bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in the stability of specific mRNA species in response to growth stage in Bacillus subtilis.
Authors: Resnekov O , Rutberg L , von Gabain A
Published: May 2006

In this study we compared the cellular concentrations and stability of the mRNA transcribed from the aprE (subtilisin) gene (a gene preferentially expressed in stationary growth phase) with those of a vegetative mRNA, succinate dehydrogenase (SDH) mRNA. The subtilisin transcript was shown to be at least 3 times more stable in early stationary phase than it is 2 hr further into stationary phase. When cells were shifted from maximum expression of the subtilisin transcript in stationary phase to physiological conditions, which allowed for the resumption of vegetative growth, the cellular concentration of the subtilisin mRNA decreased rapidly. We conclude that mRNA degradation is one of the means by which the cellular concentrations of the SDH and subtilisin transcripts are adjusted in response to growth stage.
Renaturation of complementary DNA strands mediated by purified mammalian heterogeneous nuclear ribonucleoprotein A1 protein: implications for a mechanism for rapid molecular assembly.
Authors: Pontius B W , Berg P
Published: May 2006

Purified heterogeneous nuclear ribonucleoprotein (hnRNP) A1 protein, which is found in vivo associated with heterogeneous nuclear RNA (hnRNA), promotes the rapid renaturation of nucleic acid strands. Maximal renaturation activity requires the glycine-rich carboxyl-terminal one-third of the protein, although the amino-terminal two-thirds also has activity. The A1-mediated reaction is second-order with respect to complementary DNA concentration, and the renaturation rate constant at 37 degrees C with A1 is about 3000-fold greater than in the absence of the protein. At 60 degrees C, the A1-mediated renaturation rate is even faster, and is about 300-fold greater than protein-free reactions carried out at 68 degrees C in 1 M NaCl. Provided that sufficient A1 protein is present to coat all strands in solution, the presence of nonhomologous, single-stranded DNA does not significantly inhibit the reaction. Moreover, renaturation of short strands to their complement contained in very long strands is nearly as efficient as between two short strands. These results indicate that A1 may be useful for procedures that rely on nucleic acid renaturation. We propose that A1 promotes rapid renaturation primarily by reducing the entropic barrier of bimolecular strand association through relatively transient interactions between A1-coated strands. Such interactions, mediated by flexible repeating domains, may act generally to increase the association kinetics of highly specific molecular assemblies in processes such as RNA maturation, transcription, translation, and transport.
Hunter syndrome: isolation of an iduronate-2-sulfatase cDNA clone and analysis of patient DNA.
Authors: Wilson P J , Morris C P , Anson D S , Occhiodoro T , Bielicki J , Clements P R , Hopwood J J
Published: May 2006

Iduronate 2-sulfatase (IDS, EC 3.1.6.13) is required for the lysosomal degradation of heparan sulfate and dermatan sulfate. Mutations causing IDS deficiency in humans result in the lysosomal storage of these glycosaminoglycans and Hunter syndrome, an X chromosome-linked disease. We have isolated and sequenced a 2.3-kilobase cDNA clone coding for the entire sequence of human IDS. Analysis of the deduced 550-amino acid IDS precursor sequence indicates that IDS has a 25-amino acid amino-terminal signal sequence, followed by 8 amino acids that are removed from the proprotein. An internal proteolytic cleavage occurs to produce the mature IDS present in human liver shown to contain a 42-kDa polypeptide N-terminal to a 14-kDa polypeptide. The IDS sequence has strong sequence homology with other sulfatases (such as sea urchin arylsulfatase, human arylsulfatases A, B, and C, and human glucosamine 6-sulfatase), suggesting that the sulfatases comprise an evolutionarily related family of genes that arose by gene duplication and divergent evolution. The arylsulfatases have a greater homology with each other than with the non-arylsulfatases (IDS and glucosamine 6-sulfatase). The IDS cDNA detected RNA species of 5.7, 5.4, 2.1, and 1.4 kilobases in human placental RNA and revealed structural alterations and gross deletions of the IDS gene in many of the clinically severe Hunter syndrome patients studied.
Imaging cytoskeleton--mitochondrial membrane attachments by embedment-free electron microscopy of saponin-extracted cells.
Authors: Lin A , Krockmalnic G , Penman S
Published: May 2006

Embedment-free electron microscopy images the cytoskeleton and nuclear matrix, which are very difficult to visualize in conventional electron micrographs. However, to be effective, cell structures must be depleted of soluble proteins, which otherwise shroud cell architecture. Nonionic detergents effect this extraction, releasing soluble proteins but also destroying all membranes. Saponin can permeabilize plasma membranes, releasing soluble proteins while preserving many cytoplasmic membranes. Stereoscopic electron microscopy of resinless sections shows the many connections of the cytoskeleton to mitochondrial membranes.
A genetic study of the anesthetic response: mutants of Drosophila melanogaster altered in sensitivity to halothane.
Authors: Krishnan K S , Nash H A
Published: May 2006

In an attempt to identify genes that control or encode the targets of general anesthetics, we have chemically mutagenized fruit flies and selected four lines that show an abnormal response to the volatile anesthetic halothane. Specifically, about 2-fold higher concentrations of halothane are required to induce the loss of motor control in the mutant flies. Fine mapping of two isolates indicates that they alter a previously uncharacterized gene of Drosophila. In the absence of anesthetics, these mutants display alterations of behavior that imply changes in the adult and the larval neuromuscular system.
Enzymatic aminoacylation of an eight-base-pair microhelix with histidine.
Authors: Francklyn C , Schimmel P
Published: May 2006

The major determinant for the identity of alanine tRNAs is a single base pair in the acceptor helix that is proximal to the site of amino acid attachment. A 7-base-pair microhelix that recreates the acceptor helix can be charged with alanine. No other examples of charging of small helices with specific amino acids have been reported, to our knowledge. We show here that a 13-base-pair and an 8-base-pair hairpin helix that reconstruct a domain and subdomain, respectively, of histidine tRNAs can be charged with histidine. We also show that transplantation of a base pair that is unique to histidine tRNAs is sufficient to consider histidine acceptance on a domain and subdomain of alanine tRNA. Both alanine and histidine aminoacyl-tRNA synthetases retain specificity for their cognate synthetic substrates. Alanine- and histidine-specific microhelices may resemble a system that arose early in the evolution of charging and coding.
An essential arginine residue for initiation of protein-primed DNA replication.
Authors: Hsieh J C , Yoo S K , Ito J
Published: May 2006

A group of proteins that act as primers for initiation of linear DNA replication are called DNA-terminal proteins (terminal proteins). We have found a short stretch of conserved amino acid sequence among the terminal proteins from six different sources. The location of this sequence motif is also similar among the different terminal-proteins. To determine the functional role of this terminal-protein domain in DNA replication, we have studied the bacteriophage PRD1 system. The PRD1 terminal protein and DNA polymerase genes were cloned into expression vectors, and the recombinant plasmids were used for constructing PRD1 terminal protein mutants. Site-directed mutagenesis and functional analysis showed that one of the two arginines (Arg-174) in the conserved sequence is critical for the initiation complex-forming activity of the PRD1 terminal protein. Replacement of Arg-174 by noncharged amino acids resulted in nonfunctional terminal protein. Phenylglyoxal, an alpha-dicarbonyl compound that reacts with the guanidino group of arginine, inhibits initiation complex formation between PRD1 terminal protein and dGMP. On the basis of these results, we propose that Arg-174 represents, at least in part, the binding site for phosphate groups of dGTP.
Effect of excision repair by diploid human fibroblasts on the kinds and locations of mutations induced by (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene in the coding region of the HPRT gene.
Authors: Chen R H , Maher V M , McCormick J J
Published: May 2006

(+/-)-7 beta,8 alpha-Dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE) is a direct-acting carcinogen that forms DNA adducts only with purines, predominantly (greater than 95%) with guanine. To investigate the effect of nucleotide excision repair on the kinds and locations (spectra) of mutations induced in diploid human fibroblasts by BPDE, we synchronized cells and exposed them to BPDE either at the beginning of S phase just when the target gene hypoxanthine (guanine) phosphoribosyltransferase (HPRT) is replicated or 12 hr prior to the beginning of S phase (early G1 phase). Clones resistant to 6-thioguanine were isolated, and the mRNA in lysates of 100-500 cells from each mutant clone was used to synthesize cDNA. HPRT cDNA was amplified 10(11)-fold by the polymerase chain reaction and then sequenced directly. The mutants derived from the two populations did not differ in the kinds of mutations; 19/20 of the base substitutions in cells taken from S phase and 19/19 of those from G1 phase involved G.C base pairs, predominantly G.C----T.A. However, they differed significantly in the distribution of the mutations in the coding region of the gene. In the cells from G1 phase, 29% of the mutations were clustered within a unique run of six guanine bases; in the S-phase cells, only 4% were located there. Assuming that the premutagenic BPDE-induced lesions involved purines, in the cells treated at the beginning of S phase, 24% of these lesions were located in the transcribed strand, whereas in the G1-treated cells, none were. This suggests that in the HPRT gene of diploid human cells excision repair of BPDE adducts occurs preferentially on the transcribed strand.
The mtr locus is a two-gene operon required for transcription attenuation in the trp operon of Bacillus subtilis.
Authors: Gollnick P , Ishino S , Kuroda M I , Henner D J , Yanofsky C
Published: May 2006

We have cloned and characterized the mtr operon of Bacillus subtilis. This operon encodes a presumed RNA-binding regulatory protein that is required for attenuation control of the trp operon. We have shown that the mtr operon consists of two structural genes, mtrA and mtrB, predicted to encode 22-kDa and 8-kDa polypeptides, respectively. MtrB shows homology with RegA, an RNA-binding regulatory protein of bacteriophage T4. The lesions in several mtr mutants were localized to mtrB or the putative mtr promoter. Several mtrB alleles were dominant to mtr+, suggesting that the regulatory factor is a multimeric protein. The in vivo action of the mtrA and mtrB gene products was analyzed in an E. coli strain containing a trpE-lacZ gene fusion under control of the B. subtilis trp promoter/attenuator region. Both MtrA and MtrB were necessary for regulation of beta-galactosidase production.
Allelic loss of chromosomes 16q and 10q in human prostate cancer.
Authors: Carter B S , Ewing C M , Ward W S , Treiger B F , Aalders T W , Schalken J A , Epstein J I , Isaacs W B
Published: May 2006

Recent advances in understanding the molecular genetics of common adult tumors have indicated that multiple genetic alterations including the activation of oncogenes and the inactivation of tumor suppressor genes are important in the pathogenesis of these tumors. Loss of heterozygosity is a hallmark of tumor suppressor gene inactivation and has been used to identify chromosomal regions that contain these genes. We have examined allelic loss in the most common tumor in men, prostate cancer. Twenty-eight prostate cancer specimens have been examined for loss of heterozygosity at 11 different chromosomal arms including 3p, 7q, 9q, 10p, 10q, 11p, 13q, 16p, 16q, 17p, and 18q. Fifty-four percent (13/24) of clinically localized tumors and 4 of 4 metastatic tumors showed loss of heterozygosity on at least one chromosome. Chromosomes 16q and 10q exhibited the highest frequency of loss of heterozygosity with 30% of tumors showing loss at these chromosomes. These data demonstrate that allelic loss is a common event in prostate cancer and suggest that chromosomes 16q and 10q may contain the sites of tumor suppressor genes important in the pathogenesis of human prostate cancer.
Microtubule dynamics in living dividing plant cells: confocal imaging of microinjected fluorescent brain tubulin.
Authors: Zhang D , Wadsworth P , Hepler P K
Published: May 2006

Carboxyfluorescein-labeled brain tubulin has been microinjected into stamen hair cells of Tradescantia, and its distribution during mitosis and cytokinesis was examined using confocal laser scanning fluorescence microscopy. The results show that brain tubulin incorporates into plant microtubules and is utilized throughout mitosis and cytokinesis. Microtubule structures that incorporate brain tubulin include the preprophase band, the perinuclear sheath at late prophase, the kinetochore fibers during prometaphase, metaphase, and anaphase, the interzone spindle during anaphase, and finally the phragmoplast during late anaphase and telophase. All of these microtubule-containing structures and, notably, their transitions from one to another have been observed in single live cells progressing through mitosis and cytokinesis.
Interindividual concordance of methylation profiles in human genes for tumor necrosis factors alpha and beta.
Authors: Kochanek S , Toth M , Dehmel A , Renz D , Doerfler W
Published: May 2006

The DNA in mammalian genomes is characterized by complex patterns of DNA methylation that reflect the states of all genetic activities of that genome. The modified nucleotide 5-methyldeoxycytidine (5mdC) can affect the interactions of specific proteins with DNA sequence motifs. The most extensively studied effect of sequence-specific methylations is that of the long-term silencing of eukaryotic (mammalian) promoters. We have initiated studies on the methylation status of parts of the human genome to view patterns of DNA methylation as indicators for genetic activities. In this report, analyses using both restriction enzyme--Southern blotting and the very precise genomic sequencing technique have been done. The genes for tumor necrosis factors (TNF) alpha and beta--in particular, their 5'-upstream and promoter regions--have been investigated in DNA isolated from human lymphocytes, granulocytes, and sperm. The results are characterized by a remarkable interindividual concordance of DNA methylation in specific human cell types. The patterns are identical in the DNA from one cell type for different individuals even of different genetic origins but different in the DNA from different cell types. As an example, in the DNA from human granulocytes of 15 different individuals (ages 20-48 yr, both sexes), 5mdC residues have been localized by the genomic sequencing technique in three identical sequence positions in the 5'-upstream region and in one downstream position of the gene encoding TNF-alpha. The promoter of this gene is free of 5mdC, and TNF-alpha is expressed in human granulocytes. The TNF-beta promoter is methylated in granulocytes from 9 different individuals, and TNF-beta is not expressed. In human lymphocytes, the main source of TNF-beta, the TNF-beta promoter is free of 5mdC residues. All 5'-CG-3' sites studied in the TNF-alpha and -beta genes are methylated in DNA from human sperm. In human cell lines HL-60, Jurkat, and RPMI 1788, the extent of DNA methylation in TNF-alpha and -beta genes has also been studied.
Molecular cloning of the large subunit of transforming growth factor type beta masking protein and expression of the mRNA in various rat tissues.
Authors: Tsuji T , Okada F , Yamaguchi K , Nakamura T
Published: May 2006

Masking protein (MP), which neutralizes the activity of transforming growth factor type beta 1 (TGF-beta 1), is composed of a dimeric N-terminal part of a TGF-beta 1 precursor of Mr 39,000 and an unknown large subunit of Mr 105,000-120,000. The deduced primary structure of the MP large subunit was elucidated by determining the nucleotide sequence of its cDNA. The cDNA encodes a prepro-precursor of 1712 amino acid residues with a calculated Mr of 186,596. The mature large subunit seems to be derived proteolytically from a prepro-precursor and the calculated Mr is 91,606. The precursor has seven N-linked glycosylation sites and an unusual structure containing 18 epidermal growth factor-like domains and four cysteine-rich internal repeats. The large subunit mRNA is synthesized in parallel with the expression of TGF-beta 1 mRNA in various rat tissues.
Bathoiodopsin, a primary intermediate of iodopsin at physiological temperature.
Authors: Kandori H , Mizukami T , Okada T , Imamoto Y , Fukada Y , Shichida Y , Yoshizawa T
Published: May 2006

Measurement of the primary photochemical reaction of iodopsin, a chicken red-sensitive cone visual pigment, was carried out at room temperature by using picosecond (ps) laser photolysis. Excitation of iodopsin with a ps green pulse (pulse width, 21 ps) caused the instantaneous formation of a bathochromic product, which was stable on a ps time scale. This product may correspond to "bathoiodopsin," which was detected by low-temperature spectrophotometry. Although bathoiodopsin produced at the temperature of liquid nitrogen or helium reverted to the original pigment (iodopsin) on warming (above -170 degrees C), the bathoiodopsin produced at physiological temperature decayed to all-trans-retinal and R-photopsin (the protein moiety of iodopsin) presumably through several intermediates. The absorption maximum of bathoiodopsin at room temperature was at 625 nm, a wave-length slightly shorter than that measured at low temperature (lambda max, 640 nm). The extinction coefficient of bathoiodopsin at room temperature was lower than that at low temperature and close to that of the original iodopsin at room temperature.
A nervous system-specific isotype of the beta subunit of Na+,K(+)-ATPase expressed during early development of Xenopus laevis.
Authors: Good P J , Richter K , Dawid I B
Published: May 2006

We have previously described the isolation of several genes expressed exclusively in the nervous system of adult Xenopus laevis and activated in the embryo shortly after neural induction. The sequence of one of these cDNAs, 24-15, identifies the corresponding protein as an isotype of the beta subunit of Na+,K(+)-ATPase [ATP phosphohydrolase (Na+/K(+)-transporting); EC 3.6.1.37]. This form is distinct from the previously described beta 1 subunit of Xenopus, and the protein sequence comparison suggests that it is not the frog homolog of the mammalian beta 2 subunit; therefore, we refer to the 24-15 protein as the beta 3 subunit of Na+,K(+)-ATPase of Xenopus. Antisera directed against beta 3-subunit fusion protein detected a protein in adult brain extracts with the size and properties expected for a Na+,K(+)-ATPase beta subunit. In Xenopus the beta 1 and beta 3 subunits are expressed as maternal mRNAs at similar levels; during embryogenesis rapid accumulation of beta 3 mRNA begins at stage 14 (early neurula), and the rapid accumulation of beta 1 mRNA begins at stage 23/24. In situ hybridization of antisense RNA probes to tadpole brain sections indicates that beta 3 subunit is expressed throughout the developing brain. We suggest that beta 3 is a major Na+,K(+)-ATPase beta subunit present during early nervous system development in the frog.
Recombinant yeast TFIID, a general transcription factor, mediates activation by the gene-specific factor USF in a chromatin assembly assay.
Authors: Meisterernst M , Horikoshi M , Roeder R G
Published: May 2006

The TATA box-binding transcription factor TFIID from Saccharomyces cerevisiae was tested for its ability to mediate regulatory factor functions both in a cell-free system reconstituted with other general initiation factors (purified from HeLa cells) and in a combined nucleosome assembly-transcription system. In the latter assay recombinant yeast TFIID, expressed in and purified from bacteria, was sufficient to prevent nucleosome assembly-mediated repression and to mediate transcriptional enhancement of the adenovirus major late promoter by the gene-specific activator USF. In contrast, recombinant yeast TFIID was unable to mediate activation by USF in the system reconstituted only with purified general factors. Under the same conditions a partially purified natural yeast TFIID was able to mediate activation by both USF and Sp1 (assayed with the human immunodeficiency virus promoter), but to a lesser extent than observed with a partially purified natural human TFIID. The implications of these findings are discussed with respect to the structure of the yeast and human TATA factors and the possible involvement either of specific TFIID modifications or of coactivators.
Evidence for horizontal transmission of the mobile element jockey between distant Drosophila species.
Authors: Mizrokhi L J , Mazo A M
Published: May 2006

We addressed the possibility of the horizontal transfer of long interspersed element (LINE)-like mobile elements by studying the distribution of the Drosophila melanogaster LINE-like element jockey in different Drosophila species. Outside the D. melanogaster group jockey was detected only in the distantly related species Drosophila funebris. Cloning and sequencing of this element from D. funebris revealed the existence of the two open reading frames highly similar to those of jockey from D. melanogaster. Elements from both species are transcriptionally active and contain in their promoter regions a conserved sequence important for its activity. The high degree of similarity between the D. melanogaster and the D. funebris jockey and the absence of jockey from other sibling species of the D. funebris group provide evidence for the horizontal transmission of jockey into D. funebris.
Interferon gamma response region in the promoter of the human DPA gene.
Authors: Yang Z , Sugawara M , Ponath P D , Wessendorf L , Banerji J , Li Y , Strominger J L
Published: May 2006

The interferon gamma (IFN-gamma) response region of the human class II major histocompatibility complex gene, DPA, has been localized to a 52-base-pair (bp) DNA fragment in the proximal promotor at -107 to -55 bp after transfection into HeLa cells of a series of 5', 3', and gap deletion mutants linked to a reporter gene, human growth hormone, as well as of synthetic oligonucleotides fused to the heterologous promoter thymidine kinase. The 52-mer sequence contains the X and Y box elements conserved in all class II genes; their presence is indispensable for IFN-gamma inducibility. Furthermore, an additional 5 bp immediately 5' of the X box of the DPA gene are necessary and sufficient for IFN-gamma induction. This region may contain an IFN-gamma response element. A closely related sequence has also been found in the vicinity of the critical deletion sites of three other well-studied class II gene promoters, all of which require a much longer sequence 5' of the X box. A fourth element, the W element, located about 15 bp 5' of the X box in all class II genes, is clearly of little importance in IFN-gamma inducibility of the DPA gene.
Wild-type, but not mutant, human p53 proteins inhibit the replication activities of simian virus 40 large tumor antigen.
Authors: Friedman P N , Kern S E , Vogelstein B , Prives C
Published: May 2006

Murine p53 blocks many of the replication activities of simian virus 40 (SV40) large tumor antigen (T antigen) in vitro. As murine cells do not replicate SV40 DNA, it was of interest to determine how p53 from permissive human cells functions. Recombinant baculoviruses encoding either the wild-type form of human p53 or a mutant p53 cloned from a human tumor cell line were constructed, and p53 proteins were purified from infected insect cells. Surprisingly, we found that wild-type human p53 was as inhibitory to the ability of T antigen to mediate replication of an SV40 origin-containing (ori DNA) plasmid in vitro as was murine p53. Wild-type human p53 also blocked the DNA unwinding activity of T antigen, as did its murine counterpart. In contrast to murine and wild-type human p53, the mutant human p53 did not block ori DNA replication or DNA unwinding. Murine p53 formed a complex with mutant human p53 in vivo. Furthermore, mutant human p53 reduced the inhibition of SV40 ori DNA replication by murine p53 in vitro. These results provide a model for the way in which mutant p53 proteins can affect normal functions of p53.
Primary structure of keratinocyte transglutaminase.
Authors: Phillips M A , Stewart B E , Qin Q , Chakravarty R , Floyd E E , Jetten A M , Rice R H
Published: May 2006

The nucleotide and deduced amino acid sequences of the coding regions of human and rat keratinocyte transglutaminases (protein-glutamine: amine gamma-glutamyltransferase; EC 2.3.2.13) have been determined. These yield proteins of approximately 90 kDa that are 92% identical, indicative of the conservation of important structural features. Alignments of amino acid sequences show substantial similarity among the keratinocyte transglutaminase, human clotting factor XIII catalytic subunit, guinea pig liver tissue transglutaminase, and the human erythrocyte band-4.2 protein. The keratinocyte enzyme is most similar to factor XIII, whereas the band-4.2 protein is most similar to the tissue transglutaminase. A salient feature of the keratinocyte transglutaminase is its 105-residue extension beyond the N terminus of the tissue transglutaminase. This extension and the unrelated activation peptide of factor XIII (a 37-residue extension) appear to be added for specialized functions after divergence of the tissue transglutaminase from their common lineage.
p-Aminobenzoate synthesis in Escherichia coli: purification and characterization of PabB as aminodeoxychorismate synthase and enzyme X as aminodeoxychorismate lyase.
Authors: Ye Q Z , Liu J , Walsh C T
Published: May 2006

The Escherichia coli pabA and pabB genes have been overexpressed separately and in tandem. Using purified PabB, we have confirmed the recent suggestion that PabB needs an additional protein, enzyme X, to convert chorismate and NH3 to p-aminobenzoate (PABA). With chorismate and NH3, pure PabB generates an intermediate presumed to be 4-amino-4-deoxychorismate based upon UV/visible spectroscopy and enzymatic and nonenzymatic transformations. The PabB-catalyzed interconversion of chorismate and isolated aminodeoxychorismate is readily reversible. With pure PabB as a stoichiometric assay reagent, enzyme X was purified approximately 800-fold to near homogeneity as an apparent homodimer of 50 kDa from E. coli. Enzyme X shows no activity on chorismate but quantitatively converts the preformed aminodeoxychorismate into p-aminobenzoate and pyruvate, acting thereby as an aminodeoxychorismate lyase.
A 75-kDa polypeptide, located primarily at the plasma membrane of carrot cell-suspension cultures, is photoaffinity labeled by the calcium channel blocker LU 49888.
Authors: Thuleau P , Graziana A , Canut H , Ranjeva R
Published: May 2006

Calcium channel blockers of the phenylalkylamine family bind specifically to membranes and inhibit calcium uptake in carrot protoplast. LU 49888, an azido derivative of phenylalkylamine, behaves as its unmodified homolog in terms of affinity and specificity and therefore allows us to probe the receptor by photoaffinity labeling. Upon UV irradiation, a 75-kDa peptide was specifically labeled. Incubation of microsomes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, a zwitterionic detergent, led to the solubilization of the LU 49888-binding protein. Electrophoretic analysis under denaturing conditions and gel filtration of the solubilized "receptor-ligand" complex show a 75-kDa peptide mainly located at the plasma membrane. Consequently the LU 49888-binding protein in plants differs significantly from its animal counterpart by its size and may be a primary target for external signal molecules.
Expression cloning of a cDNA encoding the mouse pituitary thyrotropin-releasing hormone receptor.
Authors: Straub R E , Frech G C , Joho R H , Gershengorn M C
Published: May 2006

Thyrotropin-releasing hormone (TRH) is an important extracellular regulatory molecule that functions as a releasing factor in the anterior pituitary gland and as a neurotransmitter/neuromodulator in the central and peripheral nervous systems. Binding sites for TRH are present in these tissues, but the TRH receptor (TRH-R) has not been purified from any source. Using Xenopus laevis oocytes in an expression cloning strategy, we have isolated a cDNA clone that encodes the mouse pituitary TRH-R. This conclusion is based on the following evidence. Injection of sense RNA transcribed in vitro from this cDNA into Xenopus oocytes leads to expression of cell-surface receptors that bind TRH and the competitive antagonist chlordiazepoxide with appropriate affinities and that elicit electrophysiological responses to TRH with the appropriate concentration dependency. Antisense RNA inhibits the TRH response in Xenopus oocytes injected with RNA isolated from normal rat anterior pituitary glands. Finally, transfection of COS-1 cells with this cDNA leads to expression of receptors that bind TRH and chlordiazepoxide with appropriate affinities and that transduce TRH stimulation of inositol phosphate formation. The 3.8-kilobase mouse TRH-R cDNA encodes a protein of 393 amino acids that shows similarities to other guanine nucleotide-binding regulatory protein-coupled receptors.
Structure and expression of cytosolic cyclophilin/peptidyl-prolyl cis-trans isomerase of higher plants and production of active tomato cyclophilin in Escherichia coli.
Authors: Gasser C S , Gunning D A , Budelier K A , Brown S M
Published: May 2006

cDNA clones encoding proteins of approximately 18 kDa in which 83% of the amino acids are conserved relative to the published sequences of mammalian cyclophilin/rotamase (CyP) have been isolated from tomato, maize, and Brassica napus. In correspondence with the mammalian genes, but in contrast with the Neurospora gene and one yeast CyP gene, the plant CyP genes encode only mature proteins lacking transit peptides. RNA blot analyses demonstrate that CyP genes are expressed in all plant organs tested. Southern blots of genomic DNA indicate that there are small families (two to eight members) of CyP-related genes in maize and B. napus. A vector was constructed for expression of the tomato cDNA in E. coli. SDS/polyacrylamide gels show that extracts of appropriately induced cells harboring this vector contain nearly 40% of the protein as a single approximately 18-kDa band. While the majority of this protein is sequestered in insoluble inclusion bodies, the soluble extracts have higher levels of peptidyl-prolyl cis-trans isomerase (rotamase) activity than extracts of wild-type cells. This additional activity is sensitive to inhibition by the cyclic undecapeptide cyclosporin A.
Cell line-specific differences in the control of cell cycle progression in the absence of mitosis.
Authors: Kung A L , Sherwood S W , Schimke R T
Published: May 2006

This paper reports that there are major differences between mammalian cell lines in the propensity to progress into subsequent cell cycles when mitosis is inhibited with agents that disrupt the assembly of the mitotic spindle apparatus (Colcemid, nocodazole, and taxol). Human HeLa S3 cells, which represent one extreme, remain arrested in mitosis, with elevated levels of cyclin B and p34cdc2 kinase activity. In Chinese hamster ovary cells, at the other extreme, the periodic rise and fall of cyclin B levels and p34cdc2 kinase activity is only transiently inhibited in the absence of mitosis. The cells progress into subsequent cell cycles, without dividing, resulting in serial doublings of cellular DNA content. In general, the propensity to progress into subsequent cell cycles in the absence of mitosis appears to be species related, such that human cell lines remain permanently blocked in a mitotic state, whereas rodent cell lines are only transiently inhibited when spindle assembly is disrupted. We interpret these results to indicate that in mammalian cell lines there exists a checkpoint which serves to couple cell cycle progression to the completion of certain karyokinetic events. Furthermore, either such a checkpoint exists in some cell lines but not in others or the stringency of the control mechanism varies among different cell lines.
A general bijective algorithm for trees.
Authors: Chen W Y
Published: May 2006

Trees are combinatorial structures that arise naturally in diverse applications. They occur in branching decision structures, taxonomy, computer languages, combinatiorial optimization, parsing of sentences, and cluster expansions of statistical mechanics. Intuitively, a tree is a collection of branches connected at nodes. Formally, it can be defined as a connected graph without cycles. Schroder trees, introduced in this paper, are a class of trees for which the set of subtrees at any vertex is endowed with the structure of ordered partitions. An ordered partition is a partition of a set in which the blocks are linearly ordered. Labeled rooted trees and labeled planed trees are both special classes of Schroder trees. The main result gives a bijection between Schroder trees and forests of small trees--namely, rooted trees of height one. Using this bijection, it is easy to encode a Schroder tree by a sequence of integers. Several classical algorithms for trees, including a combinatorial proof of the Lagrange inversion formula, are immediate consequences of this bijection.
When can ants discriminate the sex of brood? A new aspect of queen-worker conflict.
Authors: Nonacs P , Carlin N F
Published: May 2006

The stage in preimaginal ontogeny at which the sexes can first be distinguished has important implications for queen-worker conflict in social insects. If workers are unable to sex larvae at an early instar, their opportunity to control colony reproductive strategies may be limited. In addition, by concealing the sex of her sons for some portion of development, the queen could protect them from the workers' attempts to substitute their own sons or to skew the numerical sex ratio. In a series of choice experiments, workers of the carpenter ant, Camponotus floridanus, failed to discriminate the sex of several stages of larvae but did retrieve female pupae significantly more rapidly than male pupae. Our results suggest that in this species, sex may not become detectable until pupation, which is consistent with sexual deception as an aspect of queen control.
Vitamin D receptor interaction with specific DNA requires a nuclear protein and 1,25-dihydroxyvitamin D3.
Authors: Liao J , Ozono K , Sone T , McDonnell D P , Pike J W
Published: May 2006

The regulation of osteocalcin gene expression by 1,25-dihydroxyvitamin D3 is mediated by the vitamin D receptor and a cis-acting DNA response element that has been identified within the 5' region of the osteocalcin promoter. In this report, we show that vitamin D receptors derived from nuclear extracts of mammalian cells bind directly to this cis-acting element in vitro and do so in a manner requiring hormone. Vitamin D receptors derived from reticulocyte lysate translations in vitro or from extracts of a Saccharomyces cerevisiae strain that expresses the recombinant protein also bind the osteocalcin responsive element, but only when nuclear extracts of mammalian cells are provided. The vitamin-D-receptor-DNA-binding accessory factor is isolated by salt extraction, labile to temperature, and sensitive to tryptic digestion. These studies suggest that the high-affinity interaction of the vitamin D receptor with the osteocalcin vitamin D response element in vitro requires both 1,25-dihydroxyvitamin D3 and an accessory protein derived from the mammalian cell nucleus.
Cloning of cDNAs encoding amphibian bombesin: evidence for the relationship between bombesin and gastrin-releasing peptide.
Authors: Spindel E R , Gibson B W , Reeve J R , Kelly M
Published: May 2006

Bombesin is a tetradecapeptide originally isolated from frog skin; its mammalian homologue is the 27-amino acid peptide gastrin-releasing peptide (GRP). cDNAs encoding GRP have been cloned from diverse species, but little is yet known about the amphibian bombesin precursor. Mass spectrometry of HPLC-separated skin exudate from Bombina orientalis was performed to demonstrate the existence of authentic bombesin in the skin of this frog. A cDNA library was prepared from the skin of B. orientalis and mixed oligonucleotide probes were used to isolate cDNAs encoding amphibian bombesin. Sequence analysis revealed that bombesin is encoded in a 119-amino acid prohormone. The carboxyl terminus of bombesin is flanked by two basic amino acids; the amino terminus is not flanked by basic amino acids but is flanked by a chymotryptic-like cleavage site. Northern blot analysis demonstrated similarly sized bombesin mRNAs in frog skin, brain, and stomach. Polymerase chain reaction was used to show that the skin and gut bombesin mRNAs encoded the identical prohormones. Prohormone processing, however, differed between skin and gut. Chromatography showed the presence of only authentic bombesin in skin whereas gut extracts contained two peaks of bombesin immunoreactivity, one consistent in size with bombesin and one closer in size to mammalian GRP. Thus the same bombesin prohormone is processed solely to bombesin in skin but is processed to a peptide similar in size to bombesin and to a peptide similar in size to mammalian GRP in stomach.
Expression of ToxR, the transcriptional activator of the virulence factors in Vibrio cholerae, is modulated by the heat shock response.
Authors: Parsot C , Mekalanos J J
Published: May 2006

The toxR gene of Vibrio cholerae encodes a transmembrane, DNA-binding protein that positively controls transcription of the genes for cholera toxin, TCP pili, and other proteins important in cholera pathogenesis. Nucleotide sequence analysis of the toxR upstream region has revealed that the heat shock gene htpG, encoding the bacterial homologue of the eukaryotic Hsp90 protein, was located immediately upstream and was divergently transcribed from toxR. Using lacZ transcriptional fusions, we have shown that neither toxR nor htpG expression was regulated by ToxR. However, the growth temperature had a coordinate but reciprocal effect on the expression from both the toxR and htpG promoters in V. cholerae; the decrease of toxR expression between 22 degrees C and 37 degrees C was proportional to the increase of htpG expression observed within that temperature range. A similar pattern of expression of the htpG and toxR promoters was observed in the heterologous host Escherichia coli, where this regulation was controlled by the level of the E. coli rpoH (htpR) gene product, sigma-32. Consistent with the temperature-regulated expression of the V. cholerae htpG promoter in E. coli, a sequence similar to the consensus sequence of the E. coli heat shock promoters was detected upstream from the V. cholerae htpG gene. We propose a model in which the regulation of toxR expression by temperature is controlled by the level of sigma-32 (RpoH) RNA polymerase.
Characterization of a splicing mutation in group A xeroderma pigmentosum.
Authors: Satokata I , Tanaka K , Miura N , Miyamoto I , Satoh Y , Kondo S , Okada Y
Published: May 2006

The molecular basis of group A xeroderma pigmentosum (XP) was investigated by comparison of the nucleotide sequences of multiple clones of the XP group A complementing gene (XPAC) from a patient with group A XP with that of a normal gene. The clones showed a G----C substitution at the 3' splice acceptor site of intron 3, which altered the obligatory AG acceptor dinucleotide to AC. Nucleotide sequencing of cDNAs amplified by the polymerase chain reaction revealed that this single base substitution abolishes the canonical 3' splice site, thus creating two abnormally spliced mRNA forms. The larger form is identical with normal mRNA except for a dinucleotide deletion at the 5' end of exon 4. This deletion results in a frameshift with premature translation termination in exon 4. The smaller form has a deletion of the entire exon 3 and the dinucleotide at the 5' end of exon 4. The result of a transfection study provided additional evidence that this single base substitution is the disease-causing mutation. This single base substitution creates a new cleavage site for the restriction nuclease AlwNI. Analysis of AlwNI restriction fragment length polymorphism showed a high frequency of this mutation in Japanese patients with group A XP: 16 of 21 unrelated Japanese patients were homozygous and 4 were heterozygous for this mutation. However, 11 Caucasians and 2 Blacks with group A XP did not have this mutant allele. The polymorphic AlwNI restriction fragments are concluded to be useful for diagnosis of group A XP in Japanese subjects, including prenatal cases and carriers.
Cloning and expression of N-acetylglucosaminyltransferase I, the medial Golgi transferase that initiates complex N-linked carbohydrate formation.
Authors: Kumar R , Yang J , Larsen R D , Stanley P
Published: May 2006

This laboratory has previously identified a human gene encoding N-acetylglucosaminyltransferase I (GlcNAc-TI; EC 2.4.1.101) by complementation of the glycosylation defect in the Lec1 Chinese hamster ovary (CHO) cell mutant. A phage lambda library prepared from genomic DNA of a tertiary Lec1 transfectant (3 degrees T) has now been used to obtain clones encoding an active GlcNAc-TI enzyme. A small genomic DNA fragment [approximately 4.6 kilobases (kb)], isolated from an Alupositive lambda clone, conferred human GlcNAc-TI activity upon transfection into Lec1 cells. An approximately 1.3-kb probe generated from this DNA fragment detected unique but distinct DNA fragments in human and CHO genomic DNA. The probe also hybridized to a poly(A)+ RNA of approximately 2.7 kb in human and CHO cells and allowed the isolation of a full-length cDNA encoding human GlcNAc-TI activity. The overall features of the cDNA and deduced protein sequence (445 amino acids) are typical of other Golgi transferases that are type II transmembrane proteins. Northern blot analysis with the same probe showed that Lec1 mutant cells also possessed an approximately 2.7-kb poly(A)+ RNA, indicating that the lec1 mutation is a point mutation.
Substitution of amino acids Asp-85, Asp-212, and Arg-82 in bacteriorhodopsin affects the proton release phase of the pump and the pK of the Schiff base.
Authors: Otto H , Marti T , Holz M , Mogi T , Stern L J , Engel F , Khorana H G , Heyn M P
Published: May 2006

Photocycle and flash-induced proton release and uptake were investigated for bacteriorhodopsin mutants in which Asp-85 was replaced by Ala, Asn, or Glu; Asp-212 was replaced by Asn or Glu; Asp-115 was replaced by Ala, Asn, or Glu; Asp-96 was replaced by Ala, Asn, or Glu; and Arg-82 was replaced by Ala or Gln in dimyristoylphosphatidylcholine/3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate micelles at pH 7.3. In the Asp-85----Ala and Asp-85----Asn mutants, the absence of the charged carboxyl group leads to a blue chromophore at 600 and 595 nm, respectively, and lowers the pK of the Schiff base deprotonation to 8.2 and 7, respectively, suggesting a role for Asp-85 as counterion to the Schiff base. The early part of the photocycles of the Asp-85----Ala and Asp-85----Asn mutants is strongly perturbed; the formation of a weak M-like intermediate is slowed down about 100-fold over wild type. In both mutants, proton release is also slower but clearly precedes the rise of M. The amplitude of the early (less than 0.2 microseconds) reversed photovoltage component in the Asp-85----Asn mutant is very large, and the net charge displacement is close to zero, indicating proton release and uptake on the cytoplasmic side of the membrane. The data suggest an obligatory role for Asp-85 in the efficient deprotonation of the Schiff base and in the proton release phase, probably as proton acceptor. In the Asp-212----Asn mutant, the rise of the absorbance change at 410 nm is slowed down to 220 microsecond, its amplitude is small, and the release of protons is delayed to 1.9 ms. The absorbance changes at 650 nm indicate perturbations in the early time range with a slow K intermediate. Thus Asp-212 also participates in the early events of charge translocation and deprotonation of the Schiff base. In the Arg-82----Gln mutant, no net transient proton release was observed, whereas, in the Arg-82----Ala mutant, uptake and release were reversed. The pK shift of the purple-to-blue transition in the Asp-85----Glu, Arg-82----Ala, and Arg-82----Gln mutants and the similarity in the photocycle and photoelectrical signals of the Asp-85----Ala, Asp-85----Asn, and Asp-212----Asn mutants suggest the interaction between Asp-85, Arg-82, Asp-212, and the Schiff base as essential for proton release.
High-frequency nuclear transformation of Chlamydomonas reinhardtii.
Authors: Kindle K L
Published: May 2006

By using a method in which cell-wall-deficient Chlamydomonas reinhardtii cells were agitated in the presence of DNA, glass beads, and polyethylene glycol, nuclear transformation rates of approximately 10(3) transformants per micrograms of plasmid DNA were achieved. The nitrate reductase gene from wild-type Chlamydomonas was used to complement a mutation in the corresponding gene of a strain containing nit1-305. Transformants were selected by growth with nitrate as sole source of nitrogen. The transforming DNA integrated into the genome at a low-copy number in nit+ transformants. When cells carrying nit1-305 were agitated in the presence of two plasmids, one with the gene for nitrate reductase and the second with an unselected gene, the unselected gene was present in 10-50% of nit+ transformants. This high frequency of cotransformation will allow any cloned gene to be introduced into Chlamydomonas. Moreover, the overall efficiency of transformation should be high enough to permit isolation of genes from genomic libraries by complementation of stable nuclear mutants. The availability of efficient nuclear and chloroplast transformation in Chlamydomonas provides specific advantages for the study of chloroplast biogenesis, photosynthesis, and nuclear-chloroplast genome interactions.