bluuebunny/crossref_metadata_2025 huggingface dataset:
Codon choice and gene expression: synonymous codons differ in their ability to direct aminoacylated-transfer RNA binding to ribosomes in vitro.
Authors: Thomas L K , Dix D B , Thompson R C
Published: May 2006

Phe-tRNA (anticodon GAA)--polypeptide-chain elongation factor Tu-GTP ternary complexes react faster with ribosomes programmed with UUC codons than with ribosomes programmed with UUU codons. A similar preference is shown by Leu-tRNA2 (anticodon GAG) complexes, which react faster with ribosomes programmed with CUC than with those programmed with CUU. The difference is seen in the rate of ternary-complex binding to the ribosome; no differences are seen in peptide-bond formation. Highly expressed mRNAs in Escherichia coli favor codons terminating in cytosine rather than uracil when both codons are read by a single tRNA with an anticodon beginning with guanine. The results suggest that intrinsic differences between the efficiencies of synonymous codons play an important role in modulating gene expression in E. coli.
Kinetics of gamma interferon binding and induction of major histocompatibility complex class II mRNA in Leishmania-infected macrophages.
Authors: Reiner N E , Ng W , Ma T , McMaster W R
Published: May 2006

Cells of the monocyte-macrophage series must carry out discrete accessory-cell functions during the process of antigen-specific T-cell activation. One of these functions is the cell-surface expression of major histocompatibility complex (MHC) class II gene products, which are involved in the presentation of foreign antigen to T cells. Previously, we reported that murine peritoneal macrophages infected with the obligate intracellular protozoan Leishmania donovani had suppressed responses to gamma interferon (IFN-gamma) for the induction of MHC class II antigen expression. To determine the molecular basis for this suppression, we examined in the present series of experiments the interaction of this organism with cells of the murine macrophage tumor cell line P388D1. When infected with Leishmania, these cells were also markedly unresponsive to IFN-gamma for the induction of MHC class II antigen expression. This finding was not the result of a defect at the level of the IFN-gamma receptor. Thus, when 125I-labeled IFN-gamma was used, infected macrophages were found to express normal numbers of high-affinity IFN-gamma receptors, and ligand-receptor binding resulted in rapid internalization of labeled IFN-gamma. Despite normal ligand-receptor interactions, the induction in infected cells of mRNA encoding MHC (H-2) class II I-A alpha and beta chains in response to IFN-gamma was markedly suppressed. However, infected cells had normal levels of mRNA encoding the cytoskeletal protein actin. These findings indicate that Leishmania interferes with IFN-gamma induction of macrophage MHC class II antigen expression by down-regulating lymphokine induction of MHC class II mRNA. Suppression of class II expression by this intracellular parasite may prevent subsequent T-cell recognition of infected macrophages and thus favor parasite survival.
Evolution of competitive ability in Drosophila by density-dependent natural selection.
Authors: Mueller L D
Published: May 2006

The theory of density-dependent natural selection predicts that populations kept at extreme densities should evolve different competitive abilities for limited resources. These predictions have been tested with laboratory populations of Drosophila melanogaster. Six independent populations were maintained in two environments, called r and K, for 128 generations. In the r environment, population sizes were small and resources for larvae and adults were abundant. In contrast the populations in the K environment were large and crowded, and resources, such as food and space, were in short supply. The relative competitive ability for food has been estimated for each population. Populations from the K environment consume food at a rate that is 58% greater than the average rate for the r population. The differentiation of competitive abilities in these populations is due to natural selection and is consistent with predictions from the theory of evolutionary ecology.
Dominant negative protein kinase mutations that confer a G1 arrest phenotype.
Authors: Mendenhall M D , Richardson H E , Reed S I
Published: May 2006

The CDC28 gene of Saccharomyces cerevisiae encodes a protein kinase that is required for passage through the G1 phase of the cell cycle. We have used an inducible promoter fused to the CDC28 coding sequence to isolate conditionally dominant mutant alleles of CDC28. Overexpression of these dominant alleles causes arrest in the G1 phase of the cell cycle but permits the distinctive asymmetric growth that is characteristic of recessive temperature-sensitive cdc28 mutants. The dominant alleles encode products with no detectable protein kinase activity, and their phenotypic effects can be suppressed by simultaneous overproduction of the wild-type protein. DNA sequence analysis showed that the mutant site in at least one of the dominant alleles is in a residue that is highly conserved among protein kinases. These properties are best understood if the dominant mutation results in the catalytic inactivation of the protein kinase but still allows the binding of another component needed for CDC28 function. By this model, high levels of the mutant protein arrest cell division by denying the wild-type protein access to this other component. Suppressors that may encode this other component have been isolated on high-copy-number plasmids.
Regulation of interleukin 2 receptor alpha subunit (Tac or CD25 antigen) gene expression: binding of inducible nuclear proteins to discrete promoter sequences correlates with transcriptional activation.
Authors: Lowenthal J W , Böhnlein E , Ballard D W , Greene W C
Published: May 2006

Transfection of deleted forms of the human interleukin 2 receptor alpha subunit (IL-2R alpha; also called CD25 or Tac antigen) gene (IL2RA) promoter revealed a requirement for sequences 3' of base -317 for phytohemagglutinin- and phorbol 12-myristate 13-acetate (PMA)-induced promoter activation in CD4+ Jurkat T cells. In contrast, sequences 3' of base -271 were sufficient for promoter induction in CD4-/CD8- YT-1 T cells or Jurkat cells expressing the transactivator protein (tat-I) of human T-cell lymphotropic virus type I (HTLV-I). Gel retardation assays revealed that nuclear extracts from induced, but not uninduced, Jurkat and YT-1 cells mediated the formation of two specific DNA-protein complexes with oligonucleotides spanning the region of the IL2RA promoter from position -291 to -245, which contains two imperfect direct repeats (IDRs). Consistent with the different 5' sequence requirements for promoter activation in Jurkat and YT-1 cells, oligonucleotides corresponding to the region from -267 to -243 (downstream IDR and flanking region) formed only one complex with induced Jurkat extracts but two complexes with induced YT-1 extracts. Oligonucleotides containing the region of the IL2RA promoter from -293 to -270 (upstream IDR and flanking region) failed to bind protein in either cell type. In further support of the biological significance of these DNA-protein interactions, the IL2RA oligonucleotide from -291 to -245 proved to be sufficient in either orientation to confer PMA inducibility to the mitogen-insensitive thymidine kinase gene promoter in Jurkat cells. Together, these findings suggest that the interaction of inducible DNA binding proteins with the IL2RA promoter between bases -291 and -245 plays an important role in mitogen-induced changes in the transcriptional activity of this receptor gene. Furthermore, the requisite 5' sequences appear to differ in T cells depending upon the nature of the activation signal and perhaps the stage of cellular maturation.
Human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza C viruses.
Authors: Vlasak R , Luytjes W , Spaan W , Palese P
Published: May 2006

Human coronavirus OC43 and bovine coronavirus elute from agglutinated chicken erythrocytes when incubated at 37 degrees C, suggesting the presence of a receptor-destroying enzyme. Moreover, bovine coronavirus exhibits an acetylesterase activity in vitro using bovine submaxillary mucin as substrate similar to the enzymatic activity found in influenza C viruses. Furthermore, pretreatment of erythrocytes with either influenza C virus or bovine coronavirus eliminates subsequent binding and agglutination by either coronaviruses or influenza C virus, whereas binding of influenza A virus remains intact. In addition, hemagglutination by coronaviruses can be inhibited by pretreatment of erythrocytes with Arthrobacter ureafaciens or Clostridium perfringens neuraminidase or by addition of sialic acid-containing gangliosides. These results suggest that, like influenza C viruses, human coronavirus OC43 and bovine coronavirus recognize O-acetylated sialic acid or a similar derivative as cell receptor.
Dual effects of serotonin on a voltage-gated conductance in lymphocytes.
Authors: Choquet D , Korn H
Published: May 2006

The effects of serotonin (5-HT), a well-known immunomodulator and neurotransmitter, on the ionic permeability of a pre-B lymphocyte cell line was investigated with the whole-cell patch-clamp technique. We found that physiological doses of this biogenic amine regulate a voltage-gated potassium channel by activating different subsets of receptors. More specifically, 5-HT induces in the recorded cells (i) increase in the maximum potassium conductance, which is due to activation of 5-HT1-like receptors, and (ii) acceleration of the inactivation process that is under the control of 5-HT3 receptors and, accordingly, is mimicked by the 5-HT3 agonist, 2-methyl-5-HT; involvement of those two distinct categories of receptors was demonstrated by using specific antagonists that block predominantly one or the other of these two actions. These two results show that hormones can affect lymphocyte physiology through modulation of their ionic conductances in a way that might help explain some of the diverse effects of 5-HT on neuronal cells.
FLP recombinase is an enzyme.
Authors: Gates C A , Cox M M
Published: May 2006

The FLP protein of the yeast 2-microns plasmid catalyzes intermolecular site-specific recombination with a turnover number of approximately equal to 0.12 min-1 (per FLP monomer) for relaxed DNA substrates. Under conditions that enhance its stability, the protein can be used in catalytic rather than stoichiometric amounts. The reaction rate exhibits a strong dependence on FLP protein concentration even when the protein is present in excess relative to available recombination sites.
Effect of in vitro DNA methylation on beta-globin gene expression.
Authors: Yisraeli J , Frank D , Razin A , Cedar H
Published: May 2006

When the human beta-globin gene was methylated at every cytosine residue and was inserted into mouse fibroblasts by DNA-mediated gene transfer, the transcription of the gene was strongly inhibited. This methylation also prevented expression and induction of the gene in mouse erythroleukemia cells. By using partially methylated hybrid molecules, it was shown that methylation-sensitive negative regulatory elements are located in both the 5' and 3' ends of the beta-globin gene but not in the 90-base-pair region usually associated with promoter activity. To further investigate the role of DNA methylation in the regulation of the beta-globin gene, 50-base-pair poly(dG-dC) tracts were introduced into various sites in a mouse-human hybrid gene, and these inserts were methylated by means of the Hha I methylase. Heavy methylation of these artificially added sites had no effect on either transcription initiation or elongation, suggesting that DNA modification operates through fixed endogenous sites in the gene domain.
Molecular cloning of a cDNA encoding the human Sm-D autoantigen.
Authors: Rokeach L A , Haselby J A , Hoch S O
Published: May 2006

Antibodies to the Sm-D polypeptide antigen are closely associated with the rheumatic disease systemic lupus erythematosus. Sm-D exists in the cell as one of the core proteins of the small nuclear ribonucleoprotein complexes implicated in RNA processing. We have isolated a cDNA clone, D45-2, coding for the Sm-D human nuclear antigen by screening a human B-lymphocyte cDNA library with synthetic oligonucleotide probes. The 1633-base-pair clone contains an open reading frame (ORF) 357 nucleotides long, capable of encoding a 13,282-dalton polypeptide. The Sm-D coding region is initiated at an AUG codon downstream from a sequence with excellent match to the consensus for the eukaryotic ribosome-binding site. The Sm-D ORF is preceded by a 150-nucleotide-long untranslated leader and followed by a 1126-nucleotide-long untranslated region containing four putative poly(A) signals. The predicted amino acid sequence reveals a (Gly-Arg)9 repeated motif at the C terminus, which may constitute one of the Sm-D immunoreactive determinants. Moreover, this C terminus shows interesting features: (i) a good homology to protamines as expected for a nucleic acid binding protein and (ii) a striking similarity to a region in the Epstein-Barr nuclear antigen.
Thermostable DNA polymerase chain amplification of t(14;18) chromosome breakpoints and detection of minimal residual disease.
Authors: Crescenzi M , Seto M , Herzig G P , Weiss P D , Griffith R C , Korsmeyer S J
Published: May 2006

Achieving the capacity to detect minimal numbers of neoplastic cells is a major cancer diagnostic challenge. Chromosomal translocations such as the t(14;18)(q32;q21) found in follicular and some nonfollicular lymphomas provide a tumor-specific molecular marker. The 14;18 breakpoints are focused at one of six immunoglobulin heavy chain joining (JH) regions on chromosome 14 and a small major breakpoint region (MBR) of the BCL2 gene on chromosome 18. We utilized universal oligonucleotide primers of a region 5' to the BCL2 MBR and at the 3' end of JH segments to initiate a DNA polymerase chain reaction that amplified these BCL2-JH junctures. Use of thermostable DNA polymerase enabled annealing and synthesis steps at temperatures approaching the melting point of the primers, providing a sensitive and specific assay capable of detecting 1 lymphoma cell in 10(6) normal cells. This technique identified the subclinical presence of leukemic cells in all seven patients examined, including two in clinical remission. It also assessed the effectiveness of protocols designed to purge malignant cells from marrow. Moreover, this approach enabled the rapid DNA sequencing of chromosomal breakpoints without their molecular cloning. This assay markedly refines the capacity to detect minimal residual disease and should improve the ability to determine the stage of disease, stratify treatment, and evaluate therapy.
Increase in albumin, IgG, and IgM blood-nerve barrier indices in human diabetic neuropathy.
Authors: Poduslo J F , Curran G L , Dyck P J
Published: May 2006

The albumin (Alb), IgG, and IgM concentrations in the endoneurium of fascicular sural nerve biopsy samples were evaluated in controls (n = 9 or 10), diabetic patients without neuropathy (n = 6), and diabetic patients with polyneuropathy (n = 17 or 18). These values were significantly increased in diabetic patients with and without neuropathy when expressed both per endoneurial dry weight or endoneurial total protein compared to biopsy samples from healthy controls. When these concentrations, expressed per endoneurial total protein, were related to plasma concentrations similarly expressed, the resulting blood-nerve barrier (BNB) indices were significantly increased for Alb (6.1 times; P less than 0.00001), IgG (4.9 times; P = 0.00037), and IgM (2.7 times; P = 0.015). The diabetic patients without neuropathy (defined as having an index of pathology of greater than 0.65; a measure of the severity of the pathological abnormality based on morphological criteria) also had significant increases in two of these BNB indices that were intermediate between the diabetic neuropathy patients and controls (Alb, 3.9 times controls; P = 0.00002: IgG, 4.6 times controls; P = 0.00016: IgM, 1.8 times controls; not significant). No correlations were observed between the endoneurial concentrations of these plasma proteins or the BNB indices and the index of pathology, suggesting that these increases in endoneurial plasma proteins precede the pathologic alterations. The increased values for the diabetics in the absence of pathological abnormalities may prove useful in predicting neuropathic complications. The ratio of the IgG-BNB index to the Alb-BNB index was decreased 19%, and the ratio of the IgM-BNB index to the Alb-BNB index was decreased 56% in diabetic neuropathy patients compared to controls. Although the IgG and IgM concentrations are increased in the diabetic endoneurium, the Alb increase is greater and a mechanism other than size indiscriminate extravasation of plasma proteins, therefore, is suggested. Morphometric assessment of the endoneurial compartments, which would be expected to contain these plasma proteins, suggests that they are not altered in diabetic neuropathy; hence, it is hypothesized that the observed increase in endoneurial concentration of these plasma proteins results from altered transport through the endothelial or perineurial barrier, which supports an underlying vascular mechanism in the development of diabetic polyneuropathy.
Transmitter and receiver modules in bacterial signaling proteins.
Authors: Kofoid E C , Parkinson J S
Published: May 2006

Prokaryotes are capable of sophisticated sensory behaviors. We have detected sequence motifs in bacterial signaling proteins that may act as transmitter or receiver modules in mediating protein-protein communication. These modules appear to retain their functional identities in many protein hosts, implying that they are structurally independent elements. We propose that the fundamental activity characterizing these domains is specific recognition and association of matched modules, accompanied by conformational changes in one or both of the interacting elements. Signal propagation is a natural consequence of this behavior. The versatility of this information-processing strategy is evident in the chemotaxis machinery of Escherichia coli, where proteins containing transmitters or receivers are linked in "dyadic relays" to form complex signaling networks.
Isolation of a cDNA clone encoding a biologically active thyroid hormone receptor.
Authors: Koenig R J , Warne R L , Brent G A , Harney J W , Larsen P R , Moore D D
Published: May 2006

We have isolated a c-erbA cDNA clone from a GH3 cell library. The clone, denoted erb62, is 4.5 kilobases long and encodes a 461-amino acid beta-type c-erbA protein. This c-erbA protein binds 3,5,3'-triiodothyronine (T3) and T3 analogs with affinities similar to those of the authentic T3 receptor. By RNA gel blot analysis, erb62 hybridizes to a 6-kilobase RNA found in organs that express T3 receptors--e.g., heart, kidney, and brain. A COS-cell transient cotransfection system was used to show that erb62 encodes a biologically active T3 receptor. An oligonucleotide, corresponding to a portion of the rat growth hormone gene 5'-flanking region that contains a T3 response element, was inserted on the 5' side of the herpes simplex virus thymidine kinase promoter in a chloramphenicol acetyltransferase-expressing plasmid. Reporter gene expression directed by this hybrid promoter was T3 inducible only if this plasmid was cotransfected with an erb62-expressing plasmid.
Structural fluctuations of a helical polypeptide traversing a lipid bilayer.
Authors: Vogel H , Nilsson L , Rigler R , Voges K P , Jung G
Published: May 2006

Time-resolved fluorescence anisotropy (FA) measurements are reported for five helical bilayer-spanning henicosapeptides, each containing one tryptophan at sequence position 1, 6, 11, 16, or 21. The FA decay reflects two molecular processes in all cases: local internal fluctuations of the tryptophan side chain with a relaxation time of 200-500 ps, and motions of the whole polypeptide molecule with a relaxation time of 9-10 ns. The amplitudes of the fast fluctuation are largest at the helix ends and decrease toward the center of the helix. A similar observation was made for the helical polypeptides dissolved in organic solvents showing that the mobility gradient along the polypeptide sequence is an inherent property of the polypeptide helix and not due to the lipid bilayer. However, the amplitudes of the fast fluctuations can be modulated by the physical state of the lipid bilayer. With decreasing temperature, the internal mobility of the tryptophan in all positions decreases with an abrupt change at the lipid-phase transition. Concomitant molecular dynamics calculations indicate a correlation between the fast FA decay and conformational fluctuations within the helix. According to the simulation, the conformation of the polypeptide is on average predominantly helical, but actually the molecule can fluctuate between a variety of different substructures. The conformational fluctuations are largest at the helix ends and provide the free space required for rotation of the indole ring around the tryptophan side chain bonds.
Extremely large and slowly processed precursors to the
Euglena
light-harvesting chlorophyll a/b binding proteins of photosystem II
Authors: Rikin Arnon , Schwartzbach Steven D.
Published: May 2006

Antibody to the
Euglena
light-harvesting chlorophyll a/b binding protein of photosystem II (LHCPII) immunoprecipitated 207-, 161-, 122-, and 110-kDa proteins from total
Euglena
proteins pulse-labeled for 10 min with [
35
S]sulfate. The 25.6- and 27.2-kDa LHCPII were barely detectable in the immunoprecipitate. During a 40-min chase with unlabeled sulfate, the amount of radioactivity in the high molecular mass proteins decreased, and the amount of radioactivity in the 25.6- and 27.2-kDa LHCPII increased with kinetics consistent with a precursor-product relationship. The half-life of the high molecular mass proteins was ≈20 min. The major proteins immunoprecipitated from a nuclease-treated rabbit reticulocyte cell-free translation system programmed with
Euglena
whole cell or poly(A)
+
RNA had molecular masses corresponding to the molecular masses of the proteins immunoprecipitated from the pulse-labeled
in vivo
translation products. RNAs of 6.6 and 8.3 kilobases were the only
Euglena
whole cell and poly(A)
+
RNAs that hybridized to a 0.7-kilobase
Eco
RI-
Bam
HI fragment of plasmid pAB165, which contains a portion of the coding sequence for
Arabidopsis
LHCPII. RNAs of this size are more than sufficient to code for proteins of 207 kDa. Taken together, these findings demonstrate that the LHCPIIs of
Euglena
are initially synthesized as slowly processed precursors with molecular masses of 207, 161, 122, and 110 kDa.
Human p53 oncogene contains one promoter upstream of exon 1 and a second, stronger promoter within intron 1.
Authors: Reisman D , Greenberg M , Rotter V
Published: May 2006

To gain insight into how transcription of the human p53 oncogene is controlled, we characterized the regulatory regions of the gene. A 3.8-kilobase-pair (kbp) EcoRI restriction fragment encompassing the 5' end of the human p53 gene, as well as subfragments generated by restriction digests, was cloned upstream of the Escherichia coli chloramphenicol acetyltransferase (CAT) gene and CAT activity was assayed in extracts of transfected cells. Two types of CAT vectors were used: Epstein-Barr virus oriP-derived constructs that were stably introduced into the human cell lines K562, Raji, and HL-60, and pSV0-CAT-derived constructs that were transiently introduced into the monkey cell line COS. By this approach we have identified two promoters for the human p53 gene. One promoter, p53P1, is located 100-250 bp upstream of the 218-bp noncoding first exon; a second, stronger promoter, p53P2, maps within the first intron. CAT activity and expression of CAT RNA indicate that p53P2 functions up to 50-fold more efficiently than p53P1. We conclude that the expression of the human p53 gene may be controlled by two promoters and that differential regulation of these promoters may play an important role in the altered expression of the gene in both normal and transformed cells.
Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1.
Authors: Sauer B , Henderson N
Published: May 2006

The Cre protein encoded by the coliphage P1 is a 38-kDa protein that efficiently promotes both intra- and intermolecular synapsis and recombination of DNA both in Escherichia coli and in vitro. Recombination occurs at a specific site, called lox, and does not require any other protein factors. The Cre protein is shown here also to be able to cause synapsis of DNA and site-specific recombination in a mammalian cell line. A stable mouse cell line was established that expresses the Cre protein under the control of the Cd2+-inducible metallothionein I gene promoter. DNA recombination was monitored with DNA substrates containing two directly repeated lox sites. One such substrate is a circular plasmid with two directly repeated lox sites (lox2) flanking a marker gene and was introduced into cells by Ca3(PO4)2 transformation. As a second substrate we used a pseudorabies virus (a herpesvirus) containing a lox2 insertion designed to provide a sensitive detection system for recombination. In both cases, site-specific recombination in vivo is dependent on the presence of the Cre protein and occurs specifically at the 34-base-pair lox sites. These results demonstrate the controlled site-specific synapsis of DNA and recombination by a prokaryotic protein in mammalian cells and suggest that Cre-mediated site-specific recombination may be a useful tool for understanding and modulating genome rearrangements in eukaryotes.
Interaction of an immunodominant epitope with Ia molecules in T-cell activation.
Authors: Adorini L , Sette A , Buus S , Grey H M , Darsley M , Lehmann P V , Doria G , Nagy Z A , Appella E
Published: May 2006

The amino acid sequence corresponding to residues 107-116 of hen egg-white lysozyme (HEL) has been identified as containing an immunodominant T-cell epitope recognized in association with the I-Ed molecule. The immunodominance of this epitope in HEL-primed H-2d mice was demonstrated by analysis of the T-cell proliferative response induced by synthetic peptides covering almost the entire HEL sequence. All the T-cell hybridomas from H-2d mice analyzed recognize the HEL sequence 107-116 in association with the I-Ed molecule. Correlating with the restriction of T-cell recognition, HEL-(105-120)-peptide binds to I-Ed but not to I-Ad molecules. Conservative or semiconservative substitutions at positions 113 (Asn----Lys), 114 (Arg----His), or 115 (Cys----Ala) abrogate the ability of HEL-(105-120) to activate T cells. Substitutions at residues 113 and 115 affect T-cell recognition but not the binding to I-Ed molecules, whereas, as shown by binding data and competition experiments, an Arg----His substitution at position 114 profoundly impairs the capacity of the peptide to interact with I-Ed molecules. In agreement with these results, [Lys113]HEL-(105-120)-peptide but not [His114]HEL-(105-120)-peptide was found to be immunogenic in H-2d mice. Thus, a single semiconservative substitution drastically reduces binding capacity and abolishes immunogenicity, suggesting that a strict correlation exists between binding of a peptide to Ia molecules and its immunogenicity.
Single base mutation in the pro alpha 2(I) collagen gene that causes efficient splicing of RNA from exon 27 to exon 29 and synthesis of a shortened but in-frame pro alpha 2(I) chain.
Authors: Tromp G , Prockop D J
Published: May 2006

Previous observations demonstrated that a lethal variant of osteogenesis imperfecta had two altered alleles for pro alpha 2(I) chains of type I procollagen. One mutation produced a nonfunctioning allele in that there was synthesis of mRNA but no detectable synthesis of pro alpha 2(I) chains from the allele. The mutation in the other allele caused synthesis of shortened pro alpha 2(I) chains that lacked most or all of the 18 amino acids encoded by exon 28. Subclones of the pro alpha 2(I) gene were prepared from the proband's DNA and the DNA sequence was determined for a 582-base-pair (bp) region that extended from the last 30 bp of intervening sequence 26 to the first 26 bp of intervening sequence 29. Data from six independent subclones demonstrated that all had the same sequence as a previously isolated normal clone for the pro alpha 2(I) gene except that four subclones had a single base mutation at the 3' end of intervening sequence 27. The mutation was a substitution of guanine for adenine that changed the universal consensus sequence for the 3' splicing site of RNA from -AG- to -GG-. S1 nuclease experiments demonstrated that about half the pro alpha 2(I) mRNA in the proband's fibroblasts was abnormally spliced and that the major species of abnormal pro alpha 2(I) mRNA was completely spliced from the last codon of exon 27 to the first codon of exon 29. The mutation is apparently unique among RNA splicing mutations of mammalian systems in producing a shortened polypeptide chain that is in-frame in terms of coding sequences, that is used in the subunit assembly of a protein, and that contributes to a lethal phenotype.
Ion channels in synaptic vesicles from Torpedo electric organ.
Authors: Rahamimoff R , DeRiemer S A , Sakmann B , Stadler H , Yakir N
Published: May 2006

A simple method has been developed for fusing synaptic vesicles into spherical structures 20-50 micron in diameter. The method has been applied to purified cholinergic synaptic vesicles from Torpedo electric organ, and the membrane properties of these fused structures have been studied by the "cell"-attached version of the patch clamp technique. A large conductance potassium-preferring channel, termed the P channel, was consistently observed in preparations of fused synaptic vesicles. The selectivity of the channel for potassium over sodium was approximately equal to 2.8-fold. Two major conductance levels were observed during P-channel activity, and their relative proportion was dependent on the voltage applied to the membrane through the patch pipette. P channels were not seen in fused preparations of purified Torpedo lipids, nor was the frequency of their occurrence increased in preparations enriched with plasma membrane or nonvesicular membranes. We suggest, therefore, that the P channels are components of the synaptic vesicle membrane. Their function in synaptic transmission physiology is still unknown.
Discrete-time random walks on diagrams (graphs) with cycles.
Authors: Hill T L
Published: May 2006

After a review of the diagram method for continuous-time random walks on graphs with cycles, the method is extended to discrete-time random walks. The basic theorems carry over formally from continuous time to discrete time. Three problems in tennis probabilities are used to illustrate random walks on discrete-time diagrams with cycles.
Crosstalk between bacterial chemotaxis signal transduction proteins and regulators of transcription of the Ntr regulon: evidence that nitrogen assimilation and chemotaxis are controlled by a common phosphotransfer mechanism.
Authors: Ninfa A J , Ninfa E G , Lupas A N , Stock A , Magasanik B , Stock J
Published: May 2006

We demonstrate by using purified bacterial components that the protein kinases that regulate chemotaxis and transcription of nitrogen-regulated genes, CheA and NRII, respectively, have cross-specificities: CheA can phosphorylate the Ntr transcription factor NRI and thereby activate transcription from the nitrogen-regulated glnA promoter, and NRII can phosphorylate CheY. In addition, we find that a high intracellular concentration of a highly active mutant form of NRII can suppress the smooth-swimming phenotype of a cheA mutant. These results argue strongly that sensory transduction in the Ntr and Che systems involves a common protein phosphotransfer mechanism.
The hypothyroid (hyt/hyt) mouse: a model system for studying the effects of thyroid hormone on developmental changes in gene expression.
Authors: Green R P , Birkenmeier E H , Beamer W G , Maltais L J , Gordon J I
Published: May 2006

Thyroid hormone has been implicated as an important factor in rodent development. We have used a strain of mice with a recessive mutation producing congenital primary hypothyroidism (C.RF/J-hyt/+) to study the effects of thyroid hormone on developmental changes in the expression of genes encoding a number of proteins involved in lipid metabolism and transport. Total cellular RNA was prepared from the small intestine and liver of hyt/hyt mice and their unaffected littermates (+/?) at various times during postnatal development. RNA blots were probed with apolipoprotein A-I, A-II, A-IV, B, and E cDNAs plus cDNAs encoding the low density lipoprotein receptor, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and three cytoplasmic hydrophobic ligand-binding proteins (two fatty acid-binding proteins and a protein that binds all-trans-retinol). Hypothyroidism results in small changes (1.5- to 5-fold) in the concentration of many of these mRNAs in liver and small intestine between postnatal days 15 and 50. A much greater tissue-specific effect was noted on apolipoprotein B (apoB) gene expression. In euthyroid +/? animals, apoB mRNA levels fall by a factor of 30 in liver between days 20 and 35 without a comparable decrease in the small intestine. This liver-specific decrease does not occur in hyt/hyt animals. The normal decrease in hepatic apoB mRNA levels is accompanied by a decrease in plasma apoB-100 but not apoB-48. No reduction in either form of plasma apoB was noted in hyt/hyt animals. Mutant hyt/hyt mice given thyroxine from birth to 35 days had liver apoB mRNA levels comparable to those in +/? littermates. In contrast, hepatic apoB mRNA concentrations did not fall to normal levels in hyt/hyt mice given thyroxine from postnatal days 15 to 35. All treatment groups have comparable levels of plasma corticosteroids. These data suggest that (i) there is a critical period or a required response time during postnatal development for thyroid hormone action on apoB gene expression, (ii) thyroid hormone's effect on apoB is tissue specific, and (iii) the hyt/hyt mouse represents a useful system to evaluate the developmental effects of thyroid hormone on specific gene expression.
Absence of methylation of a CpG-rich region at the 5' end of the MIC2 gene on the active X, the inactive X, and the Y chromosome.
Authors: Goodfellow P J , Mondello C , Darling S M , Pym B , Little P , Goodfellow P N
Published: May 2006

We have identified and characterized a Hpa II tiny fragment (HTF) island associated with the promoter region of the pseudoautosomal gene MIC2. The MIC2 HTF island is unmethylated on both the active and inactive X chromosome and is similarly unmethylated on the Y chromosome. Unlike the majority of genes borne on the X chromosome, MIC2 fails to undergo X chromosome inactivation. HTF islands associated with X chromosome-linked genes that are inactivated are highly methylated on the inactive or transcriptionally silent homologue. The failure of MIC2 to undergo X chromosome inactivation correlates with the lack of methylation of the HTF island at the 5' end of the gene. These results provide further evidence that DNA methylation plays an important role in the phenomenon of X chromosome inactivation.
Expression of streptococcal M protein in mammalian cells.
Authors: Hruby D E , Hodges W M , Wilson E M , Franke C A , Fischetti V A
Published: May 2006

The M protein encoded by group A streptococci is a cell-wall polypeptide that has the property of enabling these organisms to evade the phagocytic cells of the human host. Therefore, the M protein plays a major role in the pathogenesis of streptococcal diseases. As an initial step toward the use of this protein as a target antigen for the production of protective anti-streptococcal immunity, a live vaccinia virus recombinant containing the M-protein gene has been constructed (VV:M6 delta). The bacterial M-protein DNA sequence is stable within this genetic context and is actively transcribed by viral RNA polymerase. Furthermore, high levels of immunoreactive M protein were detected in vivo when the VV:M6 delta recombinant was used to infect mammalian cells in culture. Thus, in addition to providing a powerful approach for dissecting the immunodominant domains of the M protein, the VV:M6 delta recombinant appears to be an excellent candidate vaccine for animal trials.
Total chemical synthesis of a 77-nucleotide-long RNA sequence having methionine-acceptance activity.
Authors: Ogilvie K K , Usman N , Nicoghosian K , Cedergren R J
Published: May 2006

Chemical synthesis is described of a 77-nucleotide-long RNA molecule that has the sequence of an Escherichia coli Ado-47-containing tRNA(fMet) species in which the modified nucleosides have been substituted by their unmodified parent nucleosides. The sequence was assembled on a solid-phase, controlled-pore glass support in a stepwise manner with an automated DNA synthesizer. The ribonucleotide building blocks used were fully protected 5'-monomethoxytrityl-2'-silyl-3'-N,N-diisopropylaminophosphoram idites. p-Nitro-phenylethyl groups were used to protect the O6 of guanine residues. The fully deprotected tRNA analogue was characterized by polyacrylamide gel electrophoresis (sizing), terminal nucleotide analysis, sequencing, and total enzyme degradation, all of which indicated that the sequence was correct and contained only 3-5 linkages. The 77-mer was then assayed for amino acid acceptor activity by using E. coli methionyl-tRNA synthetase. The results indicated that the synthetic product, lacking modified bases, is a substrate for the enzyme and has an amino acid acceptance 11% of that of the major native species, tRNA(fMet) containing 7-methylguanosine at position 47.
A DNA-binding factor specific for xenobiotic responsive elements of P-450c gene exists as a cryptic form in cytoplasm: its possible translocation to nucleus.
Authors: Fujisawa-Sehara A , Yamane M , Fujii-Kuriyama Y
Published: May 2006

Transcription of the drug-metabolizing cytochrome P-450c gene is induced by 3-methylcholanthrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Previously, we defined two xenobiotic responsive elements (XREs) of approximately equal to 15 base pairs, both of which activate transcription in cis in response to these xenobiotics. Using a gel mobility shift assay, we have identified a factor that specifically binds to the XREs. This factor appears in nuclei of mouse hepatoma cell line Hepa-1 only when the cells are treated with the xenobiotics, while the factor is undetectable in the nuclei of a 3-methylcholanthrene-treated mutant of Hepa-1 with defective function of a xenobiotic receptor. In addition, the nuclear factor bound to the XRE in the gel was found to be associated with [3H]TCDD when the cells were treated with it, suggesting that the xenobiotic receptor is at least a component of the DNA-binding factor. The cytoplasmic fraction from nontreated Hepa-1 cells also contains the factor as a cryptic form and prominently reveals its DNA-binding activity by incubation with 3-methylcholanthrene in vitro. These results not only suggest the involvement of the XRE-binding factor in transcriptional activation via XREs but also provide evidence that the binding of ligands to the preexisting factor in a cryptic form induces its XRE-binding activity, which is probably followed by its translocation from cytoplasm to nucleus.
Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli.
Authors: Huston J S , Levinson D , Mudgett-Hunter M , Tai M S , Novotný J , Margolies M N , Ridge R J , Bruccoleri R E , Haber E , Crea R
Published: May 2006

A biosynthetic antibody binding site, which incorporated the variable domains of anti-digoxin monoclonal antibody 26-10 in a single polypeptide chain (Mr = 26,354), was produced in Escherichia coli by protein engineering. This variable region fragment (Fv) analogue comprised the 26-10 heavy- and light-chain variable regions (VH and VL) connected by a 15-amino acid linker to form a single-chain Fv (sFv). The sFv was designed as a prolyl-VH-(linker)-VL sequence of 248 amino acids. A 744-base-pair DNA sequence corresponding to this sFv protein was derived by using an E. coli codon preference, and the sFv gene was assembled starting from synthetic oligonucleotides. The sFv polypeptide was expressed as a fusion protein in E. coli, using a leader derived from the trp LE sequence. The sFv protein was obtained by acid cleavage of the unique Asp-Pro peptide bond engineered at the junction of leader and sFv in the fusion protein [(leader)-Asp-Pro-VH-(linker)-VL]. After isolation and renaturation, folded sFv displayed specificity for digoxin and related cardiac glycosides similar to that of natural 26-10 Fab fragments. Binding between affinity-purified sFv and digoxin exhibited an association constant [Ka = (3.2 +/- 0.9) x 10(7) M-1] that was about a factor of 6 smaller than that found for 26-10 Fab fragments [Ka = (1.9 +/- 0.2) x 10(8) M-1] under the same buffer conditions, consisting of 0.01 M sodium acetate, pH 5.5/0.25 M urea.
Allelic sequence variation of the HLA-DQ loci: relationship to serology and to insulin-dependent diabetes susceptibility.
Authors: Horn G T , Bugawan T L , Long C M , Erlich H A
Published: May 2006

Analysis of sequence variation in the polymorphic second exon of the major histocompatibility complex genes HLA-DQ alpha and -DQ beta has revealed 8 allelic variants at the alpha locus and 13 variants at the beta locus. Correlation of sequence variation with serologic typing suggests that the DQw2, DQw3, and DQ(blank) types are determined by the DQ beta subunit, while the DQw1 specificity is determined by DQ alpha. The nature of the amino acid at position 57 in the DQ beta subunit is correlated with susceptibility to insulin-dependent diabetes mellitus. This region of the DQ beta chain contains shared peptides with Epstein-Barr virus and rubella virus.
Molecular mechanism of retinoblastoma gene inactivation in retinoblastoma cell line Y79.
Authors: Lee E Y , Bookstein R , Young L J , Lin C J , Rosenfeld M G , Lee W H
Published: May 2006

Formation of retinoblastoma, a cancer arising in the retinas of young children, is determined by mutational inactivation of an autosomal gene (RB), which has been molecularly cloned. Whereas all normal tissues and many tumor cells express an RB mRNA of 4.7 kilobases, six of six retinoblastomas were previously found either to lack RB gene expression or to have RB transcripts of abnormal (reduced) length. To further characterize the latter type of mutation, we chose to examine retinoblastoma cell line Y79, which expressed a shortened RB mRNA of about 4.0 kilobases. RB cDNA clones isolated from a library constructed with Y79 mRNA demonstrated an internal loss of 470 nucleotides near the 5' end, which corresponded to a deletion of exons 2-6. Genomic clones containing the deletion junction were isolated from a library made with Y79 DNA, which allowed precise localization and sequencing of deletion endpoints in introns 1 and 6. These regions had no apparent homology to each other or to the Alu family of repetitive sequences, implying that the deletion must have occurred by a mechanism other than recombination of homologous sequences. Deletion of exons 2-6 would interrupt the open reading frame in RB mRNA and would result in premature termination of translation. Since no normal RB protein was detected by immunoprecipitation with specific antibody, the other, apparently normal RB allele in Y79 cells was necessarily inactivated by a different mutation.
Mechanism of inhibitory effect of dextran sulfate and heparin on replication of human immunodeficiency virus in vitro.
Authors: Baba M , Pauwels R , Balzarini J , Arnout J , Desmyter J , De Clercq E
Published: May 2006

The sulfated polysaccharides dextran sulfate and heparin have proved to be potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) in vitro. Dextran sulfate (Mr 5000) and heparin (Mr 15,000) completely protected MT-4 cells against HIV-1-induced cytopathogenicity at a concentration of 25 micrograms/ml. Their 50% inhibitory concentrations were 9.1 micrograms/ml (dextran sulfate) and 7.0 micrograms/ml (heparin), respectively. No toxicity for the host cells was observed with these compounds at a concentration of 625 micrograms/ml. The anti-HIV-1 activity of heparins of various molecular weights correlated well with their anticoagulant activity. On the other hand, with dextran sulfates of low molecular weight (5000, 8000) a significant inhibitory effect on HIV-1 was achieved at a concentration that was not markedly inhibitory to the blood coagulation process. Dextran sulfate and heparin were not inhibitory to HIV-1 reverse transcriptase unless they were used at concentrations in excess of those that inhibited HIV-1 replication. They were highly effective against HIV-1 replication even when present only during the 2-hr virus adsorption period. Studies using radiolabeled HIV-1 virions indicated that dextran sulfate and heparin inhibit virus adsorption to the host cells.
Functional expression of a Drosophila gene in yeast: genetic complementation of DNA topoisomerase II.
Authors: Wyckoff E , Hsieh T S
Published: May 2006

Since DNA topoisomerase II (EC 5.99.1.3) is an essential enzyme in yeast, heterologous topoisomerase II gene expression in yeast cells can provide a system for analyzing the structure and function of topoisomerase II genes from other species. A series of yeast expression plasmids was constructed in which segments of the cDNA sequences encoding Drosophila DNA topoisomerase II were inserted under the transcriptional control of yeast GAL1 promoter. Expression of the functional form of Drosophila topoisomerase II cDNA can complement conditionally lethal, temperature-sensitive mutations in the yeast topoisomerase II gene (TOP2), as well as mutations in which the TOP2 locus was disrupted. The survival of these yeast cells depends upon the continuous expression of Drosophila topoisomerase II. Repression of Drosophila gene expression by glucose causes these yeast cells to cease dividing after a few generations. In addition to these genetic complementation data, the expression of the Drosophila topoisomerase II gene in yeast cells with a disruption in TOP2 can also be detected by immunochemical methods with an antibody specific for Drosophila topoisomerase II.
Intramolecular DNA triplexes in supercoiled plasmids.
Authors: Hanvey J C , Shimizu M , Wells R D
Published: May 2006

A series of inserts with oligopurine.oligopyrimidine mirror repeat sequences was investigated at the base pair level with specific chemical probes (OsO4 and diethylpyrocarbonate) to evaluate the in vitro existence of intramolecular triplexes. Two parent inserts in recombinant plasmids with (GAA)9 and (AG)12 sequences and three mutant inserts (containing transitions or transversions) revealed that base pair changes at one location affected the chemical reactivity 13 base pairs away. The specificity and nature of these reactions, as well as the thermal stability of the complexes, provide direct evidence for the existence of a triplex with a portion of the pyrimidine-rich strand folded back and Hoogsteen-paired in the major groove of the Watson-Crick duplex. The biological implications of this unorthodox DNA structure are discussed.