bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence that rnmB is the operator of the Escherichia coli recA gene.
Authors: Volkert M R , Margossian L J , Clark A J
Published: May 2006

rnmB281 leads to high constitutive levels of recA protein such that no increase after UV-inducing treatment occurs. The mutation maps in or near the portion of recA corresponding to the NH2-terminal end of the protein. Examination of the recA proteins from rnmB+ recA-/rnmB281 recA+ heterozygotes suggests that both rnmB alleles are cis-acting and codominant. This is the behavior expected from alleles of a regulatory gene such as an operator or promoter of recA. The possibility that rnmB mutations occur in the promoter of recA. The possibility that rnmB mutations occur in the promoter of recA, though not ruled out, seems unlikely based on the structure of the regulatory region of recA. This suggests that rnmB mutations are operator constitutive mutations of the recA gene and should be called recAo mutations. The UV-irradiation responses of recAo+ and recAo281 strains, both recA+, are compared and inferences are drawn about the roles of large amounts of recA protein in producing the responses.
Biochemical evidence for membrane disintegration in human cataracts.
Authors: Garner M H , Roy D , Rosenfeld L , Garner W H , Spector A
Published: May 2006

Biochemical evidence is presented for the disintegration of the lens fiber plasma membrane in human cataracts. The intrinsic membrane proteins are found in both the water-soluble and water-insoluble nonmembrane fractions of the cataract lens but not in the normal tissue. Furthermore, in contrast to the normal lens, not all of the lipid found in the cataractous lens is isolated with the membrane fraction. In cataracts, both the membrane and membrane fragments are involved in covalent high molecular weight aggregates with an extrinsic membrane protein (43,000 daltons) and a cytoplasmic protein (gamma-crystallin).
Gastrointestinal secretory, motor, circulatory, and metabolic effects of prosomatostatin.
Authors: Konturek S J , Tasler J , Jaworek J , Pawlik W , Walus K M , Schusdziarra V , Meyers C A , Coy D H , Schally A V
Published: May 2006

This study compares the gastrointestinal effects of somatostatin (SS) and its putative prohormone, prosomatostatin (Pro-SS), a 28-amino acid peptide isolated from the hypothalamus and the gut, in conscious dogs with chronic gastric and pancreatic fistulae. Pro-Ss suppressed the release of serum gastrin, insulin, and pancreatic polypeptide that occurs in response to feeding a meat meal in a manner similar to that seen with SS. However, in contrast to SS, which strongly reduced intestinal blood flow and oxygen consumption and stimulated intestinal motility, Pro-SS, at the doses tested, had no influence on mesenteric circulation, oxygen uptake, and intestinal motility. We conclude that Pro-SS mimics most of the gastrointestinal secretory actions of SS, but does not exhibit the intestinal circulatory, metabolic, and motor effects of SS.
Hormone-responsive expression of an endogenous proviral gene of mouse mammary tumor virus after molecular cloning and gene transfer into cultured cells.
Authors: Hynes N E , Kennedy N , Rahmsdorf U , Groner B
Published: May 2006

A recombinant lambda phage containing mouse mammary tumor virus (MMTV) proviral DNA was isolated from a gene library constructed from GR mouse liver DNA. Restriction enzyme analyses reveal that the cloned molecule contains a copy of one of the GR endogenous MMTV proviruses flanked on both sides by 2--3 kb of mouse genomic DNA. In this report we have examined the expression of the cloned MMTV provirus after cotransfection with the herpes thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase,, EC 2.7.1.21) gene and integration into mouse LTK- cells. Nine individual TK+ transformants were selected, and all were found to contain MMTV-transfected DNA. One of the TK+ transformants was chosen for further study. Total poly(A)-containing RNA was isolated from the cells, and liquid hybridization analyses with MMTV cDNA showed that it contained 0.02% MMTV-specific RNA. The sizes of the MMTV-specific species were determined and found to correspond to the 35S and 24S mRNAs synthesized in MMTV-infected cells. Glucocorticoid hormones have been shown to increase the concentration of MMTV RNA in virus-infected cultured cells. Therefore, we tested the effect of dexamethasone on the concentration of MMTV-specific RNA in cells transfected with the MMTV proviral DNA. The amount of MMTV-specific poly(A)-containing RNA found in the cells grown in the presence of hormone was 0.17%. Therefore, dexamethasone causes an 8-fold increase in the amount of MMTV-specific RNA in mouse cells containing several copies of a cloned and transfected MMTV proviral gene.
Analysis of avian myeloblastosis viral RNA and in vitro synthesis of proviral DNA.
Authors: Schulz R A , Chirikjian J G , Papas T S
Published: May 2006

Two virus-specific RNA species of 7.5 and 7.0 kilobases have been identified in avian myeloblastosis virus (AMV) by denaturing gel electrophoresis and blot hybridization analysis, and they were found to be in a 10:1 ratio. The individual RNAs direccted the cell-free synthesis of the 76,000-dalton "gag" protein and the 180,000-dalton "gal-pol" protein, thereby demonstrating 5' sequence homology of approximately 4.9 kilobases between the two species. Synthesis of these two precursor proteins by the AMV genome indicates structural differences between AMV and other avian acute leukemia viruses. The two viral RNAs were transcribed into complete cDNA copies with AMV DNA polymerase. Linear proviruses were found to be 90--100% resistant to S1 nuclease. Analysis of single-stranded transcripts demonstrated two distinct species of 2.6 and 2.3 x 10(6) daltons, and analysis of duplexes formed from the single-stranded transcripts demonstrated species of 5.2 and 4.0 x 10(6) daltons.
Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase.
Authors: Mulligan R C , Berg P
Published: May 2006

Cultured monkey (TC7) and mouse (3T6) cells synthesize an Excherichia coli enzyme, xanthine-guanine phosphoribosyltransferase (XGPRT; 5-phospho-alpha-D-ribose-1-diphosphate:xanthine phosphoribosyltransferase, EC 2.4.2.22), after transfection with DNA vectors carrying the corresponding bacterial gene, Ecogpt. In contrast to mammalian cells, which do not efficiently use xanthine for purine nucleotide synthesis, cells that produce E. coli XGPRT can synthesize GMP from xanthine via XMP. After transfection with vector-Ecogpt DNAs, surviving cells producing XGPRT can be selectively grown with xanthine as the sole precursor for guanine nucleotide formation in a medium containing inhibitors (aminopterin and mycophenolic acid) that block de novo purine nucleotide synthesis. Cells transformed for Ecogpt arise with a frequency of 10(-4) to 10(-5); they appear to be genetically stable in as much as there is no discernible decrease in XGPRT formation or loss on their ability to grow in selective medium after propagation in nonselective medium. Although several of the vector-gpt DNAs can replicate in monkey and mouse cells, none of the transformants contain autonomously replicating vector-gpt DNA. Rather, the gpt transformants contain one to five copies of the transfecting DNA associated with, and most probably integrated into, cellular DNA sequences. In several transformants, vector-coded gene products for which there was no selection are also synthesized. This suggests that recombinant DNAs containing Ecogpt as a selective marker can be useful for cotransformation of nonselectable genes.
The nucleotide sequence surrounding the replication terminus of R6K.
Authors: Bastia D , Germino J , Crosa J H , Ram J
Published: May 2006

The replication terminus of the plasmid R6K has been cloned into the single-stranded DNA phage vector M13mp5 and also into the plasmid vectors pBR313 and PBR322. By using single-stranded DNA templates prepared from the recombinant DNA clones, the sequence of 215 base pairs of DNA containing the replication terminus has been determined. The DNA sequence of the region of the terminus does not contain any 2-fold rotational symmetry. Therefore, folding of the DNA at the region of the terminus is unlikely to be a cause for replication termination. Interaction of a host-specified protein(s) with the sequence of the replication terminus is probably the basis of the mechanism of replicaion termination.
A topographic gradient of molecules in retina can be used to identify neuron position.
Authors: Trisler G D , Schneider M D , Nirenberg M
Published: May 2006

A monoclonal antibody was obtained that binds to cell membrane molecules distributed in a topographic gradient in avian retina. Thirty-five-fold more antigen was detected in dorsoposterior retina that in ventroanterior retina. Most of the antigen was associated with the synaptic layers of the retina. Less antigen was detected in cerebrum, thalamus, cerebellum, and optic tectum, but little or none was found in non-neural tissues tested. The antigen was found on most or all cell types in retina, and the concentration of antigen found is a function of the square of the circumferential distance from the ventroanterior pole of the gradient toward the dorsoposterior pole. Thus, the antigen can be used as a marker of cell position along the ventroanterior-dorsoposterior axis of the retina.
Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C.
Authors: Esmon C T , Owen W G
Published: May 2006

Perfusion of the myocardium with protein C in the presence of thrombin (EC 3.4.21.5) elicits a potent anticoagulant activity, which is identified as activated protein C on the basis of synthetic substrate hydrolysis and anticoagulant properties. The rate of activated protein C formation during the transit through the myocardium is at least 20,000 times that of thrombin-catalyzed activation of protein C in the perfusion solution. The capacity of the heart to activate protein C is maintained for at least 1 hr when thrombin is present in the perfusate, but decays (half-life approximately 30 min) once thrombin is omitted. Addition of diisopropyl-phospho-thrombin increases this decay rate more than 10-fold. Coperfusing diisopropylphospho-thrombin with active thrombin lowers the amount of protein C activation in the myocardium. Cultured monolayers of human endothelium enhance the rate of thrombin-catalyzed protein C activation. As with myocardium, the activation rate is inhibited by including diisopropylphospho-thrombin in the medium. It is proposed that the surface of vascular endothelium provides a cofactor that enhances the rate of protein C activation by thrombin.
Chemical probing of the tRNA--ribosome complex.
Authors: Peattie D A , Herr W
Published: May 2006

We probed the (Escherichia coli) tRNAPhe--ribosome interaction with the chemical reagents dimethyl sulfate and diethyl pyrocarbonate. This monitored the higher-order structure of the tRNA in this biological complex and identified critical sites in the tRNA molecule involved in binding to the ribosome. The methylation of the N-7 position of guanosine and the N-3 position of cytidine as well as diethyl pyrocarbonate attack on adenosines are sensitive to secondary and tertiary interactions. Here we identify specific bases in E. coli Phe-tRNAPhe affected by the interaction with the ribosome. The 70S ribosome protects the N-3 position of cytidine-74 and 75 in the 3'-terminal C-C-A, suggesting a strong, possibly base pairing, interaction between the ribosome and that universal sequence. The ribosome also induces strong reactivities at the N-7 positions of G-24 and G-46 in the central region of the tRNA molecule near the variable-loop domain as well as less significant reactivities at 11 other guanosines. Two of these, G-10 and G-44, are close to G-24 and G-46 in the center of the molecule; the others (guanosines 1, 5, 6, 18, 19, 63, 65, 69, and 71) are in the coaxial acceptor stem-T stem helix. All of the effects are ribosome induced and occur in the presence or absence of the messenger poly(U). Prior chemical modification of the anticodon bases as well as the two adjacent 3' purines and, less effectively, four purines in the anticodon stem prevent stable poly(U)-directed ribosome binding. Thus, we identify the 3' terminal C-C-A sequence, near the peptidyl transferase site, and the anticodon stem and loop of tRNAPhe as forming critical contacts with the ribosome. Other regions of the molecule become reactive on ribosome binding, but these do not suggest a significant conformational change being more likely due to a change of environment.
Millisecond time-resolved changes in x-ray reflections from contracting muscle during rapid mechanical transients, recorded using synchrotron radiation.
Authors: Huxley H E , Simmons R M , Faruqi A R , Kress M , Bordas J , Koch M H
Published: May 2006

Low-angle x-ray diffraction diagrams have been recorded from frog sartorius muscles by using synchrotron radiation as a high-intensity x-ray source. This has enabled changes in some of the principal reflections of interest to be followed with a time resolution of 1 ms, during small but very rapid length changes imposed on a contracting muscle. The 143-A meridional reflection, which is believed to arise from a repeating pattern of myosin cross-bridges along the length of the muscle, shows large changes in intensity in these circumstances. During both rapid releases and rapid stretches, by amounts that produce a translation of actin and myosin filaments past each other by about 100 A and that are completed in about a millisecond (i.e., before significant cross-bridge detachment would be expected), an almost synchronous decrease in 143-A intensity occurs, by 50% or more. This is followed, in the case of quick releases, by a rapid partial recovery of intensity lasting 5--6 ms (which may represent cross-bridge release and reattachment) and then by a more gradual return to the normal isometric value. Quick stretches show only the slower return of intensity. Immediately after the length change, the initial drop in 143-A intensity can be reversed if the release (or stretch) is reversed. These changes provide evidence of a more direct kind than has hitherto been available that the active sliding of actin filaments past myosin filaments during contraction is produced by longitudinal movement of attached cross-bridges.
Specific binding of solubilized adenylate cyclase to the erythrocyte cytoskeleton.
Authors: Sahyoun N E , Le Vine H , Hebdon G M , Hemadah R , Cuatrecasas P
Published: May 2006

Concepts and criteria that have been developed for the study of the molecular organization of membrane-associated proteins are employed here to investigate the interaction of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] with other membrane components. Detergent-solubilized adenylate cyclase can be shown to bind to erythrocyte-derived Triton X-100 shells containing cytoskeletal elements. This binding appears to be saturable with respect to adenylate cyclase concentration, and it is enhanced by the presence of divalent cations. Preactivation of the enzyme with 5'-guanylyl imidodiphosphate and isoproterenol, or with NaF, is a prerequisite for effective binding. Two exceptions to this general observation are noted: rat brain adenylate cyclase, which binds without prestimulation, and rat testicular cytosolic adenylate cyclase, which fails to bind under any of the conditions tried. The binding sites of the Triton X-100 shells are inactivated or released by treatment with various concentrations of trypsin or KCl. Moreover, exposure of the Triton X-100 shells to increasing temperatures results in a progressive loss of the adenylate cyclase binding capacity. On the basis of these and other findings, it is suggested that the adenylate cyclase complex possesses two principal domains that allow it to interact with both cytoskeletal elements and the lipid bilayer. The specific modulation of these interactions may be involved in the hormonal regulation of adenylate cyclase activity.
Antibodies reactive with murine mammary tumor virus in sera of patients with breast cancer: geographic and family studies.
Authors: Day N K , Witkin S S , Sarkar N H , Kinne D , Jussawalla D J , Levin A , Hsia C C , Geller N , Good R A
Published: May 2006

Sera of patients with breast cancer, of healthy women from the United States, East India, East Africa, and China, and of healthy women of American and Parsi families in which breast cancer occurred in several family members were assayed for levels of antibody reactive with the murine mammary tumor virus (MuMTV) by an enzyme-linked immunosorbent assay. Increased levels of antibody to MuMTV (absorbance greater than or equal to 0.4) were found in sera of 18.6% of American patients with breast cancer and of 2.8% of healthy American women and in 38% of patients from India and 61.9% from East Africa (healthy, 26.9%). In contrast, antibody reactive with MuMTV was found in less than 5 of women with breast cancer from mainland China (healthy Chinese, 5.0%). Differences in serum MuMTV antibody levels between breast cancer patients in the four groups were found to be significant (P less than 0.0001). Studies of two families from the United States and of one Parsi family from India with genetic propensity to breast cancer showed that high levels of antibody to MuMTV were found in 33%, 71%, and 23% of healthy family members, respectively. The antibody to MuMTV was readily absorbed with purified MuMTV and gp52. In contrast, fetal calf serum, murine type c retroviruses, or erythrocytes from various species failed to absorb the antibody.
Synthetic gene frequency maps of man and selective effects of climate
Authors: Piazza A. , Menozzi P. , Cavalli-Sforza L. L.
Published: May 2006

The world distribution of 39 independent gene frequencies in human populations is analyzed by multivariate techniques and synthetic geographic maps. Most genetic variation is associated with longitude, with South Asia showing a tendency to be central. Also latitude and, more particularly, distance from the equator play a significant role in a way that suggests that climatic factors exercise selective pressures, especially for certain genes.
Derivation of a formula for the resonance integral for a nonorthogonal basis set
Authors: Yim Yung-Chang , Eyring Henry
Published: May 2006

In a self-consistent field calculation, a formula for the off-diagonal matrix elements of the core Hamiltonian is derived for a nonorthogonal basis set by a polyatomic approach. A set of parameters is then introduced for the repulsion integral formula of Mataga-Nishimoto to fit the experimental data. The matrix elements computed for the nonorthogonal basis set in the π-electron approximation are transformed to those for an orthogonal basis set by the Löwdin symmetrical orthogonalization.
Multilayered iron complexes of [2.2]paracyclophane
Authors: Laganis Evan D. , Finke R. G. , Boekelheide V.
Published: May 2006

Recent syntheses of ruthenium complexes of [2.2]paracyclophane provided a highly interesting class of compounds, the multilayered transition metal complexes of cyclophanes. The present study discloses that arene-metal exchange procedures with ferrocene derivatives, effected either photochemically or by Lewis acid catalysis, are convenient methods for preparing the analogous multilayered iron complexes of [2.2]paracyclophane.
Gene duplication and transposition linked to antigenic variation in Trypanosoma brucei.
Authors: Pays E , Van Meirvenne N , Le Ray D , Steinert M
Published: May 2006

DNA sequence complementary to Trypanosoma brucei mRNAs coding for the synthesis of the variant-specific antigens AnTat 1.1 and AnTat 1.8 have been cloned and characterized. These sequences have been used as probes to analyze the corresponding genes in the nuclear DNA. The two genes seem to be represented in several (three to six) copies, some of which are incomplete. Transcription of one or the other of these two genes is linked to a genetic rearrangement implying duplication and transposition of the "basic" coding sequence. There is probably one additional copy of each gene, and it seems to be complete. The 3' end of each cloned sequence contains, within a 300-base-pair fragment, a genetic element that seems to be repeated and widely distributed in the genome. This repetitive sequence is variant specific. The expression-linked copy of the gene is lost in the culture (procyclic) form of the trypanosome, where the synthesis of variant-specific antigens is shut down. Comparison of two different cloned populations expressing the same serotype (AnTat 1) showed that the recurrence of a given antigenic type may be accompanied by the production of the same additional copy.
Genetic assignment of resonances in the NMR spectrum of a protein: lac repressor.
Authors: Jarema M A , Lu P , Miller J H
Published: May 2006

By using a systematic genetic approach, the resonances in the 19F NMR spectrum of 3-fluorotyrosine-substituted lac repressor protein have been assigned. The NMR data indicate that each monomer of the repressor consists of two distinct and independent domains. One domain, the NH2-terminal sixth of the primary sequence, which has been shown to be very important for DNA binding, is very mobile. The remaining COOH-terminal sequence is more rigid. Ligands of the repressor, which affect its DNA binding capability, lead to conformational changes in the COOH-terminal domain. The approach to the assignment of spectral features taken here can be extended to other systems.
Physical association of pyrimidine dimer DNA glycosylase and apurinic/apyrimidinic DNA endonuclease essential for repair of ultraviolet-damaged DNA.
Authors: Nakabeppu Y , Sekiguchi M
Published: May 2006

T4 endonuclease V [endodeoxyribonuclease (pyrimidine dimer), EC 3.1.25.1)], which is involved in repair of UV-damaged DNA, has been purified to apparent physical homogeneity. Incubation of UV-irradiated poly(dA).poly(dT) with the purified enzyme preparations resulted in production of alkali-labile apyrimidinic sites, followed by formation of nicks in the polymer. The activity to produce alkali-labile sites was optimal in a relatively broad pH range (pH 6.0-8.5), whereas the activity to form nicks had a narrow optimum near pH 6.5. By performing a limited reaction with T4 endonuclease V at pH 8.5, irradiated polymer was converted to an intermediate form that carried a large number of alkali-labile sites but only a few nicks. The intermediate was used as substrate for the assay of apurinic/apyrimidinic DNA endonuclease activity [endodeoxyribonuclease (apurinic or apyrimidinic, EC 2.1.25.2]. The two activities, a pyrimidine dimer DNA glycosylase and an apurinic/apyrimidinic DNA endonuclease, were copurified and found in enzyme preparations that contained only a 16,000-dalton polypeptide. An enzyme fraction from cells infected with bacteriophage T4v1, a mutant that is sensitive to UV radiation, was defective in both glycosylase and endonuclease activities. Moreover, occurrence of an amber mutation in the denV gene caused a simultaneous loss of the two activities, and suppression of the mutation rendered both activities partially active. These results strongly suggested that a DNA glycosylase specific for pyrimidine dimers and an apurinic/apyrimidinic DNA endonuclease reside in a single polypeptide chain coded by the denV gene of bacteriophage T4. Because the two activities exhibited different thermosensitivity, it was further suggested that conformation of the active sites for these activities may be different.
Calcium control of microfilaments: uncoupling of the F-actin-severing and -bundling activity of villin by limited proteolysis in vitro.
Authors: Glenney J R , Weber K
Published: May 2006

Villin is a major F-actin-bundling protein present in the microfilament bundle underlying the plasma membrane of the microvilli present on intestinal epithelial cells. Mild in vitro proteolysis converts villin (Mr, 95,000) into a large fragment, the villin core (apparent Mr, 90,000). Villin core has lost the F-actin-bundling activity expressed by villin in the absence of calcium but retains the micromolar Kd calcium-binding site and the calcium-dependent restriction of actin filament length (F-actin severing) of intact villin. This finding suggests a common structural and functional relatedness between the known calcium-dependent F-actin-severing proteins from different cell types, even though not all of them reveal F-actin-bundling activity.
Inhibition of fructose-1,6-bisphosphatase by fructose 2,6-biphosphate.
Authors: Van Schaftingen E , Hers H G
Published: May 2006

Fructose 2,6-bisphosphate, a known powerful stimulator of phosphofructokinase [Van Schaftingen, E., Hue, L. & Hers, H.-G. (1980) Biochem. J. 192, 897-901] was found to inhibit, at micromolar concentrations, liver and muscle fructose-1,6-biphosphate (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11). The main characteristics of this inhibition are that (i) it is much stronger at low than at high substrate concentrations, (ii) it changes the substrate saturation curve from almost hyperbolic to sigmoidal, and (iii) it is synergistic with the inhibition by AMP. This inhibition may play an important role in the stimulation of gluconeogenesis by glucagon, because this hormone is known to decrease the concentration of fructose 2,6-bisphosphate in the liver [Van Schaftingen, E., Hue, L. & Hers, H.-G. (1980) Biochem. J. 192, 887-895].
DNA breakage and closure by rat liver type 1 topoisomerase: separation of the half-reactions by using a single-stranded DNA substrate.
Authors: Been M D , Champoux J J
Published: May 2006

Circular single strands of bacteriophage phi X174 DNA are broken by rat liver DNA nicking-closing enzyme (type 1 topoisomerase) in low salt (50 mM KCl) at 37 degrees C, generating linear strands containing covalently bound enzyme [Been, M. D. & Champoux, J. J. (1980) Nucleic Acids Res. 8, 6129-6142]. The linear strands can be recircularized in the presence of 10 mM MgCl2 at 24 degrees C and 37 degrees C or 250 mM KCl at 24 degrees C. Recircularization is blocked when the hydroxyl group at the 5' terminus is phosphorylated. The linears generated by the nicking-closing enzyme can also be joined to other DNA fragments containing 5' hydroxyls, but not 5' phosphates. The linkage formed in both the intrastrand and interstrand reactions is stable to alkali. Reclosure of broken single strands is presumed to be analogous to the closure step that occurs durng nicking and closing cycles on duplex DNA.
All gene-sized DNA molecules in four species of hypotrichs have the same terminal sequence and an unusual 3' terminus.
Authors: Klobutcher L A , Swanton M T , Donini P , Prescott D M
Published: May 2006

In hypotrichous ciliates, all of the macronuclear DNA is in the form of low molecular weight molecules with an average size of approximately 2200 base pairs. Total macronuclear DNA from four hypotrichs has been shown to have inverted terminal repeats by direct sequence analysis. In Oxytricha nova, Oxytricha sp., and Stylonychia pustulata, this terminal sequence may be written as 5'-C4A4C4A4C4 ... 3'-G4T4G4T4G4T4G4T4G4 ... In Euplotes aediculatus, the sequences is similar but differs in the lengths of the duplex region (28 base pairs) and of the putative 3' extension (14 base pairs). Also in Euplotes, a second common sequence of 5 base pairs (A-A-C-T-T-T-T-G-A-A) occurs internal to the terminal repeat and a 17-base-pair heterogeneous region: 5'-C4A4C4A4C4A4C4(X)17T-T-G-A-A ... 3'-G2T4G4T4G4T4G4T4G4T4G4(X)17A-A-C-T-T ... The length of the terminal repeat sequence for O. nova was confirmed in cloned macronuclear DNA molecules.
Quiescent human diploid cells can inhibit entry into S phase in replicative nuclei in heterodikaryons.
Authors: Stein G H , Yanishevsky R M
Published: May 2006

Serum-deprived quiescent human diploid cells (HDC) were fused to replicative HDC, and DNA synthesis was monitored in the resulting heterodikaryons. Quiescent HDC had an inhibitory effect on DNA synthesis in replicative HDC nuclei in heterodikaryons. The timing of the inhibitory effect suggests that entry into S phase was inhibited but ongoing DNA synthesis was not inhibited in the replicative HDC nuclei. When quiescent HDC were fused to T98G human glioblastoma cells or SUSM-1 chemically transformed human cells, entry into S phase was similarly inhibited. However, when quiescent HDC were fused to simian virus 40-transformed human cells, adenovirus 5-transformed human cells, or HeLa cells, DNA synthesis was induced in the quiescent HDC nuclei. A simple hypothesis to explain these results is that quiescent HDC contain an inhibitor of entry into S phase. Transformed cells with a dominant replicative phenotype may have gained a factor that overrides the putative inhibitor, perhaps through viral transformation, whereas recessive transformed cells may ahve lost the normal inhibitory mechanism, perhaps through mutation. Senescent HDC behave like quiescent HDC in heterodikaryons formed with the same types of replicative cells, which suggest that senescent HDC and quiescent HDC share elements of a common mechanism for cessation of proliferation.
Microtubule-disrupting agents affect two different events regulating the initiation of DNA synthesis in Swiss 3T3 cells.
Authors: Otto A M , Ulrich M O , Zumbé A , Jimenez de Asua L
Published: May 2006

The stimulatory effect of epidermal growth factor, alone or with insulin, on the rate of initiation of DNA synthesis in Swiss 3T3 cells can be synergistically enhanced by the addition of either Colcemid or colchicine at 1 microM. However, both Colcemid and colchicine can exert the synergistic effect only when added earlier than 8 hr of the prereplicative period (lag phase). Removal of Colcemid (which allows for rapid reassembly of microtubules) earlier than 10 hr of the lag phase results in a loss of the synergistic effect. This suggests that microtubules must remain disrupted for longer times to accomplish some putative event(s) necessary for increasing the rate of initiation of DNA synthesis. Preincubation of quiescent cells with either Colcemid or colchicine for 8 hr prior to adding epidermal growth factor, alone or with insulin, shortens the lag phase by about 4 hr, irrespective of the resulting rate of initiation of DNA synthesis. These results suggest that the state of microtubules is affecting independently at least two different events involved in regulating time initiation of DNA synthesis.
Full-grown oocytes from
Xenopus laevis
resume growth when placed in culture
Authors: Wallace Robin A. , Misulovin Ziva , Etkin Laurence D.
Published: May 2006

When most full-grown, follicle cell-invested oocytes from
Xenopus laevis
are placed in an appropriate culture medium, they resume growth and remain physiologically healthy for at least 2-3 weeks. Rates of growth by full-grown oocytes
in vitro
generally approximate and can even exceed the most rapid growth rate achieved by vitellogenic oocytes
in vivo
. Resumption of oocyte growth can be correlated with the loss of investing follicle cells, which under normal conditions appear to interfere with vitellogenin and nutrient access to the oocyte. The final size reached by the oocyte within the ovary is thus not an intrinsic property of the oocyte but is extrinsically imposed by the somatic environment.
Sexual selection in
Drosophila silvestris
of Hawaii
Authors: Spiess Eliot B. , Carson Hampton L.
Published: May 2006

Previous discovery that
Drosophila melanogaster
females tend to discriminate in mating against phenotypes of earliest courting males prompted a study of the Hawaiian species
D. silvestris
. Tibial bristle variation in males from opposite coasts of the island of Hawaii functions in courtship, and the possibility that females can distinguish males differing in the tibial trait is explored. Mating tests, designed to give each female and male an alternative choice between two individuals of opposite sex every 30 min, consisted of intrapopulation tests with a strain derived from an eastern (Kilauea) population and interpopulation tests between that strain and one derived from a western (Kahuku) population. Males were given initial combat tests, with “winners” then used in mating (except one test with “loser” males). Matings (52-55%) were classified into categories according to the readiness of the female to mate and sequence of courtship. Low-threshold females (accepting the first male after less than four courtship bouts) occurred at 30-35%. Among intrapopulational tests, females (with higher threshold) accepted first- and second-courting males about equally (25:36, respectively), but for male success in mating, the winning of initial intermale combats and the uniformity of courtship effort tended to be important criteria. Among interpopulation tests, homogamic matings were nearly equal (25% each), but heterogamic matings contrasted in that Kilauea females were reluctant to mate with Kahuku males (14%), while reciprocal matings occurred most frequently (34%). Females favored males second to court, particularly when a Kilauea male (with extra tibial bristles) was the second male. Thus a morphological feature likely to be influential in mating is demonstrated to be so; and sexual selection is operating via male-male combat plus discrimination in favor of particular opposite-sex individuals in this species.
Pseudohypoparathyroidism: inheritance of deficient receptor-cyclase coupling activity.
Authors: Farfel Z , Brothers V M , Brickman A S , Conte F , Neer R , Bourne H R
Published: May 2006

Pseudohypoparathyroidism, type I (PHP-I) is an inherited disorder of primary resistance to multiple hormones that work by stimulating adenylate cyclase. In an attempt to clarify the mode of inheritance of PHP-I, we measured the activity of the N protein, a receptor-cyclase coupling component, in erythrocyte membranes. Erythrocyte N-protein activity was reduced by approximately 50% in erythrocytes of 15 PHP-I patients and was normal in 19 of their clinically normal first degree relatives. Reduced N-protein activity and the PHP-I phenotype in these families exhibited both dominant and recessive patterns of inheritance. This suggests that at least two distinct genetic loci are involved in inheritance of N-protein deficiency. In two additional families, dominant inheritance of the PHP-I phenotype was associated with normal activities of erythrocyte N protein. Thus, it appears that mutation of at least one additional genetic locus, not involving the N protein, can produce PHP-I.
Mutagen-induced resistance to mycophenolic acid in hamster cells can be associated with increased inosine 5'-phosphate dehydrogenase activity.
Authors: Huberman E , McKeown C K , Friedman J
Published: May 2006

Cell variants resistant to the cytotoxic effect of mycophenolic acid, an inhibitor of IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.2.1.14), were selected by a one-step procedure from Chinese hamster V79 cells. The frequency of these variants was increased in a dose-dependent manner after treatment with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine and after an expression time of 8 days. The degree of resistance in five of the six isolated cell variants was associated with a comparable increase in the specific activity of IMP dehydrogenase, which was 3- to 6-fold higher than that of the parent V79 cells. The IMP dehydrogenase activity from both the variants and the V79 cells had a similar affinity for the substrate IMP with a Km of about 20 microM and a similar response to mycophenolic acid with a Ki of 12-16 nM. It is suggested that cell variants with an altered regulation of IMP dehydrogenase activity may be helpful in studying the control of nucleic acid biosynthesis, cell growth, and carcinogenesis. Mycophenolic acid resistance also may be useful as a marker in short-term assays for the identification of potential chemical carcinogens.
Clinical applications of the radioimmunoassay of secretory tuberculoprotein.
Authors: Straus E , Wu N , Quraishi M A , Levine S
Published: May 2006

A radioimmunoassay that measures a specific secretory tuberculoprotein was used to detect Mycobacterium tuberculosis in 9 of 30 liquid cultures of sputum. The accumulation of immunoreactive material in liquid cultures containing isoniazid was shown to reflect in vitro susceptibility of mycobacteria to the antibiotic effects of the drug.
Von Willebrand syndrome induced by a Bothrops venom factor: bioassay for venom coagglutinin.
Authors: Brinkhous K M , Barnes D S , Potter J Y , Read M S
Published: May 2006

Hereditary deficiency of the macromolecular Factor VIII complex results in classic von Willebrand disease in man and animals, a bleeder state characterized by loss of the multiple biologic activities associated with the Factor VIII complex, including the platelet-aggregating von Willebrand factor. The bleeding time is also long. Venom coagglutinin, a Bothrops factor that causes platelet aggregation in vitro, depletes the plasma of its von Willebrand factor. The rate of platelet aggregation is a function of the amount of the coagglutinin present. Based on this observation, a sensitive and quantitative assay for the venom coagglutinin was developed. We administered the purified Bothrops factor to normal pigs and dogs and induced a von Willebrand syndrome similar to the inherited disease. The plasma von Willebrand factor was severely depleted; the antihemophilic factor and the Factor VIII-related antigen were not depleted as much. The bleeding time was normal. During the induction phase of the syndrome, transient thrombocytopenia with a long bleeding time occurred. The pig was less sensitive than the dog to the effect of coagglutinin. The severity of the syndrome is determined by the amount of venom coagglutinin administered. It is suggested that the syndrome could be induced in any mammalian species because the plasma of all mammals tested in vitro is sensitive to the venom factor. This model provides another avenue for the study of the heterogeneity of the Factor VIII complex and the pathophysiology of its components.
Distinct pharmacological properties of excitatory amino acid receptors in the rat striatum: study by Na+ efflux assay.
Authors: Luini A , Goldberg O , Teichberg V I
Published: May 2006

Specific 22Na+ efflux rates from preloaded rat striatal slices are increased in a dose-dependent manner by L-glutamate and other excitatory amino acids displaying the following order of efficiency: N-methyl-D-aspartate greater than DL-homocysteate greater than quisqualate greater than kainate greater than D-glutamate greater than L-glutamate greater than L-aspartate. Amino acid antagonists such as 2-amino-5-phosphonovalerate, gamma-D-glutamylglycine, DL-aminosuberate, DL-aminoadipate, and diethyl glutamate but not nonexcitatory amino acids such as gamma-aminobutyric acid inhibit the amino acid-induced increase in specific 22Na+ efflux rate. Increased K+ concentrations, in the presence of 2 mM Ca2+, increase the specific 22Na+ efflux. The latter and the response to N-methyl-D-aspartate, but not the responses to L-glutamate, L-aspartate, quisqualate, and kainate, are inhibited to similar extents by the same antagonists. These results suggest the release from striatal nerve terminals of a putative neurotransmitter with pharmacological properties different from those of L-glutamate or L-aspartate but similar to those of N-methyl-D-aspartate. The results of this study show that the stimulation of the 22Na+ efflux in brain slices by neuroactive amino acids and K+ ions is a valid and powerful tool for pharmacological investigations of excitatory amino acid receptors and their putative ligands.
Analogues of red and white muscle in squid mantle
Authors: Mommsen T. P. , Ballantyne J. , MacDonald D. , Gosline J. , Hochachka P. W.
Published: May 2006

In five species of squid, varying in life-style from fast-swimming pelagic predators to sluggish benthic forms, the circular muscle of the mantle was found to be metabolically and structurally differentiated into inner, middle, and outer zones. In the middle zone, mitochondrial abundance and the ratios of oxidative to glycolytic enzyme activities were low. This zone was sandwiched between thinner bands of muscle lining both the inner and outer edges of the mantle. In these bands, mitochondrial abundance and the ratios of oxidative to glycolytic enzyme activities were high. It is proposed that this metabolic differentiation is analogous to the development of red and white muscles in vertebrates and that it serves a similar function, white muscle mainly supporting burst-type swimming and red muscle sustaining steady-state oxidative work.
Establishment of the interferon-mediated antiviral state: possible role of superoxide dismutase.
Authors: Pottathil R , Chandrabose K A , Cuatrecasas P , Lang D J
Published: May 2006

Superoxide dismutase (SOD; superoxide: superoxide oxidoreductase, EC 1.15.1.1) catalyzes the dismutation of O2- free radicals formed during various enzymatic reactions or by ionizing radiation. Genes coding for SOD and sensitivity to exogenous interferon (IF) are syntenic in both mouse and man. Diethyldithiocarbamate (DDC) has been shown to inhibit SOD activity by chelating Cu2+, the metal ion essential for the catalytic activity of the enzyme. Mouse cell lines L, L929, L1210 S6, and L1210 R3 and a human cell line (WISH) pretreated with homologous IF and different concentrations of DDC for various periods of time were tested for their ability to support virus multiplication. Treatment of cells with DDC resulted in dose- and time-dependent inhibition of SOD activity and, simultaneously, in the reduction of antiviral protection by exogenous IF. Cells pretreated for 4 hr with DDC and then washed thoroughly were also resistant to IF, but DDC was without effect if the IF effect was first "established" by a 4-hr exposure to IF before addition of DDC. Under the conditions employed, DDC treatment did not result in any detectable inhibition of DNA, RNA, or protein synthesis in these cells. The data suggest that SOD or a related Cu2+-requiring enzyme may be necessary for the establishment of the IF-induced antiviral state in both human and murine cells.
A cloned cyanobacterial gene for glutamine synthetase functions in Escherichia coli, but the enzyme is not adenylylated.
Authors: Fisher R , Tuli R , Haselkorn R
Published: May 2006

The coding sequence for Anabaena 7120 glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.1] are shown to be contained within a 7.5-kilobase-pair (kbp) HindIII fragment that has been cloned by plaque hybridization. The hybridization probe for the cyanobacterial gene was a recombinant plasmid containing the glnA gene from Escherichia coli K-12. Evidence that the cloned Anabaena fragment contains the glnA gene includes complementation of a glnA deletion mutant of E. coli and immunological identity of the enzyme produced by the cloned Anabaena fragment in E. coli with glutamine synthetase purified from Anabaena 7120. Heteroduplex analysis reveals 0.65 kbp of homology between the 7.5-kbp Anabaena 7120 fragment and an 11-kbp E. coli fragment that codes for E. coli glutamine synthetase. Studies of Anabaena glnA gene activity in E. coli suggest that the cyanobacterial gene is not repressible and that the Anabaena 7120 glutamine synthetase is not adenylylated in E. coli.
Overlapping divergent genes in the maize chloroplast chromosome and
in vitro
transcription of the gene for tRNA
His
Authors: Schwarz Zsuzsanna , Jolly Setsuko O. , Steinmetz Andre A. , Bogorad Lawrence
Published: May 2006

In the presence of the S polypeptide, maize chloroplast DNA-dependent RNA polymerase preferentially transcribes sequences within the 2200-nucleotide-pair-long maize chloroplast chromosome fragment
Eco
[unk] from a supercoiled chimeric plasmid cloned in
Escherichia coli
[Jolly, S. O. & Bogorad, L. (1980)
Proc. Natl. Acad. Sci. USA
77, 822-826].
Eco
[unk] contains one gene for tRNA
His
and one for a 1.6-kilobase RNA that includes an open reading frame. These two genes overlap by at least a few nucleotides and are transcribed divergently from complementary DNA strands. This indicates possible transcriptional regulation of chloroplast DNA at the nucleotide level. The 5′ end of tRNA
His
(G-U-G) isolated from maize chloroplasts is indistinguishable from that of the transcript produced from
Eco
[unk]
in vitro
by maize chloroplast DNA-dependent RNA polymerase. This purified system initiates RNA synthesis faithfully and exhibits preference for some chloroplast genes. Maize chloroplast DNA for tRNA
His
lacks the sequence C-C-A at its 3′ terminus; it is presumably added post-transcriptionally. Maize tRNA
His
has both prokaryotic and eukaryotic features.
Stable chromosomal integration of the entire nitrogen fixation gene cluster from Klebsiella pneumoniae in yeast.
Authors: Zamir A , Maina C V , Fink G R , Szalay A A
Published: May 2006

A bacterial plasmid containing the entire nitrogen fixation (nif) gene cluster (consisting of at least 15 genes) from Klebsiella pneumoniae was used in conjunction with an Escherichia coli-yeast shuttle plasmid containing the yeast his4 gene cluster to cotransform a his4- recipient strain of Saccharomyces cerevisiae. Of 87 histidine-independent clones screened, 2 contained nif DNA. Restriction and hybridization analyses showed that two copies of the nif plasmid (46 kilobases each) are integrated in tandem in the recipient chromosome by recombination between homologous regions in the transforming plasmids. Chromosomal integration was also verified by tetrad analysis, showing that the nif DNA behaved in meiosis like a Mendelian element. During mitotic growth, one of the two copies of the nif region is frequently lost. The remaining copy of nif is stable, even after 40 generations in nonselective medium.
Antibodies specific for left-handed Z-DNA.
Authors: Lafer E M , Möller A , Nordheim A , Stollar B D , Rich A
Published: May 2006

We prepared a brominated poly(dG-dC).poly(dG-dC) which forms a stable Z-DNA helix under physiological salt conditions. Rabbits and mice were immunized with brominated and unbrominated poly(dG-dC).poly(dG-dC) complexed with methylated bovine serum albumin. Antibodies specific for Z-DNA were produced. These antibodies were found not only in the sera of animals immunized with the low-salt stabilized Z-DNA [Br-poly(dG-dC).poly(dG-dC)] but also in sera from animals immunized with the unbrominated B-DNA form of the polymer. From this it is inferred that the unbrominated poly(dG-dC).poly(dG-dC) was partially converted to Z-DNA by its combination with the basic protein methylated bovine serum albumin. In addition to specific anti-Z-DNA antibody populations, two other interesting types of antibody populations were found. One of these reacted with both the Z and B forms of poly(dG-dC).poly(dG-dC). This antibody may be converting the polymer from the B-DNA to the Z-DNA form. The other type of antibody was specific for a B form of poly(dG-dC).poly(dG-dC) and did not react at all with the Z form. The antibodies raised to Z-DNA were shown to be highly specific for Z-DNA and did not react with B-DNA, RNA, DNA.RNA hybrids, or a number of other polynucleotides. This specificity for Z-DNA will make possible their use as reagents for determining the presence of Z-DNA in biological systems. Sera of autoimmune lupus mice were also shown to have a considerable amount of naturally occurring anti-Z-DNA antibody activity.
Spurious conformational transitions in proteins?
Authors: Cooper A
Published: May 2006

Temperature-dependent dynamic processes in biological macromolecules can produce sharp and reversible transitions in spectroscopic properties that might be misinterpreted as evidence for thermally induced conformational changes. This provides a rational explanation for the paradoxical case of D-amino acid oxidase [D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3], for which a sharp fluorescence transition at 14 degrees C, not observed by sensitive calorimetry [Sturtevant, J. M. & Mateo, P. L. (1978) Proc. Natl. Acad. Sci. USA 75, 2584-2587], could be due to a dynamic quenching process of large activation energy, rather than a change in conformational state of the protein. Similar interpretations may be valid in other systems studied by experimental techniques that depend, directly or indirectly, on molecular relaxation processes.