bluuebunny/crossref_metadata_2025 huggingface dataset:
Adrenal opioid proteins of 8600 and 12,600 daltons: intermediates in proenkephalin processing.
Authors: Jones B N , Shively J E , Kilpatrick D L , Stern A S , Lewis R V , Kojima K , Udenfriend S
Published: May 2006

[Met]Enkephalin-containing proteins of 8600 and 12,600 daltons have been isolated from acid extracts of bovine adrenal medulla and purified to homogeneity, and their sequences have been determined by a combination of automated Edman degradation, tryptic mapping, and enzymatic time-course hydrolysis. The 8600-dalton protein contains one copy of the [Met]enkephalin sequence at the COOH terminus and the 12,600-dalton protein contains three copies of [Met]enkephalin, of which two are internal and the third is at the COOH terminus. They possess identical NH2-terminal amino acid sequences, suggesting that the 8600-dalton protein is derived from the 12,600-dalton protein by intracellular proteolytic processing. This is supported by results from tryptic maps of both proteins. Furthermore, chemical analysis of the tryptic peptides obtained from the 12,600-dalton protein indicates that it also contains the amino acid sequence that corresponds to a previously characterized enkephalin-containing polypeptide of 3800 daltons (peptide F) [Jones et al. (1980) Arch. Biochem. Biophys. 204, 392-395]. All three polypeptides appear to be intermediates in posttranslational processing of a still larger polyenkephalin precursor molecule, proenkephalin, and part of a biosynthetic pathway leading to smaller enkephalin-containing polypeptides and free enkephalins.
Evaluation of single-photon-counting measurements of excited-state lifetimes
Authors: Zimmerman Howard E. , Penn John H. , Carpenter Clint W.
Published: May 2006

An extremely short instrumental response function for a single-photon-counting system has been obtained by using a low-jitter photomultiplier tube, fast amplification of the single photoelectron pulse from this photomultiplier, a constant fraction discriminator with a wide bandwidth input, and a stable reference timing signal. This synchronously mode-locked laser system has been shown to be capable of measuring the fluorescence lifetimes of compounds from 66 psec to 4.5 nsec. The estimated error in these determinations is 6% or 10 psec, whichever is greater. This apparatus has proved itself to be superior to the previously used nitrogen flashlamp apparatus for the determination of subnanosecond lifetimes. The validity of using deconvolution to determine lifetimes shorter than the instrumental response function has been verified.
Combined-information protein structure refinement: Potential energy-constrained real-space method for refinement with limited diffraction data
Authors: Fitzwater Susan , Scheraga Harold A.
Published: May 2006

A potential energy-constrained real-space refinement method designed for use with x-ray diffraction data of low to moderate resolution has been developed. The number of adjustable parameters is severely restricted to ensure a reasonable ratio of data to parameters. Only dihedral angles are allowed to vary; bond lengths and bond angles are fixed at physically reasonable values. The structure of bovine pancreatic trypsin inhibitor was refined by using this method with data to only 2.5-Å resolution. Both the
R
-factor and the electron-density map improved throughout the refinement, and the final structure was a satisfactory approximation to the 1.5-Å structure.
Transcription termination at the tryptophan operon attenuator is decreased in vitro by an oligomer complementary to a segment of the leader transcript.
Authors: Winkler M E , Mullis K , Barnett J , Stroynowski I , Yanofsky C
Published: May 2006

A DNA oligomer 15 nucleotides long was used to probe the involvement of RNA secondary structure in the control of transcription termination at the attenuator of the tryptophan (trp) operon of Escherichia coli. This 15-mer is perfectly complementary to a segment of trp RNA that is thought to play a role in regulation of attenuation. When added to an in vitro transcription reaction mixture containing wild-type E. coli or Salmonella typhimurium trp operon templates, the complementary 15-mer caused a 4-fold increase in read-through transcription. By contrast, the 15-mer did not affect attenuation when a mutant E. coli template was used that does not allow formation of a crucial RNA secondary structure. Control experiments established that oligomers that were not complementary to E. coli trp leader RNA did not affect attenuation and that the 15-mer did not reduce termination when the transcript lacked a complementary region. Other experiments established that the 15-mer did not increase read-through transcription by allowing RNA polymerase molecules that might have already stopped at the attenuator to resume transcription. These findings provide direct support for the view that alternate base-paired structures control transcription termination at the trp attenuator.
Separation of two thyrotropin binding components from porcine thyroid tissue by affinity chromatography: characterization of high and low affinity sites.
Authors: Drummond R W , McQuade R , Grunwald R , Thomas C G , Nayfeh S N
Published: May 2006

Two distinct thyrotropin (TSH) binding species have been separated from solubilized porcine thyroid membranes. Membranes was solubilized with 1% Triton X-100, and the supernatant was recovered by centrifugation at 105,000 X g. Scatchard analysis of thyrotropin binding to solubilized membranes (SM) yielded a nonlinear plot with Kd values for the high and low affinity components similar to those of intact membranes. Chromatography of the SM preparation on concanavalin A-Sepharose 4B resulted in the retention of 10-20% of the binding activity. Upon elution of the column, a peak of binding material (5-7% of total activity) was eluted at 0.3 M alpha-methyl-D-mannoside. This concanavalin A (Con A) bound fraction exhibited a linear Scatchard plot with a Kd value similar to that of the high affinity component of the SM. The protein fraction that did not bind to Con A (Con A unbound) also exhibited a linear Scatchard plot, but with affinity similar to that of the low affinity component of SM. Discontinuous sucrose density gradient ultracentrifugation revealed the presence of two major binding peaks in the solubilized membrane preparation. The slowly sedimenting peak corresponded to that seen in the Con A bound fraction, whereas the rapidly sedimenting peak corresponded to that of the Con A unbound fraction. Sepharose 6B chromatography indicated that in the case of the Con A unbound fraction, a single peak of specific binding activity was eluted in the void volume, and in the case of the Con A bound fraction, one major peak with an approximate Stokes radius of 67 A and several other minor peaks were eluted. These results demonstrate the physical separation of two distinct TSH binding species from thyroid membranes and provide further support for the model of multiple classes of binding sites.
Mutant lambda phage repressor with a specific defect in its positive control function.
Authors: Guarente L , Nye J S , Hochschild A , Ptashne M
Published: May 2006

The lambda phage repressor is both a positive and a negative regulator of gene transcription. We describe a mutant lambda phage repressor that has specifically lost its activator function. The mutant binds to the lambda phage operator sites and represses the lambda phage promoters PR and PL. However, it fails to stimulate transcription from the promoter PRM. The mutation lies in that portion of repressor--namely, the amino-terminal domain--that has been shown [Sauer, R. T., Pabo, C. O., Meyer, B. J., Ptashne, M. & Backman, K. C. (1979) Nature (London) 279, 396-400] to mediate stimulation of PRM. We suggest that the mutation has altered that region of repressor which, in the wild-type, contacts RNA polymerase to activate transcription from PRM.
More than one-third of the discernible mouse polypeptides are not expressed in a Chinese hamster-mouse embryo fibroblast hybrid that retains all mouse chromosomes.
Authors: Bravo R , Schafer R , Willecke K , MacDonald-Bravo H , Fey S J , Celis J E
Published: May 2006

Gene expression at the level of polypeptide synthesis has been investigated in a somatic cell hybrid (20 BW-4) isolated after fusion of spontaneously transformed, tumorigenic Chinese hamster lung fibroblasts with mouse embryo fibroblasts. This hybrid exhibited suppression of tumorigenicity and retained--in addition to the parental Chinese hamster genome--copies of all mouse chromosomes as demonstrated by direct karyotype analysis and confirmed for 18 different mouse chromosomes by analysis of 18 different mouse isozymes. Two-dimensional gel electrophoresis of [35S]methionine-labeled polypeptides from hybrid 20 BW-4 showed that the overall polypeptide pattern corresponded to that of the hamster parent. All polypeptides detected in the hamster parental cells were also expressed in the hybrid although some of them were expressed in altered amounts. Of approximately 1200 labeled polypeptides revealed in the parental cells, 115 mouse polypeptides could be clearly distinguished from the hamster polypeptides due to their different electrophoretic mobilities. Forty-two of these (i.e., 37%) were not expressed in the hybrid 20 BW-4. These observations were confirmed by analysis of another independently isolated hybrid (2W 23) of the same parental cells that also exhibited suppression of malignancy and that retained copies of all mouse chromosomes except no. 5. The results suggest that the genome of the tumorigenic cell after hybridization can suppress expression of more than one-third of the normal parental genome. The suppressed mouse genetic information is probably located on many, if not all, different mouse chromosomes. Even if the level of genetic suppression is high the mouse genome is able to reduce the tumorigenicity of the hamster parental cell.
Isolation and characterization of yeast mutants deficient in adenylate cyclase and cAMP-dependent protein kinase.
Authors: Matsumoto K , Uno I , Oshima Y , Ishikawa T
Published: May 2006

Mutants of Saccharomyces cerevisiae that require cAMP for growth have been isolated. Some of the mutants isolated were deficient in adenylate cyclase activity and mapped at a locus, cyr1, located near the centromere of chromosome X. Growth of cells carrying the cyr1 mutation was arrested at the G1 phase of the yeast cell cycle in the absence of cAMP. The cyr1 mutation was suppressed by a secondary mutation designated bcy1. The bcy1 mutation bypassed the need for cAMP for growth. The bcy1 mutants had extremely low levels of cAMP-binding protein and cAMP-dependent protein kinase but produced a high level of cAMP-independent protein kinase. The results indicate that cAMP is an essential factor for yeast cells to proceed through the cell cycle via the activation of protein kinase.
cDNA recombinant plasmid complementary to mRNAs for light chains 1 and 3 of mouse skeletal muscle myosin.
Authors: Robert B , Weydert A , Caravatti M , Minty A , Cohen A , Daubas P , Gros F , Buckingham M
Published: May 2006

A recombinant plasmid with a cDNA sequence transcribed from mouse skeletal muscle RNA is shown to hybridize with mRNAs for myosin light chains LC1F and LC3F. The inserted fragment corresponds exclusively to the 3'-noncoding region of the mRNA. It hybridizes almost exclusively with the two light chain messengers from fast skeletal muscle RNA of adult mouse. Slight hybridization is seen with RNA from heart muscle and embryonic skeletal muscle. The implications of the conservation of the 3'-noncoding regions between the two mRNAs are discussed.
Cocaine and phencyclidine inhibition of the acetylcholine receptor: analysis of the mechanisms of action based on measurements of ion flux in the millisecond-to-minute time region.
Authors: Karpen J W , Aoshima H , Abood L G , Hess G P
Published: May 2006

The effects of cocaine and of phencyclidine and procaine on acetylcholine receptor-controlled ion flux were measured in the millisecond-to-minute time region. Chemical kinetic measurements of ion flux were made in membrane vesicles prepared from the electric organ of Electrophorus electricus and in PC-12 cells, a sympathetic neuronal cell line. A quench-flow technique was used to measure ion flux in the millisecond-to-second range in membrane vesicles. Cocaine and phencyclidine both inhibit acetylcholine receptor-controlled ion flux, but by different mechanisms. Both compounds decrease the initial rate of ion flux, an effect observed with the local anesthetic procaine. This inhibition cannot be prevented by saturating concentrations of acetylcholine (1 mM). These results from chemical kinetic experiments are consistent with electrophysiological measurements which indicate that local anesthetics act by interfering with the movement of ions through receptor-formed channels. The chemical kinetic experiments, however, give additional information about the action of phencyclidine. They indicate that phencyclidine also increases the rate of receptor inactivation (desensitization) and changes the equilibrium between active and inactive receptor conformations, effects not observed in the presence of cocaine or procaine.
Molecular cloning of the crr gene and evidence that it is the structural gene for IIIGlc, a phosphocarrier protein of the bacterial phosphotransferase system.
Authors: Meadow N D , Saffen D W , Dottin R P , Roseman S
Published: May 2006

Sugar substrates of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) normally prevent bacterial cells from utilizing sugars that are not substrates of this system (diauxic growth, "the glucose effect"). We have previously shown that this type of PTS-mediated repression can be completely reversed by a single mutation, designated crr. Two lines of evidence are presented in this report showing that crr is the structural gene for IIIGlc, one of the proteins of the PTS. First, homogeneous IIIGlc was isolated from wild-type and a crr- mutant of Salmonella typhimurium, and the proteins were compared. The preparations of IIIGlc were indistinguishable except as follows: IIIGlc from the mutant showed only 2-3% of the activity of the wild-type IIIGlc in its ability to act as a phosphocarrier protein in the in vitro phosphorylation of methyl alpha-glucoside. In addition, under certain conditions, the two proteins exhibited different behavior on gel filtration columns and in polyacrylamide gel electrophoresis. The second line of evidence was obtained by cloning the Escherichia coli crr gene, which has an estimated minimum length of 0.6 kilobase, into a high-copy-number plasmid as part of a 1.3-kilobase fragment. The plasmid transforms E. coli crr- to crr+ strains and simultaneously directs the synthesis of IIIGlc.
Polarization of the Golgi apparatus and the microtubule-organizing center in cultured fibroblasts at the edge of an experimental wound.
Authors: Kupfer A , Louvard D , Singer S J
Published: May 2006

We have used the technique of experimental wounding of confluent monolayers of normal fibroblasts to induce essentially unidirectional and synchronous cell movement at the edge of the wound. The intracellular location of the Golgi apparatus and the microtubule-organizing center was determined by double indirect immunofluorescence microscopy, using antibodies specific for the membranes of the Golgi apparatus and antibodies specific for tubulin, respectively. In cells at the wound edge, the immunolabeled Golgi apparatus and microtubule-organizing center were in close proximity to one another and located predominantly forward of the cell nucleus facing the wound. In the same cultures in cells removed from the wound, the two organelles were also coordinately located; however, they were randomly oriented with respect to the wound edge. This reorientation of the two organelles in cells at the wound edge was evident within minutes after wounding and persisted as cell extension subsequently occurred into the wound. These results suggest that both the Golgi apparatus and the microtubule-organizing center may participate in directing cell movement. The possible mechanisms involved are discussed in the light of previous hypotheses and experimental evidence concerning cell motility.
Mechanism of deoxyadenosine-induced catabolism of adenine ribonucleotides in adenosine deaminase-inhibited human T lymphoblastoid cells
Authors: Bagnara Aldo S. , Hershfield Michael S.
Published: May 2006

Loss of ATP accompanying accumulation of dATP has recently been reported to occur in the erythrocytes and lymphoblasts of patients with T lymphocytic leukemia during treatment with deoxycoformycin, an inhibitor of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) that causes the accumulation of deoxyadenosine. We have studied the mechanisms responsible for adenine ribonucleotide depletion in cultured human CEM T lymphoblastoid cells treated with deoxycoformycin and deoxyadenosine. Accumulation of dATP was accompanied by depletion of total soluble adenine ribonucleotides without change in the adenylate energy charge, by the route ATP → AMP → IMP → inosine → hypoxanthine; conversion of IMP to AMP and
de novo
purine synthesis were inhibited in these cells. ATP degradation did not occur in a mutant of CEM that was incapable of phosphorylating deoxyadenosine, or in a B cell line with very limited ability to accumulate dATP. We found that dATP and ATP were both able to stimulate markedly the deamination of AMP by lymphoblast AMP deaminase; dAMP was a poor substrate for this enzyme (
K
m
= 2.4 mM, vs. 0.4 mM for AMP). Similarly, dATP as well as ATP caused marked activation of IMP dephosphorylation by a lymphoblast cytoplasmic nucleotidase. Inhibition of intracellular AMP deaminase with coformycin prevented degradation of adenine ribonucleotides without affecting dATP accumulation. We propose that ATP-dependent phosphorylation of deoxyadenosine generates ADP and AMP. Simultaneously, dATP accumulation stimulates deamination of AMP, but not dAMP, and the dephosphorylation of IMP to inosine. Coupling of AMP degradation to ATP utilization in deoxyadenosine phosphorylation maintains the adenylate energy charge despite net depletion of cellular ATP.
Simultaneous characterization of pre- and postsynaptic neuron contact sites in brain.
Authors: Palkovits M , Léránth C , Jew J Y , Williams T H
Published: May 2006

The combined application of electron microscopic study of nerve degeneration and immunocytochemical techniques enables both pre- and postsynaptic structures of central nervous system synapses to be characterized simultaneously. Information obtained from a model experiment is presented to indicate the usefulness of the method. After the surgical transection of the ventral noradrenergic bundle in rats, degenerating boutons of brainstem axons were demonstrated on luteinizing hormone-releasing hormone-positive and adrenocorticotropic hormone-positive neurons in the hypothalamic arcuate nucleus.
Carcinogen- and radiation-transformed C3H 10T1/2 cells contain RNAs homologous to the long terminal repeat sequence of a murine leukemia virus.
Authors: Kirschmeier P , Gattoni-Celli S , Dina D , Weinstein I B
Published: May 2006

Carcinogen- or radiation-transformed C3H 10T1/2 murine fibroblasts transcribe a set of poly(A)+RNAs that contain sequences homologous to the long terminal repeat (LTR) sequence of Moloney murine sarcoma virus. These LTR-containing RNAs consist of a series of discrete bands ranging in size from about 38 to 18 S. The higher molecular weight molecules (30-38 S) in this set of RNAs also contain sequences homologous to the gag, pol, and env genes of a murine leukemia virus. A 24S RNA contains sequences homologous to the env gene of murine leukemia virus. A 20S and an 18S RNA also share homology with the LTR probe but fail to hybridize to the gag, pol, or env probes or to a probe for the U3 region of the LTR sequence. Thus, the latter transcripts do not appear to arise from a known endogenous murine leukemia virus genome. Although this entire set of RNAs is absent from normal C3H 10T1/2 cells (or is present at an extremely low level), these RNAs are induced by BrdUrd or 5-azacytidine. The presence of these RNAs may provide highly sensitive molecular markers of transformation of murine cells.
Escherichia coli single-strand DNA binding protein from wild type and lexC113 mutant affects in vitro proteolytic cleavage of phage lambda repressor.
Authors: Resnick J , Sussman R
Published: May 2006

In Escherichia coli, the single-strand DNA-binding protein (SSB) is required for DNA replication. A mutation of the ssb gene, lexC113, imparts to the cells UV sensitivity and inability to induce lambda prophage and to amplify recA protein, indicating participation of SSB in DNA repair and viral induction processes. We report the effect of purified SSB, isolated from wild-type and lexC113 strains, on the recA-mediated proteolysis of lambda repressor in vitro. (i) These proteins abolished the inhibition produced by excess single-strand DNA and (ii) in the presence of the binding proteins, the apparent stoichiometry--1 monomer of recA to 6 nucleotides of single-strand DNA [Craig, N. L. & Roberts, J. W. (1980) Nature (London) 283, 26-30] was no longer observed. (iii) At the optimal concentration--1 protein monomer to 8 nucleotides--they increased the rate and extent of repressor cleavage at all single-strand DNA concentrations, including that observed at the apparent optimal DNA concentration. (iv) At binding protein/nucleotide ratios greater than or equal to 1:3, SSB from lexC113 inhibited repressor cleavage while that from wild type did not. (v) These results are consistent with the notion that SSB is probably involved in the induction of prophages in vivo.
Complete amino acid sequence of the delta heavy chain of human immunoglobulin D.
Authors: Takahashi N , Tetaert D , Debuire B , Lin L C , Putnam F W
Published: May 2006

We have determined the amino acid sequence of the variable (V) region of the delta heavy (H) chain of human IgD isolated from the plasma of myeloma patient WAH. This V region is unusual in its amino end group (arginine) and in its length (129 residues). The length is due to 10 insertions in the third complementarity-determining region (CDR3). A computer search showed that no reported CDR3-joining region (-JH) sequences are identical and that they appear to be unrelated to the constant (C) region sequences of immunoglobulins. The V region sequence together with our previous results for the C region give the complete sequence of the human delta chain WAH, which has 512 amino acid residues and a Mr congruent to 65,000. The human delta chain has four domains (V, C delta 1, C delta 2, and C delta 3) and a long hinge region; by comparison, the mouse delta chain lacks a continuous segment of 135 residues, including half the hinge region and the entire C delta 2 domain. The human and mouse delta chains also differ in the number, kind, and location of GlcN and GalN glycans and probably in conformation and quaternary structure. These and other considerations suggest that there may be multiple forms of both secreted and membrane-bound IgD that differ in size, structure, and function.
Optical properties of etiolated plant tissues
Authors: Mandoli D. F. , Briggs W. R.
Published: May 2006

Etiolated tissues of several plants are multiple bundles of fiber optics capable of coherent transfer of light over at least 20 mm. The acceptance angles (the angles at which light can be intercepted and then internally reflected longitudinally) for mung beans, oats, and corn are 47°, 59°, and 52°-54°, respectively. The shapes of the curves that describe the acceptance angles are the same for various tissues of the same plant but differ between species. The pattern of light transmitted longitudinally through a tissue is dependent on the angle at which the light intercepts the side of the tissue and is strongly influenced by the tissue geometry. When 0.5 mm of the tip is irradiated, the amount of light traveling down the “shaded” side of the coleoptile is equal to or 2- to 3-fold greater than the amount traveling down the “lighted” side.
In vivo
recombination of cauliflower mosaic virus DNA
Authors: Lebeurier Geneviève , Hirth Léon , Hohn Barbara , Hohn Thomas
Published: May 2006

Ligation and recombination of the DNA of cauliflower mosaic virus (CaMV) is demonstrated by the following experiments: (
i
) Ligation: Different noninfectious fragments of the CaMV genome (obtained after insertion into plasmid pBR322 followed by enzymatic excision) regained infectivity when mixtures of them were used to inoculate their host. The symptom appearance was delayed by comparison with a typical CaMV infection, and only the newly formed leaves were affected. (
ii
) Recombination: Pairs of noninfectious recombinant full-length CaMV genomes (integrated into pBR322 at different restriction endonuclease sites) regained infectivity upon simultaneous inoculation of a sensitive host. The symptomatology of the resulting infection was indistinguishable from that of a typical CaMV infection. We show that progeny DNA had the same characteristics (size, structure, restriction endonuclease digestion pattern) as
bona fide
CaMV DNA, and that the vector pBR322 had been completely eliminated. A cloned tandem dimer of CaMV DNA with a partial deletion similarly was infectious in the plant assays. This system should be useful to study the expression of mutant genomes, thus allowing characterization of the CaMV genes.
Adult hemoglobins are synthesized in erythroid colonies in vitro derived from murine circulating hemopoietic progenitor cells during embryonic development.
Authors: Wong P M , Clarke B J , Carr D H , Chui D H
Published: May 2006

Circulating peripheral blood cells and disaggregated yolk sac cells were obtained from normal mouse embryos as early as day 9 of gestation, prior to the formation of the fetal liver. These were cultured in vitro in plasma clots or methylcellulose, in the presence of either embryonic fluid or adult spleen cell-conditioned medium, with or without added erythropoietin. Large erythroid colonies were observed by the sixth day of culture. In all instances, these erythroid colonies synthesized adult hemoglobins. These results indicate that erythroid progenitor cells committed to adult hemoglobin synthesis are present in early embryonic circulation.
Esterase 6 in Drosophila melanogaster: reproductive function of active and null males at low temperature.
Authors: Gilbert D G , Richmond R C
Published: May 2006

Esterase 6 is a polymorphic carboxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1) localized to the reproductive tract of male Drosophila melanogaster and transferred to females at copulation. The reproductive fitness of males is strongly affected by temperature and the esterase 6 alleles (active versus null) that they carry. Low temperature (18 degrees C) dramatically changes the character of reproductive functions relative to optimal 25 degrees C. Males with active esterase 6 mate sooner, copulate for a shorter time, and produce more progeny per mating than do esterase 6 null males at 18 degrees C, in contrast to equal progeny production at 25 degrees C. A male esterase 6 effect on the remating speed of females at 25 degrees C is absent at 18 degrees C. Previous work suggests that male esterase 6 promotes ejaculate transfer, sperm storage, and use in females. The differences in reproductive fitness at 18 degrees C noted here may follow from the effects of esterase 6 on sperm transfer and utilization. The esterase 6 effect on productivity is consistent for different female types, even though the components of productivity that were affected, egg laying and egg fertility, changed with female type. These data lead to the hypothesis that the wide-spread polymorphism for esterase 6 may be adaptively significant.
DNA rearrangements in MPC-11 immunoglobulin heavy chain class-switch variants.
Authors: Eckhardt L A , Tilley S A , Lang R B , Marcu K B , Birshtein B K
Published: May 2006

Immunoglobulin heavy chain class switching has been observed in vitro. In the IgG2b-producing MPC-11 mouse myeloma cell line, IgG2a-producing cells arise at a high frequency. In some cases, switch variants producing normal-sized (Mr 55,000) gamma 2a heavy chains have arisen spontaneously from a mutagen-induced "intermediate" (ICR 9.7.1) that produces an unusually large (Mr 75,000) heavy chain. Other switch variants have been isolated directly from the parent cell line. The expressed and unexpressed gamma 2b genes of MPC-11 can be distinguished in restriction endonuclease digests of total genomic DNA so that DNA rearrangements detected in MPC-11 variants can be directly associated with one or the other of these two genes. We describe here DNA rearrangements occurring on the expressed heavy chain chromosome of several MPC-11 gamma 2a switch variants and on the expressed chromosome of the ICR 9.7.1 intermediate. Our data indicate that all of these variants express the parental heavy chain variable region (VH) gene, supporting previous protein studies. We provide mapping data for the expressed gene of both ICR 9.7.1 and one of the IgG2a-producing variant cell lines (ICR 9.9.2.1) derived from it and discuss the advantages of an in vitro switching system for examining the dynamics of the immunoglobulin heavy chain class switch.
Cyclic GMP mimics the muscarinic response in Xenopus oocytes: identity of ionic mechanisms.
Authors: Dascal N , Landau E M
Published: May 2006

Acetylcholine (AcCho) elicits four distinct membrane responses in Xenopus oocytes; the responses can be studied by using the voltage clamp technique. The fastest of the responses, a transient inward current (D1 response), is muscarinic, being evoked by oxotremorine and blocked by atropine but not by curare or hexamethonium. The action of AcCho is cooperative, three transmitter-receptor complexes being required to cause a membrane conductance change, and the dose-response curve in most cases can be fitted by an equation assuming the existence of two binding sites with an affinity ratio of about 11. Guanosine 3',5'-cyclic monophosphate and the 8-bromo and dibutyryl derivatives cause a response similar to D1 in both its time course and the underlying ionic mechanism. The nucleotide-generated response has a smaller amplitude than the AcCho-generated D1.
Cloning of the Vibrio harveyi luciferase genes: use of a synthetic oligonucleotide probe.
Authors: Cohn D H , Ogden R C , Abelson J N , Baldwin T O , Nealson K H , Simon M I , Mileham A J
Published: May 2006

A mixed-sequence synthetic oligonucleotide probe was used to isolate a clone containing the gene encoding the alpha subunit of bacterial luciferase from Vibrio harveyi and part of the gene coding for the beta subunit. DNA sequence analysis has allowed us to determine that the genes are closely linked on the bacterial chromosome and transcribed in the same direction. Comparison of the sequences in the regions preceding the two structural genes has revealed considerable homology and has identified sites that may be involved in the expression of the genes. Identification of a clone from a clone bank of total genomic DNA from this organism shows that mixed probes can be successfully used to isolate a gene of interest from any bacterium provided some protein sequence for the gene product is available.
Modulator sequences mediate oxygen regulation of CYC1 and a neighboring gene in yeast.
Authors: Lowry C V , Weiss J L , Walthall D A , Zitomer R S
Published: May 2006

Three transcripts from Saccharomyces cerevisiae--CYC1 mRNA (transcribed from the iso-I cytochrome c gene) and two RNAs of unknown function, designated tr-1 and tr-2-were identified by reverse Southern blot analysis and found to be regulated in response to oxygen. CYC1 mRNA and tr-1 accumulation occurred only in the presence of oxygen while tr-2 appeared only under anaerobic conditions. tr-2 was transcribed from a region approximately 1 kilobase 5' from the CYC1 coding sequence and in the opposite direction. tr-1 showed homology to the same region as tr-2 but was transcribed from elsewhere in the genome. Expression of tr-2 and CYC1 was observed to be normal in cells transformed with centromeric plasmids carrying the two genes. Mutant transforming plasmids were constructed in which a 400-base-pair region between tr-2 and CYC1 was either deleted or inverted. The deletion led to low-level nearly unregulated expression of both the CYC1 and tr-2 genes, suggesting that sequences upstream from both genes are important for their expression and regulation. The inversion mutation produced a reversed pattern of CYC1 regulation in which the mRNA was present in anaerobically grown cells but absent in the presence of oxygen, mimicking wild-type tr-2 regulation and suggesting that the CYC1 transcription unit is under the control of the translocated tr-2 modulator sequences. Models for the function of these modulators are discussed.
Construction of a
Rhizobium japonicum
gene bank and use in isolation of a hydrogen uptake gene
Authors: Cantrell Michael A. , Haugland Richard A. , Evans Harold J.
Published: May 2006

A gene bank of
Rhizobium japonicum
DNA was constructed by using the broad host range conjugative cosmid pLAFR1. Eighty-three percent of the clones in the bank contained cosmids with insert DNA averaging 22.6 kilobase pairs in length. A series of cosmids containing a hydrogen uptake (
hup
) gene was identified by transferring the gene bank into a H
2
uptake-negative (Hup
-
)
R. japonicum
point mutant (PJ17nal) and screening tetracycline-resistant colonies for the ability to grow chemolithotrophically and to reduce methylene blue in a recently devised colony assay. Hup
+
transconjugants arose at a frequency of approximately 6 × 10
-3
. Plasmid DNAs from II of the Hup
+
transconjugants were isolated and used to transform
Escherichia coli. Eco
RI digests of all plasmids isolated from Hup
+
transconjugants had three DNA fragments in common. Eight of the
E. coli
transformants containing
hup
gene cosmids were conjugated with PJ17nal and another Hup
-
point mutant, PJ18nal. All PJ17nal transconjugants were Hup
+
. The frequency of Hup
+
transconjugants with PJ18nal was approximately 10
-3
. The results indicate that the
hup
gene cosmids may contain one gene and a portion of another.
Several mechanisms can account for defective E alpha gene expression in different mouse haplotypes.
Authors: Mathis D J , Benoist C , Williams V E , Kanter M , McDevitt H O
Published: May 2006

The murine Ia antigens, encoded by the I region of the major histocompatibility complex, are cell-surface glyco-proteins (consisting of alpha and beta polypeptides) thought to be involved in the control of immune responsiveness. Mice of haplotypes b, s, q, and f fail to express one of the Ia antigen complexes, the E complex, on the cell surface. We have attempted to determine at the molecular level how such a defect (or defects) might be generated. By using I-region E alpha and A alpha gene probes for analyses of RNA and DNA structure, it was possible to conclude that at least three mechanisms can operate. Mice of haplotypes b and s bear a deletion in the E alpha gene, f haplotype mice synthesize predominantly an E alpha mRNA of aberrant size, and mice of the q haplotype seem to have a defect in RNA processing or a problem with mRNA stability, or both.
Metabolism and mechanism of action of formycin B in Leishmania.
Authors: Rainey P , Santi D V
Published: May 2006

Formycin B is a potent inhibitor of growth of the promastigote forms of Leishmania tropica, L. mexicana, L. braziliensis, and L. donovani. The metabolic products formed in these organisms are formycin B 5'-monophosphate and formycin A 5'-mono-, di-, and triphosphates, with formycin A 5'-triphosphate predominating. In addition, formycin A is extensively incorporated into RNA. From the metabolic profile, we conclude that formycin B is first converted to the 5'-monophosphate by the nucleoside phosphotransferase found in Leishmania and is subsequently converted to cytotoxic adenosine nucleotide analogs of formycin A that become incorporated into RNA. The metabolism and mechanism of formycin B appears to be qualitatively similar to that of allopurinol riboside, but quantitative differences and species selectivity suggest that these agents may have a different spectrum of activity as potential anti-leishmanial agents.
Double-stranded RNA-dependent phosphorylation of protein P1 and eukaryotic initiation factor 2 alpha does not correlate with protein synthesis inhibition in a cell-free system from interferon-treated mouse L cells.
Authors: Jacobsen H , Epstein D A , Friedmann R M , Safer B , Torrence P F
Published: May 2006

The double-stranded RNAs (I)n X (C)n and (A)n X (dUfl)n (dUfl is 2'-fluoro-2'-deoxyuridylic acid) have been compared as inhibitors of translation in cell-free systems from interferon-treated mouse L cells and from rabbit reticulocytes. In the interferon-treated mouse L-cell system, both double-stranded RNAs stimulated kinase activity, leading to phosphorylation of protein P1 and eukaryotic initiation factor 2 alpha (eIF-2 alpha), but only (1)n X (C)n activated the (2'-5')-oligoadenylate synthetase. Moreover, in this system, (I)n X (C)n, but not (A)n X (dUfl)n, inhibited translation. Both (A)n X (dUfl)n and (I)n X (C)n also activated the rabbit reticulocyte kinase to phosphorylate protein P1 and eIF-2 alpha, but, in contrast to mouse L-cell systems, both (A)n X (dUfl)n and (I)n X (C)n were potent inhibitors of translation in reticulocyte lysates. These results indicate that protein P1 and eIF-2 alpha phosphorylation are not sufficient to cause inhibition of protein synthesis in interferon-treated mouse L-cell extracts. They further suggest that protein synthesis inhibition by (I)n X (C)n in extracts of interferon-treated L cells correlates better with activation of (2'-5')-oligoadenylate synthetase than with activation of the protein P1 and eIF-2 alpha kinase.
Discriminatory inhibition of protein synthesis in cell-free systems by vaccinia virus transcripts.
Authors: Coppola G , Bablanian R
Published: May 2006

The effect of vaccinia virus early transcripts on cellular (globin, HeLa, Chinese hamster ovary) and viral (vaccinia, encephalomyocarditis) mRNA function was studied in reticulocyte and wheat germ cell-free protein-synthesizing systems. Vaccinia virus transcripts of two size classes (8-10 S and 4-7 S), generated in vitro by viral cores, inhibited function of cellular and encephalomyocarditis virus mRNA but not that of vaccinia virus in reticulocyte lysate systems. Mild alkaline hydrolysis or micrococcal nuclease treatment of vaccinia virus in vitro transcripts resulted in a loss of their ability to inhibit protein synthesis directed by HeLa cell RNA. Vaccinia virus in vitro transcripts also selectively inhibited HeLa cell protein synthesis in wheat germ systems, suggesting that double-stranded RNA is not involved in this inhibition of protein synthesis. The addition, to the reticulocyte translating system, of cytoplasmic RNA obtained from infected cells in conjunction with cellular mRNA (globin, HeLa) resulted in the inhibition of synthesis of the globin or HeLa polypeptides with little or no effect on the translation of the vaccinia virus proteins. RNA extracted from vaccinia virions inhibited cellular but not vaccinia virus mRNA function when added to the reticulocyte lysate systems with uninfected or infected HeLa cell cytoplasmic RNA.
Pseudomonas exotoxin A: toxoid preparation by photoaffinity inactivation.
Authors: Marburg S , Tolman R L , Callahan L T
Published: May 2006

A method for toxoid preparation has been developed in which toxins expressing enzymatic activity can be detoxified by photoaffinity labeling techniques. In the case of Pseudomonas aeruginosa exotoxin A, the method relies on the affinity of azido-substituted analogs of the substrate (NAD) for the proenzyme form of the toxin. Photolysis of the putative toxin-analog complex results in irreversible inactivation of the toxin without loss of antigenic character. It is proposed that this occurs by nitrene insertion into a chemical bond on the toxin molecule. This affinity photoinactivation process should be applicable to other ADP-ribosylating toxins.
Lack of Z-DNA conformation in mitomycin-modified polynucleotides having inverted circular dichroism.
Authors: Tomasz M , Barton J K , Magliozzo C C , Tucker D , Lafer E M , Stollar B D
Published: May 2006

Poly(dG-dC) . poly(dG-dC) and Micrococcus lysodeikticus DNA were modified by exposure to reductively activated mitomycin C, an antitumor antibiotic. The resulting covalent drug-polynucleotide complexes displayed varying degrees of CD inversions, which are strikingly similar to the inverted spectrum observed with Z-DNA. The following criteria have been used to establish, however, that the inverted CD pattern seen in mitomycin C-polynucleotide complexes does not reflect a Z-DNA conformation. (i) The ethanol-induced transition of poly(dG-dC) . poly(dG-dC) from B to Z conformation is not facilitated but rather is inhibited by mitomycin C modification. This may be due to the presence of crosslinks, (ii) Radioimmunoassay indicated no competition for Z-DNA-specific antibody by any of the mitomycin C-modified polynucleotides, (iii) 31P NMR of the complexes yielded a single relatively narrow resonance, which is inconsistent with the dinucleotide repeat characteristic of Z-DNA. Alternative explanations for the inverted CD pattern include a drug-induced left-handed but non-Z conformational change or the superposition of an induced CD onto the CD of B-DNA due to drug-base electronic interactions. These results illustrate the need for caution in interpreting CD changes alone as an indication of Z-DNA conformation.
Selective accumulation of cytosol CDP-choline as an isolated erythrocyte defect in chronic hemolysis.
Authors: Paglia D E , Valentine W N , Nakatani M , Rauth B J
Published: May 2006

Erythrocytes from a young woman with chronic hemolytic anemia were found to contain 0.31-0.45 mM CDP-choline, concentrations that are 15-25 times those in normal erythrocytes and equivalent to 20-30% of the total adenine nucleotide content. Accumulation of CDP-choline has been reported only in erythrocytes from subjects with severe (homozygous) pyrimidine nucleotidase deficiency. In the latter syndrome, however, pyrimidine nucleotidase activity is very low and a spectrum of uridine- and cytidine-containing nucleotides is present along with epiphenomena involving glutathione and ribosephosphate pyrophosphokinase. By contrast, selective accumulation of CDP-choline was the only abnormality demonstrable in proband erythrocytes. Membrane phospholipids were quantitatively and qualitatively normal, compatible with the observation that mature erythrocytes maintain membrane phospholipids largely by passive exchange with plasma components or by acylation of lysophospholipids. Although the presence of small amounts of other CDP-containing cofactors, such as CDP-ethanolamine, could not be entirely excluded, the cytidine/choline ratio closely approximated 1:1 in all studies. These data are compatible with the view that choline phosphotransferase and ethanolamine phosphotransferase are separate enzymes in erythroid cells. Selective accumulation of CDP-choline in proband erythrocytes is also compatible with an inherited deficiency of choline phosphotransferase in erythroid precursors, though this hypothesis remains unproved.
Serotype-specific glycoprotein of simian 11 rotavirus: coding assignment and gene sequence.
Authors: Both G W , Mattick J S , Bellamy A R
Published: May 2006

Cloned DNA copies of the double-stranded RNA genomic segments of simian 11 rotavirus have been used to determine the coding assignment for VP7, the type-specific antigen of this virus. Translation of hybrid-selected mRNAs in an in vitro system supplemented with canine pancreatic microsomes permitted VP7 to be assigned to segment 9 and the two nonstructural viral proteins NCVP4 and NCVP3, to segments 7 and 8, respectively. Hybridization of cloned DNA probes for segments 7-9 with the corresponding segments of human rotavirus Wa confirmed these assignments. The complete nucleotide sequence of gene 9 has been determined. The deduced amino acid sequence reveals VP7 to be 326 amino acids in length with two NH2-terminal hydrophobic regions and a single glycosylation site at residues 69-71.
L
2
Cohomology of the Bergman metric
Authors: Donnelly Harold , Fefferman Charles
Published: May 2006

The
L
2
cohomology of the Bergman metric is infinite dimensional in the middle degree and vanishes for all other degrees. Asymptotic expansions are given for the Schwartz kernels of the corresponding projections onto harmonic forms.
Control of DNA ligase molecular forms in nucleocytoplasmic combinations of axolotl and Pleurodeles.
Authors: Signoret J , David J C , Lefresne J , Houillon C
Published: May 2006

A light form of DNA ligase (EC 6.5.1.2), the only form present in oocytes of the axolotl (Ambystoma mexicanum), has been shown to be replaced by a heavy form of the enzyme when the egg enters cleavage. This early biochemical event has been assumed to rely on direct nuclear input. Sucrose gradient analysis permits discrimination between enzymes from axolotl and the sharp-ribbed salamander (Pleurodeles waltlii) for both heavy and light enzymatic forms of DNA ligase. Genetic activity of blastula nuclei transplanted in activated cytoplasm has been tested by determination of the enzymatic forms and specific types of DNA ligases when the implanted egg enters cleavage. A blastula nucleus of Pleurodeles in axolotl cytoplasm determines a heavy ligase of the Pleurodeles type. Conversely, a haploid androgenetic nucleus of Pleurodeles in axolotl cytoplasm controls a light ligase of the Pleurodeles type. Reciprocal experiments give homologous results. To our knowledge, this is the earliest nucleus-dependent synthesis revealed in development for any system. The heavy ligase of one species may coexist with the light form of the other species but not with the light form of its own specific type. Inhibition of the production of the heavy form for one genome results in the expression of the light form. We conclude that genetic control of DNA ligase in very early development involves structural genes for heavy and light forms of enzyme, with an exclusion process operating an alternative expression of corresponding genes. This exclusion relationship between nonallelic genes is species specific.
Deletions and single base pair changes in the yeast mating type locus that prevent homothallic mating type conversions.
Authors: Weiffenbach B , Rogers D T , Haber J E , Zoller M , Russell D W , Smith M
Published: May 2006

Several cis-acting mutations that prevent homothallic mating type conversions in Saccharomyces cerevisiae have been examined. Deletions within the mating type (MAT) locus were obtained by selecting for survivors among homothallic MAT alpha cells carrying the rad52 mutation. The survivors were unable to switch mating type, even in RAD+ derivatives. The deletions varied in size from fewer than 50 to more than 750 base pairs. All of the deletions removed a Hha I site at the border between the alpha-specific sequences (Y alpha) and the adjacent Z region. We also examined several spontaneous inc mutations that prevent MAT switching. Two of these mutations were cloned in recombinant DNA plasmids and their sequences were determined. The MAT alpha-inc 3-7 mutation proved to have an altered Hha I site at the Y alpha/Z border, by virtue of a single base pair substitution G . C leads to A . T in the second base pair of the Z region (Z2). Restriction fragment analysis showed that two other independently isolated strains with MAT alpha-inc mutations had altered the same Hha I site. The MAT a-inc 4-28 mutation contains a single base pair substitution C . G leads to T . A at position Z6. A base pair difference at position Z11 in two MATa strains does not affect MATa conversions. We conclude that the region near the Y/Z border is essential for the efficient switching of MAT alleles and constitutes an enzyme recognition site for a specific nucleolytic cleavage of MAT DNA.