bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamic filtering by two-dimensional 1H NMR with application to phage lambda repressor.
Authors: Weiss M A , Eliason J L , States D J
Published: May 2006

Flexible regions of proteins play an important role in catalysis, ligand binding, and macromolecular interactions. Because of its enhanced sensitivity to motional narrowing, two-dimensional coupling constant J-correlated 1H NMR may be used to observe these regions selectively. Dynamic filtering is an intrinsic feature of this experiment because cross-peak amplitude decays rapidly as linewidths approach the coupling constant. We demonstrate here the flexibility of the NH2-terminal arm of phage lambda repressor, which is thought to wrap around the double helix in the repressor-operator complex. The assignment of arm resonances is made possible by the construction of mutant repressor genes containing successive NH2-terminal deletions.
Topological repression of gene activity by a transposable element.
Authors: Stokes H W , Hall B G
Published: May 2006

The ebgA (evolved beta-galactosidase) gene of Escherichia coli was isolated as part of a 9.6-kilobase (kb) sequence cloned into plasmid pBR322. The position of the ebgA gene within that 9.6-kilobase sequence was identified by insertional inactivation by means of the transposon gamma-delta. In addition to the gamma-delta insertions that inactivate ebgA by disrupting the coding sequence, seven additional gamma-delta insertions reduce expression of the gene by a factor of greater than 200 by insertions elsewhere into the replicon. One of these insertions is into the pBR322 sequence itself. This action at a distance to reduce expression requires that gamma-delta is cis with respect to the ebgA gene. The effect is independent of the orientation or position of gamma-delta within the replicon, but it does depend both upon the orientation of the ebgA-bearing sequence within the replicon and upon the total size of the replicon. Transcription readthrough (promoter occlusion) does not explain this phenomenon, and we suggest that the presence of gamma-delta may alter the local supercoiling in the region of the ebgA promoter in such a way as to inhibit transcription. This repression by a transposable element appears to represent a novel mechanism for altering gene expression.
Structural analysis of human adult and fetal alkaline phosphatases by cyanogen bromide peptide mapping.
Authors: Vockley J , D'Souza M P , Foster C J , Harris H
Published: May 2006

The adult and fetal forms of human intestinal alkaline phosphatase (ALPase; orthophosphoric-monoester phosphohydrolase, EC 3.1.3.1) are indistinguishable by a variety of analytical procedures. However, they differ electrophoretically and can be differentiated by binding studies with monoclonal antibodies. In this report, these two enzymes along with placental and liver ALPases are compared by the technique of CNBr peptide mapping, and the role of carbohydrate in generating these patterns is investigated. NaDodSO4/PAGE of CNBr digests of radiolabeled ALPases from fetal and adult intestine shows that these two isozymes share five of seven common-sized CNBr fragments. Placental ALPase shares only one common-sized fragment with either intestinal enzyme. Liver ALPase has no CNBr fragments in common with any of the others. These data indicate that fetal intestinal ALPase is not a heterodimer of one subunit each of intestinal ALPase and placental ALPase as has been postulated. CNBr digests of neuraminidase-treated enzymes reveal a change of mobility of only one CNBr band in each of fetal intestinal, placental, and liver ALPases, indicating the presence of sialic acid residues in these fragments. Periodic acid/Schiff reagent staining (specific for carbohydrate) of CNBr digests of fetal and adult intestinal ALPases reacts with only one band in each enzyme, which is the same band from the fetal enzyme shown to contain sialic acid. However, fetal and adult intestinal ALPases each contain at least one CNBr fragment of unique size that is apparently nonglycosylated.
Autoradiographic assay of mutants resistant to diphtheria toxin in mammalian cells in vitro.
Authors: Ronen A , Gingerich J D , Duncan A M , Heddle J A
Published: May 2006

Diphtheria toxin kills mammalian cells by ribosylating elongation factor 2, a protein factor necessary for protein synthesis. The frequency of cells able to form colonies in the presence of the toxin can be used as an assay for mutation to diphtheria toxin resistance. We report here that resistance to diphtheria toxin can also be detected autoradiographically in cells exposed to [3H]leucine after treatment with the toxin. In cultures of Chinese hamster ovary cells, the frequency of such resistant cells is increased by exposure of the cells to gamma-rays, ultraviolet light, ethylnitrosourea, mitomycin c, ethidium bromide, and 5-bromo-2'-deoxyuridine in a dose- and time-dependent manner. The resistant cells form discrete microcolonies if they are allowed to divide several times before intoxication, which indicates that they are genuine mutants. The assay is potentially adaptable to any cell population that can be intoxicated with diphtheria toxin and labeled with [3H]leucine, whether or not the cells can form colonies. It may be useful, therefore, for measuring mutation rates in slowly growing or nondividing cell populations such as breast, brain, and liver, as well as in cells that do divide but cannot be readily cloned, such as the colonic epithelium.
Envelope proteins of human T-cell leukemia virus: expression in Escherichia coli and its application to studies of env gene functions.
Authors: Kiyokawa T , Yoshikura H , Hattori S , Seiki M , Yoshida M
Published: May 2006

The DNA fragments of the 5' and 3' halves of the putative env gene predicted from the DNA sequence of human T-cell leukemia virus (HTLV) provirus were inserted into expression vectors pORF2 and pORF1, respectively, and two hybrid proteins composed of env polypeptides and beta-galactosidase were efficiently produced in Escherichia coli. The hybrid proteins containing the NH2-terminal (EH9) and COOH-terminal (EA1) halves were both immunologically reactive with sera from adult T-cell leukemia patients, demonstrating the utility of the hybrid proteins for diagnosis of HTLV infection. Rabbit antisera against these hybrid proteins detected the two glycoproteins gp62 and gp46, which were previously identified as HTLV env gene products. With these rabbit antisera, two properties of the env gene products were studied. (i) The antisera inhibited syncytia formation of cat S+L- cells induced by HTLV, suggesting that one or both of the env gene products of HTLV, gp62 and gp46, are involved in induction of cell fusion. (ii) The env product gp62 or gp46 or both products are exposed on the surface of HTLV-infected cells and might modulate the proliferation of HTLV-infected T cells in the host because the antisera against the hybrid proteins were cytotoxic on HTLV-producing cell lines. The latter conclusion also is supported by the fact that adult T-cell leukemia patients and healthy HTLV carriers have antibodies to the env gene products.
Determination of the promoter region of mouse ribosomal RNA gene by an in vitro transcription system.
Authors: Yamamoto O , Takakusa N , Mishima Y , Kominami R , Muramatsu M
Published: May 2006

Sequences required for a faithful and efficient transcription of a cloned mouse ribosomal RNA gene (rDNA) are determined by testing a series of deletion mutants in an in vitro transcription system utilizing two kinds of mouse cellular extract. Deletion of sequences upstream of -40 or downstream of +52 causes only slight reduction in promoter activity as compared with the "wild-type" template. For upstream deletion mutants, the removal of a sequence between -40 and -35 causes a significant decrease in the capacity to direct efficient initiation. This decrease becomes more pronounced when the deletion reaches -32 and the sequence A-T-C-T-T-T, conserved among mouse, rat, and human rDNAs, is lost. Residual template activity is further reduced as more upstream sequence is deleted and finally becomes undetectable when the deletion is extended from -22 down to -17, corresponding to the loss of the conserved sequence T-A-T-T-G. As for downstream deletion mutants, the removal of the sequence downstream of +23 causes some (and further deletions up to +11 cause a more) serious decrease in template activity in vitro. These deletions involve other conserved sequences downstream of the transcription start site. However, the removal of the original transcription start site does not abolish the transcription initiation completely, provided that the whole upstream sequence is intact.
Avian sarcoma virus gag and env gene structural protein precursors contain a common amino-terminal sequence.
Authors: Ficht T A , Chang L J , Stoltzfus C M
Published: May 2006

The initiation site for translation of the avian sarcoma virus glycoprotein precursor, Pr63env, has been determined by analyzing the amino-terminal peptides of Pr63env and the polyprotein precursor Pr76gag encoded by the viral gag gene. The acceptor splice junction used to form the env gene mRNA has also been identified. Hybrid-selected virus-specific mRNAs were translated in vitro in the presence of either L-[35S]methionine to label at every methionine residue or L-[35S]methionine-tRNAMeti to label specifically at the amino-terminal methionine residues. Tryptic peptide maps of Pr63env labeled at every methionine residue contain all of the peptides, plus one additional peptide, present in the map of Pr57env, a nonglycosylated env-encoded polypeptide of molecular weight 57,000 immunoprecipitated from tunicamycin-treated cells. Specific amino-terminal labeling of the in vitro-synthesized polypeptides showed that the peptide missing from Pr57env corresponds to the amino-terminal tryptic peptide of Pr63env, which is removed in vivo as part of the amino-terminal signal peptide. Comparison of the amino-terminal tryptic peptides of Pr63env and Pr76gag showed that they are identical. In contrast, the chymotryptic amino-terminal peptides of Pr76gag and Pr63env are not identical. The location of the acceptor-splice junction in the env mRNA of the Prague A strain of avian sarcoma virus was determined by mung bean nuclease mapping to be at nucleotide 5,078. Fusion of the gag and env gene sequences during splicing results in use of the same AUG codon to initiate synthesis of Pr76gag and Pr63env. This sequence is contained within the 397-nucleotide 5' terminal leader that is spliced to the body of the env mRNA. The possible significance of these results for the regulation of avian sarcoma virus synthesis and translation is discussed.
Molecular hybridization under conditions of high stringency permits cloned DNA segments containing reiterated DNA sequences to be assigned to specific chromosomal locations.
Authors: Fisher J H , Gusella J F , Scoggin C H
Published: May 2006

Identifying the specific DNA sequences involved in the chromosomal abnormalities in developmental and neoplastic diseases may be essential to understanding the molecular biology of these disorders. The use of recombinant DNA techniques in conjunction with rodent-human hybrid cells makes it possible to assign chromosomal locations to specific DNA sequences. However, the ubiquitous presence of reiterated DNA species often complicates the application of straightforward molecular hybridization. To accelerate the mapping of cloned sequences to specific chromosomal locations, we investigated the possibility that cloned sequences containing reiterated DNA might be used without isolating unique sequences. By varying conditions of hybridization (specifically temperature) and using restricted DNA samples from human genomic DNA, Chinese hamster ovary-human chromosome 11 hybrids, and non-chromosome 11 hybrids, we have been able to assign cloned DNA sequences containing reiterated sequences to their chromosome of origin. By hybridization under the high-stringency condition of 55 degrees C, specific banding was produced with both human genomic DNA and the human-chromosome-containing hybrid from which the probe was prepared. Furthermore, using a panel of chromosome 11 deletion mutants, we have been able to assign a cloned sequence to a specific chromosomal location. We believe that this approach will accelerate gene mapping procedures and facilitate identification of DNA sequences involved in chromosomal abnormalities.
Modulation of chloroplast movement in the green alga Mougeotia by the Ca2+ ionophore A23187 and by calmodulin antagonists.
Authors: Serlin B S , Roux S J
Published: May 2006

The Ca2+ ionophore A23187 can induce chloroplast rotation within a single nonirradiated Mougeotia cell. The induced turning was dependent on the position of ionophore application and Ca2+ in the external medium. The role of calmodulin in mediating light-induced chloroplast rotation in the alga Mougeotia was investigated by using the paired calmodulin-antagonist drugs W5-W7 and W12-W13. In each pair, the antagonist with the greater affinity for calmodulin had the greater inhibitor effect on the phytochrome-controlled light response. These results support the hypothesis that calcium functions as a chemical messenger to couple the stimulus of phytochrome photoactivation with physiological responses in plants.
Evidence for translational regulation of the activator of general amino acid control in yeast.
Authors: Hinnebusch A G
Published: May 2006

The GCN4 gene encodes a positive regulator of unlinked amino acid biosynthetic genes in yeast. I present evidence that the GCN4 gene is itself regulated by amino acid availability and that the regulation occurs at the translational level. A GCN4-lacZ fusion was used as a measure of the expression of GCN4 gene product. Starvation for histidine leads to derepression of the fusion enzyme in the wild type but not in a gcn2- strain. The gcn2- mutation does not reduce fusion transcript levels relative to wild type, suggesting that the product of GCN2 functions as an activator of GCN4 translation. The GCN4 transcript has a 5' leader that is approximately equal to 600 nucleotides long and contains four small open reading frames. A deletion of the small open reading frames results in constitutive derepression of fusion enzyme levels as the result of an approximately equal to 10-fold increase in the efficiency of translation of the fusion transcript. The deletion suppresses the requirement for GCN2 function. These results suggest that the GCN4 5' leader acts in cis to repress GCN4 translation and that GCN4 translation increases in response to amino acid starvation as the result of GCN2 antagonism of the repressing sequences in the GCN4 5' leader.
Adenosine selectively blocks parallel-fiber-mediated synaptic potentials in rat cerebellar cortex.
Authors: Kocsis J D , Eng D L , Bhisitkul R B
Published: May 2006

Electrophysiological techniques were used to study the efficacy of adenosine in modulating synaptic transmission mediated from convergent parallel- and climbing-fiber inputs to Purkinje cells. Our results indicate that adenosine application leads to selective blocking of parallel fiber-mediated synaptic activity but not of climbing fiber activity. Adenosine does not alter the action-potential excitability properties of the parallel fibers. However, application of gamma-aminobutyric acid (GABA), which directly affects Purkinje cell dendritic membranes [Malenka, R. C. & Kocsis, J. D. (1982) J. Neurophysiol. 48, 608-621], leads to reduction of both parallel- and climbing-fiber synaptic activity. These results support the proposals that adenosine receptors in the cerebellar cortex are selectively localized on the nonmyelinated parallel fibers and that the blocking action of adenosine is the result of a mechanism other than direct alteration of axon excitability.
Characterization of the genes specifying two metacyclic variable antigen types in Trypanosoma brucei rhodesiense.
Authors: Lenardo M J , Rice-Ficht A C , Kelly G , Esser K M , Donelson J E
Published: May 2006

Bloodstream trypanosomes evade the immune system of their mammalian host by sequentially expressing a large number of different variable surface glycoproteins (VSGs). In contrast, metacyclic trypanosomes, the final developmental stage in the tsetse fly, express a much more restricted set of VSGs. These metacyclic VSGs are the first to be exposed to the immune system of the mammalian host after infection and may offer the potential for the eventual development of a vaccine. We have identified cDNAs for two VSGs in cDNA libraries prepared from amplified metacyclic populations of Trypanosoma brucei rhodesiense and show that they correspond to two different metacyclic serotypes. Determination of the cDNA sequences shows that metacyclic VSG mRNAs are similar to VSG mRNAs expressed during the bloodstream stage. Southern blots demonstrate that the metacyclic VSG genes are located near chromosomal telomeres. No evidence of gene rearrangement associated with expression of these VSGs was found.
A viable simian virus 40 variant that carries a newly generated sequence reiteration in place of the normal duplicated enhancer element.
Authors: Swimmer C , Shenk T
Published: May 2006

A segment comprising the transcriptional enhancer elements was deleted from a recombinant plasmid carrying the simian virus 40 genome. The mutated viral chromosome was excised from the plasmid and propagated through several cycles of growth in monkey kidney cells. A variant was obtained that carried reiterations of sequences that span both sides of the deleted enhancer region. The mutant virus, dup1495, displays a lag in its growth kinetics as compared to its parent, but it ultimately generates wild-type yields. The mutant virus expresses early mRNAs at near-normal levels, and the reiterated sequence functioned in cis to enhance transformation of mouse cells by the herpesvirus thymidine kinase gene. Thus dup1495 reiterated segments encode enhancer activity even though their primary sequence is radically different from that of the normal simian virus 40 enhancer.
A purified fraction containing RNA polymerase I that can accurately transcribe rat ribosomal RNA gene.
Authors: Kurl R N , Rothblum L I , Jacob S T
Published: May 2006

Transcription of a cloned rat rDNA containing the transcription initiation region was studied using purified fractions derived from whole cell extract. The cell extract obtained from rat mammary adenocarcinoma cells was fractionated successively on DEAE-Sephadex and heparin-Sepharose columns. The fractions eluted by 175 mM (NH4)2SO4 (DE-B) from the DEAE-Sephadex column and those eluting with RNA polymerase I on the subsequent heparin-Sepharose column (HS-B) were used in a run-off transcription assay using Xho I-cleaved cloned rat rDNA. Both fractions that contained greater than 90% of the RNA polymerase I activity produced the anticipated 635-nucleotide-long transcript. Fractionation of fraction DE-B on a phosphocellulose column also resulted in a RNA polymerase I-containing complex that, by itself, could transcribe the rDNA accurately. The major polypeptides obtained after NaDodSO4/PAGE of fraction HS-B had Mr values of 190,000, 120,000, 65,000, 42,000, and 25,000, corresponding to the major subunits of RNA polymerase I. Purified nuclear RNA polymerase I did not produce the correct transcript. These studies have shown that a purified fraction containing RNA polymerase I from whole cells can support accurate transcription of rDNA.
Evidence that a major class of mouse endogenous long terminal repeats (LTRs) resulted from recombination between exogenous retroviral LTRs and similar LTR-like elements (LTR-IS).
Authors: Schmidt M , Glöggler K , Wirth T , Horak I
Published: May 2006

Two endogenous retroviral long terminal repeats (LTRs) were sequenced and compared to LTR-IS (a family of insertion-element-like sequences with structural features of solitary retroviral LTRs) and to Moloney murine leukemia virus DNA. The sequence comparisons revealed that the major difference between these two endogenous LTRs is a 190-base-pair segment which is also present in LTR-IS elements. Hybridization analysis of DNAs from several mouse species using specific probes shows linkage of the 190-base-pair segment to a LTR-IS specific fragment. It is concluded that the major class of endogenous LTRs has been generated by recombination between exogenous retroviral LTRs and LTR-IS sequences.
Synthesis and glycosylation of the common alpha subunit of human glycoprotein hormones in mouse cells.
Authors: Ramabhadran T V , Reitz B A , Tiemeier D C
Published: May 2006

The synthesis and the post-translational modification of the alpha subunit of human glycoprotein hormones have been studied in a mouse cell. A full-length cDNA coding for the human alpha subunit has been expressed in mouse C127 cells under the control of mouse metallothionein regulatory sequences, using a bovine papilloma virus vector. Stable clones secreting the alpha subunit into the medium have been obtained. Two intracellular forms of 22,000 Da and 21,000 Da have been detected. Pulse-chase experiments suggest that the 22,000-Da form is exported, while the 21,000-Da form appears to remain intracellular. The secreted form of the alpha subunit migrates as a broad peak between 22,000 and 30,000 Da, suggesting further modification of the intracellular form prior to secretion. Both the secreted and the intracellular forms incorporate glucosamine label, indicating that at least a portion of the modification observed here is in the form of glycosylation.
Inheritance of a secondary sexual character in Drosophila silvestris.
Authors: Carson H L , Lande R
Published: May 2006

Reciprocal crosses were carried out between laboratory stock specimens obtained from two races of Drosophila silvestris from the island of Hawaii that differ in a quantitative secondary sexual character. The race from the Hilo side of the island has a novel attribute, consisting of an extra row of cilia on the tibia of males, which is used during courtship. With regard to this character, sex-linked genes contribute about 30% of the difference, and the remaining 70% of the difference between the races is produced by genes on at least two autosomes. The novel character appears to have been the outcome of altered sexual selection in the Hilo-side race. In an altered genetic environment, resulting from a founder event or random genetic drift, sexual selection may take a new direction. Such a shift may serve as a model for incipient speciation.
Regulation of polyomavirus transcription by large tumor antigen.
Authors: Farmerie W G , Folk W R
Published: May 2006

We have analyzed the regulation of viral transcription by the large tumor antigen in cells infected by several viable deletion and insertion mutants of polyomavirus. We find that deletion of the early promoter "TATA box" and associated large tumor antigen binding site has only a small effect on the balance of early and late mRNAs. Furthermore, transcription of a polyomavirus containing a heterologous adenovirus promoter in place of the normal TATA box and cap sites is regulated by the large tumor antigen. We conclude that repression of polyomavirus early transcription cannot occur simply by binding of the large tumor antigen to DNA sequences at the site of transcription initiation and must involve the interaction of the large tumor antigen binding at other sites.
Sequence of the bacteriophage SP01 gene coding for transcription factor 1, a viral homologue of the bacterial type II DNA-binding proteins.
Authors: Greene J R , Brennan S M , Andrew D J , Thompson C C , Richards S H , Heinrikson R L , Geiduschek E P
Published: May 2006

The Bacillus subtilis phage SP01, whose DNA contains 5-hydroxymethyluracil (hmUra) in place of thymine, codes for an abundant, small, basic protein called TF1. TF1 binds preferentially to hydroxymethyluracil-containing DNA and thereby selectively inhibits transcription of such DNA in vitro. The gene for TF1 has been sequenced. We find that this viral protein is a homologue of the ubiquitous bacterial type II DNA-binding proteins. The three-dimensional structure of one of these bacterial proteins has recently been determined. We are able to discern common as well as distinctive features in the amino acid sequence and the three-dimensional structure of the homologous viral protein.
Isometric embeddings of graphs
Authors: Graham R. L. , Winkler P. M.
Published: May 2006

We prove that any finite undirected graph can be canonically embedded isometrically into a maximum cartesian product of irreducible factors.
Detection of superoxide generated by endothelial cells.
Authors: Rosen G M , Freeman B A
Published: May 2006

Superoxide and lipid free-radical generation in cultured endothelial cells treated with menadione or nitrazepam were measured using electron paramagnetic resonance spectroscopy. Superoxide was detected both intracellularly and extracellularly. Extracellular generation of superoxide and hydrogen peroxide was also measured, either by spectrophotometric measurement of succinoylated cytochrome c reduction or by polarography. Extracellular superoxide was generated due to reduced menadione diffusing across the plasma membrane and reacting with oxygen to generate superoxide in the medium. Increased intracellular oxygen tension favored intracellular oxidation of reduced menadione, thus decreasing diffusion of reduced menadione from the cells and, hence, decreasing extracellular superoxide production. The nitro anion free radical of reduced nitrazepam, which cannot cross the plasma membrane, did not generate detectable extracellular superoxide. Our results show that intracellular superoxide can be spin-trapped using 5,5-dimethyl-1-pyrroline-1-oxide and that secondary free-radical injury to membrane lipids, due to excess production of partially reduced species of oxygen by intact cells, can be detected by spin-trapping lipid free radicals with phenyl N-tert-butylnitrone.
Isolated uterine nuclei and cytosol receptors of aged rats exhibit impaired estrogenic stimulation of RNA polymerase II.
Authors: Haji M , Chuknyiska R S , Roth G S
Published: May 2006

An in vitro cell-free system of uterine nuclei and cytosol receptors has been used to analyze the effects of aging on estrogen stimulation of RNA polymerase II activity. By using fixed concentrations of nuclei and cytoplasmic receptor--estrogen complexes (R-E2), it was found that mature nuclei are 3 times more efficient (155% vs. 57%) than old ones for stimulation of polymerase activity by mature R-E2. Meanwhile, mature R-E2 are 5 times more efficient (155% vs. 31%) than old ones in supporting such stimulation in mature nuclei. Stimulation by old cytosol R-E2 is so poor that it is essentially unaffected by nuclear age (31% with both mature and old nuclei). Finally, equimolar mixtures of mature and old cytosol R-E2 stimulate polymerase II activity in mature nuclei by 77%, a value intermediate between mature and old cytosols used separately. These results indicate that both nuclei and cytosol from old uteri are deficient in their ability to support estrogenic stimulation of RNA polymerase II.
Intact proenkephalin is the major enkephalin-containing peptide produced in rat adrenal glands after denervation.
Authors: Fleminger G , Howells R D , Kilpatrick D L , Udenfriend S
Published: May 2006

We have shown previously that enkephalin-containing peptides of high molecular weight increase 10- to 15-fold 3 days after adrenal denervation and have suggested that much of this material may represent newly synthesized proenkephalin. In this paper we report that most of the material appearing after denervation is indeed intact proenkephalin. The putative proenkephalin was partially purified by gel filtration chromatography and HPLC. A molecular size of about 25 kDa was estimated by gel filtration. On HPLC, the putative proenkephalin was eluted at a much higher propanol concentration than the 18.2-kDa enkephalin-containing peptide isolated previously. Treatment of this putative proenkephalin with endoproteinase Lys-C showed it contain [Met]-enkephalin, [Leu]enkephalin, [Met]enkephalin-Arg6-Phe7, and [Met]enkephalin-Arg6-Gly7-Leu8 in the same ratios as are found in proenkephalin as deduced from sequencing of proenkephalin cDNA.
Transcripts of human HLA gene fragments lacking the 5'-terminal region in transfected mouse cells.
Authors: Yoshie O , Schmidt H , Lengyel P , Reddy E S , Morgan W R , Weissman S M
Published: May 2006

Clones of mouse L cells transfected with a human HLA-B7 gene fragment lacking the 5' segment of exon 2 and all upstream sequences express HLA-specific transcripts of various lengths. These include species that correspond in size to full-length HLA-B7 mRNA. The level of these transcripts is increased in cells treated with interferon. It is probable that the full-length transcripts arise as a result of the linkage of the HLA-B7 gene fragments with DNA segments providing transcription initiation or polyadenylylation signals.
Receptor-mediated activation of a phospholipase A2 in rabbit neutrophil plasma membrane.
Authors: Bormann B J , Huang C K , Mackin W M , Becker E L
Published: May 2006

Using the exogenous substrate [1-14C]oleate-labeled autoclaved Escherichia coli, we have demonstrated that the chemotactic factors fMet-Leu-Phe, complement component C5a, and leukotriene B4 [(5S,12R)-dihydroxy-6-cis,8-trans,11-trans,14-cis-icosatetraenoic acid] stimulate a phospholipase A2 of isolated plasma membranes of rabbit peritoneal neutrophils. Each of the chemotactic factors shows a biphasic concentration dependence with the optimal concentrations occurring at 1, 10, and 0.1 nM, respectively. The specific antagonists of fMet-Leu-Phe binding, carbobenzoxy-Phe-Met and t-butoxycarbonyl-Phe-Leu-Phe, effectively block the stimulation by fMet-Leu-Phe, indicating that the activation is receptor mediated. delta 6-trans-leukotriene [(5S-12R)-dihydroxy-all-trans-6,8,10,14-icosatetraenoic acid], a biologically inactive stereoisomer of leukotriene B4, does not stimulate phospholipase activity, suggesting that the enhancement by leukotriene B4 is also receptor mediated. The unstimulated and activated phospholipase exhibit a broad range of maximal activity between pH 7.0 and pH 8.5, both with an optimal pH of 8.5. The activation of the phospholipase by fMet-Leu-Phe is completely calcium dependent; no increase in activity is demonstrable if fMet-Leu-Phe is added in the absence of exogenous calcium or in the presence of EGTA. In contrast, the unstimulated plasma membrane activity of the phospholipase, as well as the activity arising after stimulation, is relatively insensitive to the concentration of calcium, being inhibited by less than 50% in the presence of 10 mM EGTA. The phospholipase hydrolyzes 1-[1-14C]palmitoyl-2-acyl-sn-glycerophosphoethanolamine to form only radioactive lysophosphatidylethanolamine as the product, indicating that the enzyme has an A2 specificity.
Direct observation of delta-crystallin accumulation by laser light-scattering spectroscopy in the chicken embryo lens.
Authors: Sun S T , Tanaka T , Nishio I , Peetermans J , Maizel J V , Piatigorsky J
Published: May 2006

By using the technique of laser light-scattering spectroscopy, direct observation has been made on the intracellular accumulation of a crystallin protein within the cells of chicken embryo lens during the process of development. Appearance of delta-crystallin has been detected as early as day 4, and its concentration reaches a plateau at day 19. The measurements constitute a noninvasive determination of accumulation of protein molecules that specifically characterize the process of cell differentiation.
Origin of the beta S-globin gene in blacks: the contribution of recurrent mutation or gene conversion or both.
Authors: Antonarakis S E , Boehm C D , Serjeant G R , Theisen C E , Dover G J , Kazazian H H
Published: May 2006

In order to investigate the origin(s) of the mutation(s) leading to the beta S-globin gene in North American populations of African ancestry, we analyzed DNA polymorphisms in the beta-globin gene cluster in a large number of both beta A- and beta S-globin gene-bearing chromosomes in U.S. and Jamaican Blacks. We found 16 different haplotypes of polymorphic sites associated with 170 beta S-globin gene-bearing chromosomes. The three most common beta S haplotypes, which account for 151/170 of the beta S-globin gene-bearing chromosomes, are only rarely seen in the chromosomes bearing the beta A-globin gene in these populations (6/47). Two observations suggest multiple origins or interallelic gene conversion, or both, of the beta S mutation. First, the mutation is present in all three beta-globin gene frameworks. Second, the beta S haplotypes can be divided into four groups, each of which cannot be derived from any other by less than two crossing-over events. In summary, our observation of the beta S mutation on 16 different haplotypes in African populations can be best explained by (i) a number of simple recombination events 5' to the beta-globin gene and (ii) up to four independent mutations and/or interallelic gene conversions.
Inhibition of opiate receptor-mediated signal transmission by rabies virus in persistently infected NG-108-15 mouse neuroblastoma-rat glioma hybrid cells.
Authors: Koschel K , Münzel P
Published: May 2006

Acute and persistent rabies virus infection of mouse neuroblastoma-rat glioma hybrid cells (NG-108-15) results in a loss of the normal inhibiting function of opiates via the opiate receptor on hormone-stimulated adenylate cyclase activity. Previous studies of these persistently infected cells have shown a decrease in the affinity of the opiate receptors for agonists without any change in the number of these receptors. We now demonstrate that persistently infected cells are unable to couple the opiate receptors to the inhibitory regulatory protein Ni of the adenylate cyclase, as measured by the loss of stimulation of the GTPase activity of this protein. However, the unstimulated basal GTPase activities of the regulatory components Ni and Ns are unchanged in the persistently infected cells. These studies also reveal a disorder of the stimulation of the adenylate cyclase by GTP or fluoride via the stimulating regulatory G/F protein (Ns) in persistently infected cells, whereas direct stimulation of the catalytic subunit of the adenylate cyclase by forskolin remains unchanged. Therefore, there are different points of dysfunction caused by the persistent rabies infection in the signal pathway from the opiate receptor to the adenylate cyclase and from the stimulating Ns protein to the enzyme: (i) opiate receptor binding is reduced by a decrease of agonist affinity (previously published data), (ii) the stimulation of GTPase activity of the inhibiting regulatory component Ni of the adenylate cyclase system is inhibited, and (iii) the signal pathway from the stimulating regulatory component of the adenylate cyclase system to the unchanged activity of the catalytic subunit is defective.
3' editing of mRNAs: sequence requirements and involvement of a 60-nucleotide RNA in maturation of histone mRNA precursors.
Authors: Birchmeier C , Schümperli D , Sconzo G , Birnstiel M L
Published: May 2006

In vitro-synthesized transcripts of the sea urchin histone H2A gene with 3' extensions are efficiently and rapidly processed to H2A mRNA with faithful 3' ends in Xenopus laevis oocyte nuclei. Processing requires the presence of a histone-specific dyad symmetry element and of H2A-proximal spacer sequences in the precursor RNA. In DNA injection experiments with a processing-deficient H2A mutant, the transcription products appear to terminate heterogeneously in the first 100-200 base pairs of the post-H2A spacer. Processing of synthetic H3 RNA precursors requires the prior injection of a 60-nucleotide RNA from sea urchin embryos that seems to be a component of a small nuclear ribonucleoprotein.
Electron nuclear double resonance evidence supporting a monomeric nature for P700
+
in spinach chloroplasts
Authors: O'Malley Pádraig J. , Babcock Gerald T.
Published: May 2006

Proton electron nuclear double resonance (ENDOR) spectra of P700
+
in spinach chloroplasts and in photosystem I particles have been obtained and compared with the corresponding ENDOR spectrum of monomeric chlorophyl
a
+
(Chl
a
+
) cation radical. The hyperfine couplings for P700
+
can be interpreted in terms of those expected for a monomer Chl
a
+
radical. The reduction in α-carbon spin densities observed for the
in vivo
species when compared to the
in vitro
radical is attributed to differences in the composition of the ground-state orbital for the two systems. For P700
+
, a mixture of 75% D
0
/25% D
1
, in which D
0
and D
1
represent the ground-and first excited-state orbitals calculated by Petke
et al.
for Chl
a
+
[Petke, J. D., Maggiora, G. M., Shipman, L. L. & Christoffersen, R. E. (1980)
Photochem. Photobiol.
31, 243-257], gives good agreement between calculated and experimental spin-density reduction factors. Interaction of the pigment ion with its protein environment such as through ligation of the central Mg atom, hydrogen bonding to the 9-keto-carbonyl group, and electrostatic interactions with charged amino acid residues are proposed as factors responsible for the lowering in energy of the D
1
level
in vivo
. Combined with similar previous proposals for P680
+
of photosystem II, the data suggest that both primary donor cation radicals of green plant photosynthesis can be viewed as monomeric Chl
a
+
species in which the D
1
orbital makes a significant contribution to the spin-density distribution.
Specificity of polyamine requirements for the replication and maintenance of different double-stranded RNA plasmids in Saccharomyces cerevisiae.
Authors: Tyagi A K , Wickner R B , Tabor C W , Tabor H
Published: May 2006

We have shown previously that the M1 double-stranded (ds) RNA (i.e., the killer plasmid [KIL-k1]) that codes for a protein toxin requires spermidine or spermine for its replication. We now report that replication of two other ds RNA plasmids of yeast also requires polyamines: (i) M2 ds RNA [( KIL-k2]) and (ii) L-A-E, a ds RNA plasmid carrying the non-Mendelian genetic element [EXL]. Putrescine alone is sufficient to maintain L-A-E but is not sufficient to maintain either M1 ds RNA or M2 ds RNA, which require either spermidine or spermine. Once M1 or M2 or L-A-E is lost, it cannot be restored by the addition of polyamines. In contrast, L-A-HN, a ds RNA molecule that carries the cytoplasmic genes [HOK] and [NEX], is not lost during polyamine deprivation. It is striking that polyamine deprivation differentially affects L-A-E and L-A-HN, even though these two ds RNA molecules have more than 99% homology. L-C, which is the same size as L-A but very different in sequence, is also not lost on polyamine starvation.
Post-translational activation introduces a free radical into pyruvate formate-lyase.
Authors: Knappe J , Neugebauer F A , Blaschkowski H P , Gänzler M
Published: May 2006

Pyruvate formate-lyase (formate acetyltransferase; EC 2.3.1.54) of Escherichia coli cells is post-translationally interconverted between inactive and active forms. Conversion of the inactive to the active form is catalyzed by an Fe2+-dependent activating enzyme and requires adenosylmethionine and dihydroflavodoxin. This process is shown here to introduce a paramagnetic moiety into the structure of pyruvate formate-lyase. It displays an EPR signal at g = 2 with a doublet splitting of 1.5 mT and could comprise an organic free radical located on an amino acid residue of the polypeptide chain. Hypophosphite was discovered as a specific reagent that destroys both the enzyme radical and the enzyme activity; it becomes covalently bound to the protein. The enzymatic generation of the radical, which is linked to adenosylmethionine cleavage into 5'-deoxyadenosine and methionine, possibly occurs through an Fe-adenosyl complex. These results suggest a radical mechanism for the catalytic cycle of pyruvate formate-lyase.
Rhizobium
free-living nitrogen fixation occurs in specialized nongrowing cells
Authors: Ludwig Robert A.
Published: May 2006

A model for free-living N
2
fixation by
Rhizobium
sp. RC3200 is presented that asserts that this process occurs in nongrowing cells. Cultures containing mixed populations of cell types, N
2
-fixing and vegetative, grow cooperatively. In nitrogen-limited liquid suspension cultures, cooperative growth occurs by means of ammonium that is produced and exported by nongrowing, N
2
-fixing cells and transported to vegetative cells. This model implies prokaryotic differentiation: the creation of metabolically specialized cells, terminally nonviable, that functionally cooperate in a higher cell order. Here, the switch to a Rhizobium N
2
-fixing cell state is regulated by both O
2
and utilizable nitrogen.
Natural fibroin genes purified without using cloning procedures from fibroin-producing and -nonproducing tissues reveal indistinguishable structure and function.
Authors: Tsujimoto Y , Suzuki Y
Published: May 2006

Natural fibroin genes were purified from total DNA extracted from the fibroin-producer cells (posterior silk gland) and -nonproducer cells (middle silk gland or pupa) by two cycles of CsCl/actinomycin D centrifugation followed by sucrose density gradient centrifugation. Purity of the final samples was greater than 14%. DNA sequences of these natural genes between positions -171 and +104 were identical and showed no sign of base modification as assayed by the method of Maxam and Gilbert. The determined sequence includes the promoter and a major part of the modulator. When assayed in an in vitro transcription system prepared from middle silk gland, template activities of the purified natural fibroin genes from the producer and the nonproducer were indistinguishable from that of cloned fibroin DNA. Digestion and blotting of total genomic DNAs with several restriction enzymes that recognize methylation changes on DNA revealed no difference of hybridization pattern of fibroin DNAs in a region from -650 to +326 between the producer and nonproducer. Thus, it is unlikely that the differential transcription of the fibroin gene is controlled by a change of base modification in the regions of transcription signals.
Albumin extinction without methylation of its gene.
Authors: Ott M O , Sperling L , Weiss M C
Published: May 2006

In earlier work we identified at the 5' end of the rat albumin gene an Msp I site whose undermethylation appears to be necessary but not sufficient for stable expression of the gene in rat hepatoma cells. Here, we ask whether the block to expression of albumin production, which occurs when rat hepatoma cells are hybridized with cells that do not produce the protein, could be the result of de novo methylation of this site. In two types of somatic hybrids, rat hepatoma-mouse L cell fibroblasts, and rat hepatoma-dedifferentiated variant rat hepatoma cells, extinction occurs and is maintained during the first 5-15 generations after fusion. During this time the Msp I site of the now inactive rat albumin gene remained unmethylated.
Differential methylation of hypoxanthine phosphoribosyltransferase genes on active and inactive human X chromosomes.
Authors: Yen P H , Patel P , Chinault A C , Mohandas T , Shapiro L J
Published: May 2006

Previous theoretical considerations and some experimental data have suggested a role for DNA methylation in the maintenance of mammalian X chromosome inactivation. The isolation of specific X-encoded sequences makes it possible to investigate this hypothesis directly. We have used cloned fragments of the human hypoxanthine phosphoribosyltransferase (HPRT) gene and methylation-sensitive restriction enzymes to study methylation patterns in genomic DNA of individuals with different numbers of X chromosomes and in somatic cell hybrid lines containing human X chromosomes that are either active or inactive or have been reactivated by treatment with 5-azacytidine. The results of these analyses show that there is hypomethylation of active X chromosomes relative to inactive X chromosomes in the 5' region of this gene. In the middle region of the gene, however, a site that is consistently undermethylated on inactive X chromosomes was identified. Taken together, the data suggest that the overall pattern of methylation, rather than methylation of specific sites, plays a role in the maintenance of X chromosome inactivation.
Are DNA spacers relics of gene amplification events?
Authors: Wong W M , Abrahamson J L , Nazar R N
Published: May 2006

The genome of a thermophilic fungus, Thermomyces lanuginosus, contains a tandemly arranged cluster of sequences that are approximately equal to 50% homologous with the cytoplasmic 5S RNA and that are selectable by hybridization techniques. Unlike typical pseudogenes, these sequences are not truncated; rather, they bear a limited sequence homology with the entire length of the 5S RNA and are oriented end to end without significant intervening sequences. We suggest that these are gene relics that were duplicated by a rolling circle-like mechanism and that have evolutionarily drifted to become gene spacers. Accordingly, we raise the possibility that this offers a fortuitous glimpse at the origins for many of the gene spacers in the eukaryotic genome.
Chromosomal location of the co-expressed human skeletal and cardiac actin genes.
Authors: Gunning P , Ponte P , Kedes L , Eddy R , Shows T
Published: May 2006

We have examined the relationship between chromosomal location and regulation of the two human genes encoding the sarcomeric muscle actins. The human genes encoding skeletal alpha-actin and cardiac alpha-actin are co-expressed in both human skeletal muscle and heart. We have subcloned a single-copy DNA fragment from an intervening sequence in the human cardiac alpha-actin gene and a single-copy DNA sequence from the 3' untranslated region of a human skeletal alpha-actin cDNA. Using these two gene-specific probes, we examined DNA isolated from human-mouse somatic cell hybrid lines segregating human chromosomes. We observed the segregation of restriction endonuclease-generated DNA cleavage fragments that hybridize to the two probes. The two striated muscle genes do not co-segregate and are on different autosomes. The human cardiac alpha-actin gene (ACTC) is on chromosome 15 in the q11----qter region whereas the skeletal alpha-actin gene (ACTSK) is on chromosome 1 in the p21----qter region. The co-expression of these two genes is not a function of chromosomal linkage. Neither of these muscle genes can be the primary target resulting in X-linked muscular dystrophies.
Behavioral and neurochemical differentiation of specific projections in the septal-hippocampal cholinergic pathway of the rat.
Authors: Blaker W D , Peruzzi G , Costa E
Published: May 2006

In the rat, an intraseptal injection of the gamma-aminobutyric acid (GABA) agonist muscimol decreases the turnover rate of acetylcholine in the hippocampus and, during extinction of a food-reinforced lever-press response, increases extinction responding in a dose-dependent manner. Intraseptal beta-endorphin decreases the turnover rate of hippocampal acetylcholine through activation of septal GABAergic interneurons and increases extinction responding. On the other hand, intraseptal substance P, which decreases the turnover rate of hippocampal acetylcholine in a manner unrelated to septal GABAergic mechanisms, fails to increase extinction responding. The turnover rate of acetylcholine in various hippocampal regions after intraseptal injection of muscimol and substance P was also studied. Muscimol decreases the acetylcholine turnover rate only in the ventral hippocampus, whereas substance P decreases it only in the dorsal hippocampus. We hypothesize that a lowering in the cholinergic input to the ventral hippocampus is capable of increasing extinction responding, whereas a decrease in the input to the dorsal hippocampus is without such an effect. Hence, the cholinergic projections to the two hippocampal areas are modulated by different transmitter systems and have different physiological functions.