opioid receptors, we have previously demonstrated the occurrence of positive immunostaining in several structures of mouse brain. We describe here the neuroanatomical distribution patterns of DADTI-immunoreactive neuronal bodies, axons, and tanycytes in rat brain. Positive neuronal somata were localized mainly in the ventral mesencephalon, including the ventral tegmental area and the pars compacta of the substantia nigra. A minor population of positive somata was found in the pars reticulata and pars lateralis of the substantia nigra, raphe nuclei, supramammillary nucleus, and retrorubral reticular nucleus. All these regions, except for the supramammillary nucleus, contain dopamine cell bodies. Intensely stained positive nerve fibers could be traced along the medial forebrain bundle. Dense positive terminals were seen in the neostriatum, nucleus accumbens shell, olfactory tubercle, septal areas, cingulate, and medial prefrontal cortex. Double-immunostaining study revealed that, in the substantia nigra, almost all (97.8%) DADTI-positive neurons colocalized with tyrosine hydroxylase (TH), and the doubly stained cells occupied about one-third (29.1%) of the total population of TH-positive neurons. Only a few DADTI/TH-positive cells also stained for 28-kDa calbindin D, although many neurons double-stained for 28-kDa calbindin D and TH. In contrast, the supramammillary nucleus contained a number of DADTI-positive cells, which nearly always stained positively for 28-kDa calbindin D but did not stain for TH. The association of DADTI-like immunoreactivity with certain dopaminergic pathways seems of particular interest. A small population of DADTI-immunostained tanycytes was present in the ventral part of the third ventricle wall.
Cloning and properties of the Caenorhabditis elegans TATA-box-binding protein.
Authors: Lichtsteiner S , Tjian R
Published: May 2006

The nematode Caenorhabditis elegans has become an organism of choice for the study of developmental processes at the genetic level. We have undertaken to develop an in vitro system to study transcription in C. elegans. As a first step we report here the cloning of the cDNA encoding the C. elegans TATA-box-binding protein (CeTBP). We used "touch-down PCR" to generate a specific DNA probe derived from the C-terminal region conserved in all TBP genes cloned to date. Several clones encoding an extended open reading frame were isolated from a phage lambda cDNA library. The complete amino acid sequence of CeTBP deduced from the cDNA reveals a protein of 37 kDa with an extended sequence similarity in the C-terminal region with all other TBP cDNAs sequenced so far. The N-terminal region of CeTBP (amino acids 1-153), however, does not show any homology with TBPs from other organisms. Interestingly, the N-terminal portion of the molecule contains three short direct repeats. Purified recombinant CeTBP binds specifically to the TATA box sequence, interacts with transcription factors TFIIA and TFIIB, and is able to substitute for the TFIID basal activity when assayed by in vitro transcription in both HeLa and C. elegans nuclear extracts. CeTBP is therefore a basal transcription factor.
An unusual active hexose transport system in human and mouse preimplantation embryos.
Authors: Chi M M , Manchester J K , Basuray R , Mahendra S , Strickler R C , McDougal D B , Lowry O H
Published: May 2006

In a metabolic study of human and mouse preimplantation embryos (preembryos), we measured glucose uptake and phosphorylation with nonradioactive 2-deoxyglucose (DG) as tracer. Initial experiments indicated an active hexose transport capacity, a property thought to be restricted in mammals to intestinal villi and kidney tubules [Baly, D. L. & Horuk, R. (1988) Biochim. Biophys. Acta 947, 571-590]. Significant findings are as follows: (i) During a 60-min incubation with a low level of DG, mouse blastocyst DG rose to levels up to 30 times that of the medium. (The intestinal active system does not transport DG [Crane, R. K. (1960) Physiol. Rev. 40, 789-825].) (ii) Active preembryo transport was not blocked (as it would have been in the intestine) by phlorizin [Alvarado, F. & Crane, R. K. (1962) Biochem. Biophys. Acta 56, 170-172 and Sacktor, B. (1989) Kidney Int. 36, 342-350] or by replacement of Na+ with choline+ or K+ [Crane (1960) and Sacktor (1989)]. (iii) Transport of DG was blocked by cytochalasin B (which is not true for the intestinal transporter). We conclude that a distinct active hexose transporter and at least one facilitated transporter are present in preembryos, perhaps appearing in tandem on different membranes during formation of the increasingly complex preembryo structure.
Identification and characterization of hematopoietic stem cells from the yolk sac of the early mouse embryo.
Authors: Huang H , Auerbach R
Published: May 2006

The yolk sac is the first site of hematopoiesis in the mammalian embryo. However, little is known about the initial stem cells in the yolk sac. We have isolated hematopoietic stem cells from early mouse embryonic yolk sac by using a sequential protocol of nonadherence to plastic, density gradient centrifugation, immunocytoadherence, and cell sorting. Isolated, nonadherent, density < 1.077-g/cm3, surface antigen AA4.1+, wheat germ agglutinin bright (WGAbright) cells give rise to multiple lineages, including T cells, B cells, and myeloid cells, as detected by using fetal thymus organ culture, S17 stromal feeder layers, or methylcellulose culture colony-forming cells, respectively. AA4.1+, WGAbright cells expressed high levels of heat-stable antigen (HSA) and CD45 (Ly-5) but did not significantly express major histocompatibility complex antigens, CD44, or Sca-1. Peak stem cell concentration is reached by day 11, before stem cells can be found in the liver, omentum, or thymus. In vivo long-term reconstitution of lethally irradiated mice was effected by as few as 720 AA4.1+, WGAbright yolk sac cells, but it required addition of a subset of bone marrow cells capable of providing immediate (short-term) radiation protection. Yolk sac donor-derived T cells, B cells, and macrophages were readily identified 6 months after transfer of yolk sac-derived stem cells. We suggest that, because of their cell surface phenotype as well as their capacity to differentiate in vitro and in vivo, the cells isolated from the mouse embryonic yolk sac may include the most primitive hematopoietic pluripotential stem cells yet identified.
Identification of an embryonic isoform of myelin basic protein that is expressed widely in the mouse embryo.
Authors: Mathisen P M , Pease S , Garvey J , Hood L , Readhead C
Published: May 2006

We have identified a myelin basic protein (MBP) isoform in mouse embryos that includes an exon upstream of the usual transcription initiation site. This isoform, embryonic-neonatal MBP (E-MBP), is expressed at the protein level in the embryonic nervous system at a time when other MBP isoforms are not detected. In addition to the central and peripheral nervous systems of the embryo and neonate, the thymus, spleen, and testes also express E-MBP at the protein level. The expression of E-MBP in cell types distinct from the nervous system strongly suggests that this MBP isoform has a role apart from the formation of myelin.
beta-Amyloid precursor protein binds to the neurite-promoting IKVAV site of laminin.
Authors: Kibbey M C , Jucker M , Weeks B S , Neve R L , Van Nostrand W E , Kleinman H K
Published: May 2006

We previously characterized a 110-kDa membrane-associated laminin-binding protein (LBP110) from brain which binds the laminin A chain -Ile-Lys-Val-Ala-Val-(IKVAV) site and increases in injury. Here we demonstrate that antisera directed against different epitopes of beta-amyloid precursor protein (APP) recognize LBP110 and that APP is recognized by LBP110 antiserum. APP specifically binds IKVAV and not another biologically active laminin-derived peptide containing the amino acid sequence -Tyr-Ile-Gly-Ser-Arg-. PC-12 cells transfected with antisense APP RNA produce less APP and LBP110, and they form fewer processes when cultured on either laminin or the IKVAV peptide. Thus, LBP110 is a member of the APP family and a function for APP in neurite outgrowth is now defined.
Borrelia burgdorferi is clonal: implications for taxonomy and vaccine development.
Authors: Dykhuizen D E , Polin D S , Dunn J J , Wilske B , Preac-Mursic V , Dattwyler R J , Luft B J
Published: May 2006

The chromosomal genes fla and p93 and the ospA gene from a linear plasmid were sequenced from up to 15 isolates of Borrelia burgdorferi, which causes Lyme borreliosis in man. Comparison of the gene trees provides no evidence for genetic exchange between chromosomal genes, suggesting B. burgdorferi is strictly clonal. Comparison of the chromosomal gene trees with that of the plasmid-encoded ospA reveals that plasmid transfer between clones is rare. Evidence for intragenic recombination was found in only a single ospA allele. The analysis reveals three common clones and a number of rare clones that are so highly divergent that vaccines developed against one are unlikely to provide immunity to organisms from others. Consequently, an understanding of the geographic and genetic variability of B. burgdorferi will prove essential for the development of effective vaccines and programs for control. While the major clones might be regarded as different species, the clonal population structure, the geographic localization, and the widespread incidence of Lyme disease suggest that B. burgdorferi should remain the name for the entire array of organisms.
Further evidence for function of the Drosophila Notch protein as a transmembrane receptor.
Authors: Lyman D , Young M W
Published: May 2006

N locus mutations associated with unusual mutant phenotypes were found to alter the structure of the encoded protein. Two mutations, NCo and N60g11, eliminate much of the cytoplasmic domain. NCo can act as a null allele or as a competitive inhibitor of N+ function, whereas N60g11 produces dominant gain of function in some cell types. This difference in function can be attributed to retention of cdc10/SWI6 repeats in the Notch60g11 protein. The results suggest a role for these repeats in intracellular signaling and are consistent with action of Notch as a receptor. nd3 and l(1)NB alter extracellular epidermal growth factor-like and lin-12/Notch elements, respectively. nd3 eliminates a conserved cysteine residue, so the mutation may result in complete loss of function for a single Notch epidermal growth factor element. N60g11 and l(1)NB produce related gain-of-function phenotypes. It is proposed that l(1)NB produces an extracellular modification of the protein that stimulates aberrant intracellular signaling by the Notch cytoplasmic domain.
Subunit structure of a class A aspartate transcarbamoylase from Pseudomonas fluorescens.
Authors: Bergh S T , Evans D R
Published: May 2006

The class A aspartate transcarbamoylase (ATCase, EC 2.1.3.2) from Pseudomonas fluorescens was purified to homogeneity with retention of full catalytic and regulatory functions. Careful determinations under conditions that minimized proteolysis showed that the molecule is a 1:1 stoichiometric complex of two polypeptide chains of 34 and 45 kDa. Pyridoxal phosphate is a competitive inhibitor of the enzyme (Ki = 1 microM). Reduction of the pyridoxal phosphate enzyme adduct with sodium boro[3H]hydride showed that the active site is located on the 34-kDa polypeptide. Affinity labeling with 5'-[p-(fluorosulfonyl)benzoyl]adenosine, an ATP analog, suggested that the regulatory site is also located on the 34-kDa species. While the function of the 45-kDa subunit is unknown, neither carbamoyl phosphate synthetase nor dihydroorotase activities are associated with the ATCase. The molecular mass of the enzyme was determined by gel filtration, sedimentation velocity, and electron microscopy to be 464 kDa. Thus the enzyme is composed of six copies of the 34-kDa polypeptide and six copies of the 45-kDa polypeptide. The molecule has a Stokes' ratio of 70.9 A and a frictional ratio of 1.37, suggesting a compact globular shape. We propose that the P. fluorescens ATCase is composed of two trimers of 34-kDa catalytic chains and is likely to be a D3 dodecamer with an arrangement of subunits analogous to that of the class B ATCase molecules.
Local breathing and global unfolding in hydrogen exchange of barnase and its relationship to protein folding pathways.
Authors: Clarke J , Hounslow A M , Bycroft M , Fersht A R
Published: May 2006

We have measured the rate constants for exchange of amide protons in 15N-labeled wild-type barnase and a disulfide mutant that is more stable by 2 kcal.mol-1. The relative rate constants for exchange for wild type and mutant should reflect the changes in the equilibrium constants for local or global unfolding. The values for regions whose structure has been shown to be unaffected by the mutation fall into three subsets: those that are essentially unaffected by the mutation and so presumably exchange by local breathing; those where the energies change by close to 2 kcal.mol-1 and so presumably require global unfolding for exchange; and intermediate values that probably reflect a mixture of local and global unfolding in wild-type barnase. Amide protons that require the full change in unfolding energy are predominantly in the beta-sheet, which forms early in folding, but also include two that are involved in tertiary interactions that are known not to be formed until late in the folding pathway. Exchange in the major helix, which is known to form early, is largely unaffected by mutation and so exchanges by local breathing. There is thus no direct relationship between hydrogen-exchange behavior and the protein folding pathway. However, experiments on mutants of varying stability may provide further evidence on the sequence of events in folding.
Mutations that alter the third cytoplasmic loop of the a-factor receptor lead to a constitutive and hypersensitive phenotype.
Authors: Boone C , Davis N G , Sprague G F
Published: May 2006

The STE3 gene of Saccharomyces cerevisiae encodes a G protein-coupled receptor that is specific for the mating pheromone a-factor. The ste3L194Q mutation, which leads to the substitution of glutamine for leucine-194 within the third cytoplasmic loop of the receptor, resulted in a 20-fold increase in pheromone sensitivity and also caused partial constitutive activation of the response pathway. Moreover, other amino acid substitutions at the 194 position and several deletion mutations that collectively remove most of the third cytoplasmic loop resulted in hyperactive receptors. Therefore, we suggest that one role of the third cytoplasmic loop is to function as a negative regulatory domain involved in the maintenance of a nonsignaling state of the receptor. The constitutive activity and the pheromone hypersensitivity of ste3L194Q cells were recessive, suggesting that the wild-type receptor can antagonize the signal associated with the activated receptor. The ste3 delta 306 mutation, which results in truncation of most of the C-terminal domain of the receptor, led to a 20-fold increase in pheromone sensitivity, indicating that this domain also mediates negative regulation of the receptor. The ste3L194Q and ste3 delta 306 mutations appear to affect receptor activity independently, because the double mutant was associated with a 400-fold increase in pheromone sensitivity.
Glycosyl-phosphatidylinositol-anchored or integral membrane forms of CD14 mediate identical cellular responses to endotoxin.
Authors: Lee J D , Kravchenko V , Kirkland T N , Han J , Mackman N , Moriarty A , Leturcq D , Tobias P S , Ulevitch R J
Published: May 2006

Endotoxin stimulates leukocytes to release cytokines that initiate septic shock in humans and animals. CD14, a glycosyl-phosphatidylinositol-anchored membrane glycoprotein, is an endotoxin receptor on leukocytes, and endotoxin binding to CD14 induces cytokine production. Here we show that glycosyl-phosphatidylinositol-anchored or integral membrane CD14 mediates identical cellular responses to endotoxin, including NF-kappa B activation and protein tyrosine phosphorylation. We also show that an anti-CD14 monoclonal antibody that does not block endotoxin binding to CD14 nonetheless inhibits cell activation by endotoxin. These findings suggest that binding of endotoxin to cell-surface CD14 is followed by subsequent interactions of the endotoxin-CD14 complex with additional membrane component(s) that enable transmembrane signaling. This function of CD14 may be prototypic for other members of the glycosyl-phosphatidylinositol-anchored family of proteins that do not play a primary role in signal transduction but rather are the principal ligand-binding units of membrane-bound receptor complexes.
Hepatocyte nuclear factor 3/fork head or "winged helix" proteins: a family of transcription factors of diverse biologic function.
Authors: Lai E , Clark K L , Burley S K , Darnell J E
Published: May 2006

A family of transcription factors, first identified as hepatocyte nuclear factors (HNF-3 alpha, -3 beta, and -3 gamma) and as a homeotic Drosophila mutant, fork head, has been intensively studied for the past 4 years. Important findings have emerged about the structure of the DNA-binding portion of the proteins as well as biologic discoveries about the diversity of the family and its implied role in early development.
Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface.
Authors: Francisco J A , Campbell R , Iverson B L , Georgiou G
Published: May 2006

We have expressed a single chain Fv (scFv) antibody fragment, consisting of the variable heavy and variable light domains from two separate anti-digoxin monoclonal antibodies, on the external surface of Escherichia coli by fusing it to an Lpp-OmpA hybrid previously shown to direct heterologous proteins to the cell surface. This scFv fusion was expressed at a high level and was shown to bind the hapten with high affinity and specificity. Whole cell ELISAs, fluorescence microscopy, protease sensitivity, and flow cytometry all confirmed that the scFv was anchored on the outer membrane and was accessible on the surface. Utilizing fluorescence-activated cell sorting, we were able to specifically enrich scFv-producing cells from a 10(5)-fold excess of control cells in only two steps. The expression of antibody fragments on the surface of E. coli is being evaluated as an attractive method for the in vitro production and selection of useful antibody fragments.
Identification of barriers to rotation of DNA segments in yeast from the topology of DNA rings excised by an inducible site-specific recombinase.
Authors: Gartenberg M R , Wang J C
Published: May 2006

Controlled excision of DNA segments to yield intracellular DNA rings of well-defined sequences was utilized to study the determinants of transcriptional supercoiling of closed circular DNA in the yeast Saccharomyces cerevisiae. In delta top1 top2ts strains of S. cerevisiae expressing Escherichia coli DNA topoisomerase I, accumulation of positive supercoils in intracellular DNA normally occurs upon thermal inactivation of DNA topoisomerase II because of the simultaneous generation of positively and negatively supercoiled domains by transcription and the preferential relaxation of the latter by the bacterial enzyme. Positive supercoil accumulation in DNA rings is shown to depend on the presence of specific sequence elements; one likely cause of this dependence is that the persistence of oppositely supercoiled domains in an intracellular DNA ring requires the presence of barriers to rotation of the DNA segments connecting the domains. Analysis of the S. cerevisiae 2-microns plasmid partition system by this approach suggests that the plasmid-encoded REP1 and REP2 proteins are involved in forming such a barrier in DNA containing the REP3 sequence.
A second hepatitis C virus-encoded proteinase.
Authors: Grakoui A , McCourt D W , Wychowski C , Feinstone S M , Rice C M
Published: May 2006

Host and viral proteinases are believed to be required for the production of at least nine hepatitis C virus (HCV)-specific polyprotein cleavage products. Although several cleavages appear to be catalyzed by host signal peptidase or the HCV NS3 serine proteinase, the enzyme responsible for cleavage at the 2/3 site has not been identified. In this report, we have defined the 2/3 cleavage site and obtained evidence which suggests that this cleavage is mediated by a second HCV-encoded proteinase, located between aa 827 and 1207. This region encompasses the C-terminal portion of the 23-kDa NS2 protein, the 2/3 cleavage site, and the serine proteinase domain of NS3. Efficient processing at the 2/3 site was observed in mammalian cells, Escherichia coli, and in plant or animal cell-free translation systems in the absence of microsomal membranes. Cleavage at the 2/3 site was abolished by alanine substitutions for NS2 residues His-952 or Cys-993 but was unaffected by several other substitution mutations, including those that inactivate NS3 serine proteinase function. Mutations abolishing cleavage at the 2/3 site did not block cleavage at other sites in the HCV polyprotein. Cotransfection experiments indicate that the 2/3 site can be cleaved in trans, which should facilitate purification and further characterization of this enzyme.
Isolation of a shark immunoglobulin light chain cDNA clone encoding a protein resembling mammalian kappa light chains: implications for the evolution of light chains.
Authors: Greenberg A S , Steiner L , Kasahara M , Flajnik M F
Published: May 2006

The time of emergence of immunoglobulin kappa and lambda light (L) chains in evolution is unknown. An L chain cDNA clone was isolated from the nurse shark (Ginglymostoma cirratum), a cartilaginous fish, whose predicted variable (V) region amino acid sequence has up to 60% sequence identity to mammalian V kappa domains. Genomic analyses suggest a cluster-type gene organization for this L chain locus, similar to the shark lambda-like immunoglobulin L chain loci rather than mammalian kappa loci. We propose that divergence of the ancestral L chain into isotypes likely occurred before the emergence of elasmobranchs 400-450 million years ago. Similarities in gene organization between the two isotypes in sharks may reflect the gene organization utilized by the ancestral L chain.
Identification of a hexapeptide that mimics a conformation-dependent binding site of acetylcholine receptor by use of a phage-epitope library.
Authors: Balass M , Heldman Y , Cabilly S , Givol D , Katchalski-Katzir E , Fuchs S
Published: May 2006

Monoclonal antibody (mAb) 5.5 is directed against the ligand-binding site of the nicotinic acetylcholine receptor. The epitope for this antibody is conformation-dependent, and the antibody does not react with synthetic peptides derived from the receptor sequence. We have identified a ligand peptide that mimics this conformation-dependent epitope from a phage-epitope library composed of filamentous phage displaying random hexapeptides. Among 38 positive phage clones, individually selected from the library, 34 positive clones carried the sequence Asp-Leu-Val-Trp-Leu-Leu (DLVWLL), 1 positive clone had the sequence Asp-Ile-Val-Trp-Leu-Leu (DIVWLL), and 3 positive clones expressed the sequence Leu-Ile-Glu-Trp-Leu-Leu (LIEWLL), none of which are significantly homologous with the nicotinic acetylcholine receptor alpha subunit sequence. All of these phages bind specifically to mAb 5.5. The synthetic peptide DLVWLL inhibits binding of mAb 5.5 to the related peptide-presenting phage and to the nicotinic acetylcholine receptor in a concentration-dependent manner; the IC50 value is of the order of 10(-4) M. Bioactivity of the peptide "mimotope" DLVWLL was demonstrated in vivo in hatched chickens by inhibition of the mAb 5.5 effect by the peptide. The neuromuscular block and myasthenia gravis-like symptoms that are induced in chicken by passive transfer of mAb 5.5 were specifically abolished by DLVWLL. This study shows the potential of a random peptide phage-epitope library for selecting a mimotope for an antibody that recognizes a folded form of the protein, where peptides from the linear amino acid sequence of the protein are not applicable.
Social control of primary sex differentiation in the Midas cichlid.
Authors: Francis R C , Barlow G W
Published: May 2006

Sexual differentiation in teleost fishes is characteristically labile. The most dramatic form of sexual lability is postmaturational sex change, which is common among teleosts although rare or absent in other vertebrate taxa. In many cases this process is regulated by social cues, particularly dominance interactions. Here we show that in the Midas cichlid, Cichlasoma citrinellum, these same sorts of social interactions affect much earlier stages of sexual differentiation. In this species, males are larger than females. By manipulating relative size in juveniles, we show that this sex-based size difference does not arise from endogenous factors associated with sex. Rather, sex is determined by relative size as a juvenile. We argue that this mode of sex determination, which may be common among teleosts, is a heterochronic variant of postmaturational sex change, one in which some individuals are deflected from a default female trajectory before maturation, as a result of social signals. The size-advantage model, which specifies the optimal size for sex change in hermaphroditic species, can be extended to account for the decision whether to mature as a male or a female in the Midas cichlid.
An examination of the generation-time effect on molecular evolution.
Authors: Ohta T
Published: May 2006

By using DNA sequences of 17 mammalian genes, the generation-time effect is estimated separately for synonymous substitutions and nonsynonymous substitutions. Star phylogenies composed of rodentia, artiodactyla, and primates are examined. The generation-time effect is found to be more conspicuous for synonymous substitutions than for non-synonymous substitutions, by using the methods of (i) Nei and Gojobori, (ii) Li, and (iii) Ina. The proportion of accepted amino acid substitutions in evolution is estimated to be about twice as large in the primate lineage as in the rodent lineage. This result is in accord with the nearly neutral theory of molecular evolution.
Resolution of the HLA-DRB6 puzzle: a case of grafting a de novo-generated exon on an existing gene.
Authors: Mayer W E , O'hUigin C , Klein J
Published: May 2006

HLA-DRB6, one of the human major histocompatibility complex genes, lacks exon 1, which normally codes for the leader and the first four amino acid residues of the mature protein. Because it also lacks the HLA promoter, it was surprising to find that the gene is transcribed at a low level in a chimpanzee B-lymphoblastoma cell line, in which the DRB6 homolog is truncated as in humans. The study designed to resolve the paradox has revealed that a retrovirus related to the mouse mammary tumor viruses was inserted into intron 1 of DRB6 > 23 million years ago. The insertion was either accompanied or followed by the deletion of exon 1 and the promoter region of DRB6. In the 3' long terminal repeat of the retrovirus, however, an open reading frame for a new exon arose, which codes for a sequence of mostly hydrophobic amino acid residues; the sequence could function as a leader for the truncated DRB6 gene. This new exon has a functional donor splice site at its 3' end, which enables it to be spliced in register with DRB6 exon 2. Upstream from the new exon is a promoter enabling transcription of the DRB6 gene. Besides providing an example of a de novo generation of an exon, the study suggests a potential mechanism for generating new genes through the replacement of old exons with newly generated ones.
Intragenic recombination at the human phosphoglucomutase 1 locus: predictions fulfilled.
Authors: Takahashi N , Neel J V
Published: May 2006

In 1982, we advanced a phylogeny that attributed eight alleles of the phosphoglucomutase 1 locus (PGM1) to three independent mutations in a primal allele, followed by four intragenic recombination events involving these mutants [Takahashi, N., Neel, J. V., Satoh, C., Nishizaki, J. & Masunari, N. (1982) Proc. Natl. Acad. Sci. USA 79, 6636-6640]. The recent description of a cDNA probe for this locus [Whitehouse, D. B., Putt, W., Lovegrove, J. U., Morrison, K., Hollyoake, M., Fox, M. F., Hopkinson, D. A. & Edwards, Y. H. (1992) Proc. Natl. Acad. Sci. USA 89, 411-415] now renders it possible to test the validity of this phylogeny. cDNAs of PGM1 reverse-transcribed from mRNAs obtained from Japanese individuals possessing eight different electrophoretically defined alleles (PGM1*1+, PGM1*1-, PGM1*2+, PGM1*2-, PGM1*3+, PGM1*3-, PGM1*7+, PGM1*7-) were amplified by PCR and the sequences were determined. Only three different base substitutions were identified when PGM1*1+ was taken as the reference allele, as follows: an A to T transversion at residue 265, a C to T transition at residue 723, and a T to C transition at residue 1320. The second of these substitutions creates a Bgl II restriction enzyme site and the third creates a Nla III site. At the amino acid level, these substitutions alter amino acid 67 from Lys to Met, amino acid 220 from Arg to Cys, and amino acid 419 from Tyr to His, respectively. These mutations resulted in the electrophoretic properties defining PGM1*7+, the PGM1*2+, and the PGM1*1- alleles, respectively. Subsequent intragenic recombinational events resulted in the remaining four alleles. For two of these latter alleles (PGM1*7- and PGM1*3-), more than one type of intragenic crossover can produce the allele. These findings verify the predicted phylogeny and provide a case study in the evolution of complexity at a genetic locus.
Intraarticular expression of biologically active interleukin 1-receptor-antagonist protein by ex vivo gene transfer.
Authors: Bandara G , Mueller G M , Galea-Lauri J , Tindal M H , Georgescu H I , Suchanek M K , Hung G L , Glorioso J C , Robbins P D , Evans C H
Published: May 2006

Gene therapy offers a radical different approach to the treatment of arthritis. Here we have demonstrated that two marker genes (lacZ and neo) and cDNA coding for a potentially therapeutic protein (human interleukin 1-receptor-antagonist protein; IRAP or IL-1ra) can be delivered, by ex vivo techniques, to the synovial lining of joints; intraarticular expression of IRAP inhibited intraarticular responses to interleukin 1. To achieve this, lapine synoviocytes were first transduced in culture by retroviral infection. The genetically modified synovial cells were then transplanted by intraarticular injection into the knee joints of rabbits, where they efficiently colonized the synovium. Assay of joint lavages confirmed the in vivo expression of biologically active human IRAP. With allografted cells, IRAP expression was lost by 12 days after transfer. In contrast, autografted synoviocytes continued to express IRAP for approximately 5 weeks. Knee joints expressing human IRAP were protected from the leukocytosis that otherwise follows the intraarticular injection of recombinant human interleukin 1 beta. Thus, we report the intraarticular expression and activity of a potentially therapeutic protein by gene-transfer technology; these experiments demonstrate the feasibility of treating arthritis and other joint disorders with gene therapy.
Isolation of cDNA of an auxin-regulated gene encoding a G protein beta subunit-like protein from tobacco BY-2 cells.
Authors: Ishida S , Takahashi Y , Nagata T
Published: May 2006

The addition of 2,4-dichlorophenoxyacetic acid to tobacco BY-2 cells that had been cultured in modified Linsmaier and Skoog medium deprived of auxin for 3 days induced cell division, whereas without 2,4-dichlorophenoxy-acetic acid application, no such induction of cell division was seen. When differential cDNA screening for auxin was done at 4 hr after the addition of 2,4-dichlorophenoxyacetic acid, the cDNA of an auxin-responsive gene designated arcA was isolated. The predicted gene product of arcA is a polypeptide with a M(r) of 35,825. arcA, thus, is a plant hormone-regulated gene that encodes a protein structurally related to the beta subunit of a guanine nucleotide-binding regulatory protein, which is composed of seven repetitive segments of Trp-Asp 40-aa repeats. The possibility that arcA gene products induce cell division is discussed.
A ubiquitous protein is the source of naturally occurring peptides that are recognized by a CD8+ T-cell clone.
Authors: Udaka K , Tsomides T J , Walden P , Fukusen N , Eisen H N
Published: May 2006

We previously isolated from mouse spleen an octapeptide (LSPFPFDL) that in association with the class I major histocompatibility complex protein Ld is recognized by the antigen-specific receptor of an alloreactive CD8+ T-cell clone (2C). Guided by an assay dependent upon the same 2C T-cell receptor, we have now isolated from the same source another naturally occurring peptide. The second peptide (VAITRIEQLSPFPFDL) includes the entire octapeptide sequence and preliminary evidence suggests that it may be a natural precursor of the octapeptide. On finding extensive sequence homology between the 16-mer and part of human 2-oxoglutarate dehydrogenase, we determined the cDNA sequence of mouse 2-oxoglutarate dehydrogenase and found that the deduced amino acid sequence matches precisely the two naturally occurring peptides, indicating their origin by cellular processing of this ubiquitous self protein.
Nitric oxide synthase in the rat anterior pituitary gland and the role of nitric oxide in regulation of luteinizing hormone secretion.
Authors: Ceccatelli S , Hulting A L , Zhang X , Gustafsson L , Villar M , Hökfelt T
Published: May 2006

By using immunohistochemistry and in situ hybridization, we have demonstrated that the nitric oxide (NO)-synthesizing enzyme NO synthase is present in gonadotrophs and in folliculo-stellate cells of the anterior pituitary gland of male and female rats. A marked increase in levels of NO synthase protein and mRNA was observed after gonadectomy. In vitro studies on dispersed anterior pituitary cells suggest that NO inhibits gonadotropin-releasing-hormone-stimulated luteinizing hormone release. An inhibitory effect of NO has also been shown on growth-hormone-releasing-hormone-stimulated release of growth hormone [Kato, M. (1992) Endocrinology 131, 2133-2138]. Thus these findings support a dual mechanism for NO in the control of anterior pituitary hormone secretion, an autocrine mediation of luteinizing hormone release on gonadotrophs, and a paracrine effect on growth hormone secretion involving folliculo-stellate cells closely related to somatotrophs. We speculate that NO may participate in producing the pulsatile secretion patterns of these two pituitary hormones.
A peptide motif that recognizes A.T tracts in DNA.
Authors: Reardon B J , Winters R S , Gordon D , Winter E
Published: May 2006

The DAT1 gene of Saccharomyces cerevisiae encodes a DNA binding protein that specifically interacts with nonalternating oligo(A).oligo(T) tracts (A.T tracts). Deletion analysis of DAT1 coding information showed that the amino-terminal 36 residues are sufficient for specific DNA binding activity. Furthermore, a 35-residue synthetic peptide corresponding to amino acids 2-36 bound to A.T tracts with an equilibrium dissociation constant of 4 x 10(-10) M. Within this region the pentad Gly-Arg-Lys-Pro-Gly is repeated three times. Mutational analysis revealed that the Arg side chains are required for high-affinity binding, whereas the other pentad side chains are dispensable. Chemical interference experiments showed that the DAT1 protein interacts with the minor groove of the double helix. The data suggest that the pentad arginines interact in a cooperative manner with a repeated minor groove feature of A.T tract DNA to achieve high-affinity recognition. Amino acid similarities with other DNA binding proteins suggest that the DAT1 protein pentad represents a specialized example of a widespread motif used by proteins to recognize A.T base pairs.
Charged histidine affects alpha-helix stability at all positions in the helix by interacting with the backbone charges.
Authors: Armstrong K M , Baldwin R L
Published: May 2006

To determine whether a charged histidine side chain affects alpha-helix stability only when histidine is close to one end of the helix or also when it is in the central region, we substitute a single histidine residue at many positions in two reference peptides and measure helix stability and histidine pKa. The position of a charged histidine residue has a major effect on helix stability in 0.01 M NaCl: the helix content of a 17-residue peptide is 24% when histidine is at position 3 compared to 76% when it is at position 17. This dependence of helix content on histidine position decreases sharply in 1 M NaCl, as expected for counterion screening of the charge-helix dipole interaction. Results at interior positions indicate that the position of a charged histidine residue affects helix stability at these positions. Unexpectedly high values of the helix content are found when either neutral or charged histidine is at one of the last three C-terminal positions, suggesting that either form can stabilize an isolated helix by hydrogen bonding to a main-chain CO group.
Selective repression of transcriptional activators at a distance by the Drosophila Krüppel protein.
Authors: Licht J D , Ro M , English M A , Grossel M , Hansen U
Published: May 2006

The Krüppel (Kr) protein, bound at kilobase distances from the start site of transcription, represses transcription by RNA polymerase II in mammalian cells. Repression is monotonically dependent on the dose of Kr protein and the presence of Kr binding site(s) on the DNA. These data suggest an inhibitory protein-protein interaction between the Kr protein and proximal transcription factors. Repression by Kr depends on the specific activator protein driving transcription. In particular, Kr protein selectively represses transcription mediated by the Sp1 glutamine-rich activation domain, tethered to the promoter by a GAL4 DNA-binding domain, but does not repress transcription stimulated by the acidic GAL4 activator. We believe this represents repression by a quenching interaction between DNA-bound Kr protein and the activation region of Sp1, rather than competition between Sp1 and Kr for a limiting transcriptional component. Selective, context-related repression affords an added layer of combinatorial control of gene expression by sequence-specific transcription factors.
Amino acid substitutions in the floor of the putative antigen-binding site of H-2T22 affect recognition by a gamma delta T-cell receptor.
Authors: Moriwaki S , Korn B S , Ichikawa Y , van Kaer L , Tonegawa S
Published: May 2006

We have previously identified a self-reactive gamma delta T-cell clone (KN6) specific for the H-2T region gene product T22b. Now we have investigated by an in vitro mutagenesis analysis of the T22b gene the possibility that the interaction between the KN6 gamma delta T-cell receptor and T22b involves a peptide. The results demonstrate that mutations at the floor of the putative antigen-binding groove of T22b affect recognition by the gamma delta T-cell receptor. Furthermore, we have shown that KN6 cells react with cells that are deficient in the class I peptide transporter TAP1/TAP2. These results suggest that peptide is involved in the interaction of the KN6 T-cell receptor with T22 and that loading of T22 with the putative peptide is TAP1/TAP2-independent.