bluuebunny/crossref_metadata_2025 huggingface dataset:
Calibration of DNA curvature and a unified description of sequence-directed bending.
Authors: Koo H S , Crothers D M
Published: May 2006

Chemically synthesized duplex oligodeoxynucleotides having different average numbers of adenine tracts (A6) per helix turn were ligated into multimers and analyzed by electrophoresis on polyacrylamide gels. The magnitude of the anomaly in gel mobility is found to be a quadratic function of the curvature of the DNA molecule. Parameters that describe intrinsic DNA bending, expressed as the tilt and roll components of the helix-axis deflection at the junctions between the adenine tract and adjacent B-DNA, were adjusted to fit the measured relative curvature of regularly repeated DNA bending sequences known from other studies and synthesized for this study. The model developed here retains the predominance of bending in the direction of tilt at the junctions but incorporates an appreciable roll component at the 5' end of an adenine tract, opening the minor groove there. This feature is consistent with chemical "footprinting" experiments on molecules containing adenine tracts. The overall direction of bending is effectively toward the minor groove, viewed from the center of an A5 or A6 tract. A possible underlying structure, which can also be described by a wedge bending model, is that derived from fiber diffraction studies of poly(dA).poly(dT). However, alternative models for the adenine tract, such as propeller twisted DNA, cannot be eliminated, although they do not lead to the correct direction of bending. The results permit calculation of the helix-axis trajectory of natural DNA molecules containing adenine-tract bends.
Human apolipoprotein B (apoB) mRNA: identification of two distinct apoB mRNAs, an mRNA with the apoB-100 sequence and an apoB mRNA containing a premature in-frame translational stop codon, in both liver and intestine.
Authors: Higuchi K , Hospattankar A V , Law S W , Meglin N , Cortright J , Brewer H B
Published: May 2006

Human apolipoprotein B (apoB) is present in plasma as two separate isoproteins, designated apoB-100 (512 kDa) and apoB-48 (250 kDa). ApoB is encoded by a single gene on chromosome 2, and a single nuclear mRNA is edited and processed into two separate apoB mRNAs. A 14.1-kilobase apoB mRNA codes for apoB-100, and the second mRNA, which codes for apoB-48, contains a premature stop codon generated by a single base substitution of cytosine to uracil at nucleotide 6538, which converts the translated CAA codon coding for the amino acid glutamine at residue 2153 in apoB-100 to a premature in-frame stop codon (UAA). Two 30-base synthetic oligonucleotides (nucleotides 6523-6552 of apoB mRNA), designated apoB-Stop and apoB-Gln, were synthesized containing the complementary sequence to the stop codon (UAA) and glutamine codon (CAA), respectively. Analysis of intestinal apoB mRNA by hybridization with apoB-Stop and apoB-Gln probes and sequence analysis of apoB clones in two independent human small intestinal cDNA libraries established that intestinal apoB mRNA contained both the apoB mRNA that codes for apoB-100 and the apoB mRNA containing the premature in-frame stop codon, which codes for apoB-48. Investigation of hepatic apoB mRNA and two hepatic cDNA libraries by hybridization with the apoB-Stop and apoB-Gln synthetic probes as well as by cDNA sequencing revealed that liver apoB mRNA also contains both the apoB-100 mRNA and the apoB-48 mRNA containing the stop codon. The combined results from these studies establish that both human intestine and liver contain the two distinct apoB mRNAs, an mRNA that codes for apoB-100 and an apoB mRNA that contains the premature stop codon, which codes for apoB-48. The premature in-frame stop codon is not tissue specific and is present in both human liver and intestine.
RecA-mediated cleavage activates UmuD for mutagenesis: mechanistic relationship between transcriptional derepression and posttranslational activation.
Authors: Nohmi T , Battista J R , Dodson L A , Walker G C
Published: May 2006

The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli. It has been shown that the UmuD protein shares homology with LexA, the repressor of the SOS genes. In this paper we describe a series of genetic experiments that indicate that the purpose of RecA-mediated cleavage of UmuD at its bond between Cys-24 and Gly-25 is to activate UmuD for its role in mutagenesis and that the COOH-terminal fragment of UmuD is necessary and sufficient for the role of UmuD in UV mutagenesis. Other genetic experiments are presented that (i) support the hypothesis that the primary role of Ser-60 in UmuD function is to act as a nucleophile in the RecA-mediated cleavage reaction and (ii) raise the possibility that RecA has a third role in UV mutagenesis besides mediating the cleavage of LexA and UmuD.
arcA (dye), a global regulatory gene in Escherichia coli mediating repression of enzymes in aerobic pathways.
Authors: Iuchi S , Lin E C
Published: May 2006

In Escherichia coli the levels of numerous enzymes associated with aerobic metabolism are decreased during anaerobic growth. In an arcA mutant the anaerobic levels of these enzymes are increased. The enzymes, which are encoded by different regulons, include members that belong to the tricarboxylic acid cycle, the glyoxylate shunt, the pathway for fatty acid degradation, several dehydrogenases of the flavoprotein class, and the cytochrome o oxidase complex. Transductional crosses placed the arcA gene near min O on the chromosomal map. Complementation tests showed that the arcA gene corresponded to the dye gene, which is also known as fexA, msp, seg, or sfrA because of various phenotypic properties [Bachmann, B. (1983) Microbiol. Rev. 47, 180-230]. A dye-deletion mutant was derepressed in the aerobic enzyme system. The term modulon is proposed to describe a set of regulons that are subject to a common transcriptional control.
Acetylcholine receptor-inducing factor from chicken brain increases the level of mRNA encoding the receptor alpha subunit.
Authors: Harris D A , Falls D L , Dill-Devor R M , Fischbach G D
Published: May 2006

A 42-kDa glycoprotein isolated from chicken brain, referred to as acetylcholine receptor-inducing activity (ARIA), that stimulates the rate of incorporation of acetylcholine receptors into the surface of chicken myotubes may play a role in the nerve-induced accumulation of receptors at developing neuromuscular synapses. Using nuclease-protection assays, we have found that ARIA causes a 2- to 16-fold increase in the level of mRNA encoding the alpha subunit of the receptor, with little or no change in the levels of gamma- and delta-subunit messengers. ARIA also increases the amount of a putative nuclear precursor of alpha-subunit mRNA, consistent with an activation of gene transcription. These results suggest that the concentration of alpha subunit may limit the rate of biosynthesis of the acetylcholine receptors in chicken myotubes. They also indicate that neuronal factors can regulate the expression of receptor subunit genes in a selective manner. Tetrodotoxin, 8-bromo-cAMP, and forskolin also increase the amount of alpha-subunit mRNA, with little change in the amount of gamma- and delta-subunit mRNAs. Unlike, ARIA, however, these agents have little effect on the concentration of the alpha-subunit nuclear precursor.
Some retinoblastomas, osteosarcomas, and soft tissue sarcomas may share a common etiology.
Authors: Weichselbaum R R , Beckett M , Diamond A
Published: May 2006

DNA and RNA were extracted from primary human osteosarcomas and soft tissue sarcomas obtained from patients without retinoblastoma and were analyzed by hybridization with a cDNA probe for RB mRNA; absence or alterations of the RB gene are associated with development of retinoblastoma. Most of the osteosarcomas or soft tissue sarcomas examined by us did not express detectable levels of RB mRNA, whereas normal cells and epithelial tumor cells did. One osteosarcoma expressed a 2.4-kilobase transcript in addition to a normal 4.7-kilobase species. Our data suggest that transcriptional inactivation or post-transcriptional down-regulation of the RB gene may be important in the etiology of some osteosarcomas and soft tissue sarcomas as well as retinoblastomas.
Isolation and sequencing of a cDNA coding for the human DF3 breast carcinoma-associated antigen.
Authors: Siddiqui J , Abe M , Hayes D , Shani E , Yunis E , Kufe D
Published: May 2006

The murine monoclonal antibody (mAb) DF3 reacts with a high molecular weight glycoprotein detectable in human breast carcinomas. DF3 antigen expression correlates with human breast tumor differentiation, and the detection of a cross-reactive species in human milk has suggested that this antigen might be useful as a marker of differentiated mammary epithelium. To further characterize DF3 antigen expression, we have isolated a cDNA clone from a lambda gt11 library by screening with mAb DF3. The results demonstrate that this 309-base-pair cDNA, designated pDF9.3, codes for the DF3 epitope. Southern blot analyses of EcoRI-digested DNAs from six human tumor cell lines with 32P-labeled pDF9.3 have revealed a restriction fragment length polymorphism. Variations in size of the alleles detected by pDF9.3 were also identified in Pst I, but not in HindIII, DNA digests. Furthermore, hybridization of 32P-labeled pDF9.3 with total cellular RNA from each of these cell lines demonstrated either one or two transcripts that varied from 4.1 to 7.1 kilobases in size. The presence of differently sized transcripts detected by pDF9.3 was also found to correspond with the polymorphic expression of DF3 glycoproteins. Nucleotide sequence analysis of pDF9.3 has revealed a highly conserved (G + C)-rich 60-base-pair tandem repeat. These findings suggest that the variation in size of alleles coding for the polymorphic DF3 glycoprotein may represent different numbers of repeats.
Poly(A) site choice rather than splice site choice governs the regulated production of IgM heavy-chain RNAs.
Authors: Galli G , Guise J , Tucker P W , Nevins J R
Published: May 2006

Alternative processing of the immunoglobulin mu primary transcript results in regulated production of mRNAs encoding the secreted (microseconds) and membrane-bound (micro m) form of IgM heavy chain during B-cell development. To elucidate the basis for this control, we analyzed the expression of altered forms of the mu transcription unit. Deletion of intron sequence between the microseconds and micro m exons, which reduces the distance between the two poly(A) sites as well as the distance between micro m splice sites, enhances production of micro m RNA. Correct expression is restored by insertion of heterologous sequences, demonstrating that spacing is indeed the critical aspect. The altered spacing appears to affect poly(A) site usage rather than splice site usage, since it was the distance between the poly(A) sites rather than the distance between splice sites that was found to be decisive. Finally, removal of either the C mu 4 splice donor or the m1 splice acceptor, thus eliminating normal micro m splicing, does not increase usage of the microseconds poly(A) site. We therefore conclude that the major factor in determining the ratio of microseconds to micro m is a poly(A) site choice rather than a splicing choice.
Evidence that glutamic acid 167 is an active-site residue of Shiga-like toxin I.
Authors: Hovde C J , Calderwood S B , Mekalanos J J , Collier R J
Published: May 2006

Escherichia coli Shiga-like toxin I, a close relative of Shiga toxin and a distant relative of the ricin family of plant toxins, inhibits eukaryotic protein synthesis by catalyzing the depurination of adenosine 4324 in 28S rRNA. By comparing the crystallographic structure of ricin with amino acids conserved between the Shiga and ricin toxin families, we identified seven potential active-site residues of Shiga-like toxin I. The structural gene encoding Shiga-like toxin I A chain (Slt-IA), the enzymatically active subunit, was engineered for high expression in E. coli. Oligonucleotide-directed mutagenesis of the gene for Slt-IA was used to change glutamic acid 167 to aspartic acid. As measured by an in vitro assay for inhibition of protein synthesis, the specific activity of mutant Slt-IA was decreased by a factor of 1000 compared to wild-type Slt-IA. Immunoblots showed that mutant and wild-type Slt-IA were synthesized as full-length proteins and were processed correctly by signal peptidase. Both proteins were equally susceptible to trypsin digestion, suggesting that the amino acid substitution did not produce a major alteration in Slt-IA conformation. We conclude that glutamic acid 167 is critical for activity of the Shiga-like toxin I A chain and may be located at the active site.
Evidence for tektins in centrioles and axonemal microtubules.
Authors: Steffen W , Linck R W
Published: May 2006

Affinity purified, polyclonal antibodies were prepared against three tektins (tektins A, B, and C) isolated from sea urchin sperm axonemal microtubules. These antibodies (anti-tektins) were used to localize tektins in axonemes, basal bodies, and centrioles. By immunofluorescence microscopy it could be demonstrated that in sperm tails from Lytechinus pictus all three anti-tektins stain all nine axonemal doublet microtubules and A-tubule extensions along their entire length. In addition to staining doublet microtubules, anti-tektin C weakly labeled the central-pair microtubules in sperm tails from Patiria miniata. The anti-tektin staining revealed also a clear cross-reaction with basal bodies of sea urchin sperm and with centrioles of cells from hamsters, humans, and pigs. These data provide evidence of tektin or tektin-like proteins in basal bodies and centrioles and suggest that centriole microtubules are constructed according to the same principles as flagellar microtubules.
Heart and bone tumors in transgenic mice.
Authors: Behringer R R , Peschon J J , Messing A , Gartside C L , Hauschka S D , Palmiter R D , Brinster R L
Published: May 2006

Tissue-specific tumorigenesis can be induced in transgenic mice by the directed expression of simian virus 40 (SV40) large tumor (T) antigen. In an attempt to determine the susceptibility of haploid, round spermatids to neoplastic transformation by this oncogene, transgenic mice were generated that harbored a chimeric gene composed of the SV40 T-antigen genes fused to the 5' and 3' flanking sequences of the mouse protamine 1 gene. The transgene was expressed in round spermatids and, surprisingly, in the heart and temporal bone as well. Expression in the heart resulted in rhabdomyosarcomas that always appeared in the right atrium. Bilateral osteosarcomas developed within the petrous portion of the temporal bone. No testicular pathology was observed. T-antigen immunostaining was readily detected in tumor tissue but not in the testis. In addition, SV40 transcripts were processed differently in testis and tumor tissue. Transgenic mouse lines were established that routinely develop these tumors, and they should provide a valuable resource for studies involving cardiac and bone physiology and neoplasia. The atrial tumor cells can be maintained in vitro and some continue to display a cardiac muscle phenotype.
Granulocyte-macrophage colony-stimulating factor enhances neutrophil function in acquired immunodeficiency syndrome patients.
Authors: Baldwin G C , Gasson J C , Quan S G , Fleischmann J , Weisbart R , Oette D , Mitsuyasu R T , Golde D W
Published: May 2006

We conducted a clinical trial of human recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) in leukopenic patients with acquired immunodeficiency syndrome (AIDS) and analyzed neutrophil function before, during, and after in vivo administration of rGM-CSF. Prior to GM-CSF infusion, AIDS patients' neutrophil superoxide generation and neutrophil antibody-dependent cell-mediated cytotoxicity were enhanced normally by in vitro exposure to GM-CSF. Neutrophil phagocytosis and intracellular killing of Staphylococcus aureus were also normal in the majority of these patients. Two patients, however, had discrete neutrophil functional defects: one in phagocytosis and one in intracellular killing. During the period of GM-CSF infusion, these abnormalities were corrected. The number of circulating neutrophils increased in all patients treated with GM-CSF in a dose-dependent manner. Neutrophils produced in vivo in response to GM-CSF administration functioned normally and there was evidence for neutrophil priming and activation in vivo. We conclude that GM-CSF treatment of AIDS patients leads to the production of functionally active neutrophils, suggesting therapeutic potential for GM-CSF in the treatment of patients with impaired host defense.
Heterogeneity of genomic fusion of BCR and ABL in Philadelphia chromosome-positive acute lymphoblastic leukemia.
Authors: Rubin C M , Carrino J J , Dickler M N , Leibowitz D , Smith S D , Westbrook C A
Published: May 2006

Philadelphia chromosome-positive acute lymphoblastic leukemia occurs in two molecular forms, those with and those without rearrangement of the breakpoint cluster region on chromosome 22. The molecular abnormality in the former group is similar to that found in chronic myelogenous leukemia. To characterize the abnormality in the breakpoint cluster region-unrearranged form, we have mapped a 9;22 translocation from the Philadelphia chromosome-positive acute lymphoblastic leukemia cell line SUP-B13 by using pulsed-field gel electrophoresis and have cloned the DNA at the translocation junctions. We demonstrate a BCR-ABL fusion gene on the Philadelphia chromosome. The breakpoint on chromosome 9 is within ABL between exons Ia and II, and the breakpoint on chromosome 22 is approximately equal to 50 kilobases upstream of a breakpoint cluster region in an intron of the BCR gene. This upstream BCR breakpoint leads to inclusion of fewer BCR sequences in the fusion gene, compared with the BCR-ABL fusion gene of chronic myelogenous leukemia. Consequently, the associated mRNA and protein are smaller. The exons from ABL are the same. Analysis of leukemic cells from four other patients with breakpoint cluster region-unrearranged Philadelphia chromosome-positive acute lymphoblastic leukemia revealed a rearrangement on chromosome 22 close to the breakpoint in SUP-B13 in only one patient. These data indicate that breakpoints do not cluster tightly in this region but are scattered, possibly in a large intron. Given the large size of BCR and the heterogeneity in breakpoint location, detection of BCR rearrangement by standard Southern blot analysis is difficult. Pulsed-field gel electrophoresis should allow detection at the DNA level in every patient and thus will permit clinical correlation of the breakpoint location with prognosis.
Interaction of origin binding protein with an origin of replication of herpes simplex virus 1.
Authors: Elias P , Lehman I R
Published: May 2006

Herpes simplex virus 1 encodes a protein that binds specifically to a viral DNA replication origin (oriS). This origin-binding protein has been purified to homogeneity by means of sequence-specific DNA affinity chromatography. The purified origin-binding protein, which has a molecular weight of 83,000, interacts with both parts of the oriS dyad; however, the affinity for the two sites differs by an order of magnitude. The sequence TTCGCA occurs as a nearly perfect direct repeat within the two sites and may constitute the recognition sequence for the origin-binding protein.
Drosophila homolog of the murine Int-1 protooncogene.
Authors: Uzvölgyi E , Kiss I , Pitt A , Arsenian S , Ingvarsson S , Udvardy A , Hamada M , Klein G , Sümegi J
Published: May 2006

We have isolated phage clones from Drosophila melanogaster genomic and cDNA libraries containing a sequence homologous to the murine Int-1 protooncogene. The Drosophila gene is represented by a single locus at position 28A1-2 on chromosome 2. The gene is expressed as a 2.9-kilobase-long polyadenylylated mRNA in embryo, larval, and pupal stages. It is hardly detectable in adult flies. The longest open reading frame of the cDNA clone corresponds to a protein 469 amino acids long. Alignment of the predicted amino acid sequences shows that the Drosophila protein is 86 amino acids longer than its murine counterpart. In spite of the difference in length, the two proteins are highly conserved with an overall sequence homology of 54%. Both Drosophila and murine Int-1 proteins begin with a hydrophobic leader sequence and contain cysteine residues and sites for glycosylation (four in the murine protein and one in the Drosophila protein) in conserved positions, suggesting that they play important functional roles.
Chloramphenicol induction of cat-86 requires ribosome stalling at a specific site in the leader.
Authors: Alexieva Z , Duvall E J , Ambulos N P , Kim U J , Lovett P S
Published: May 2006

The plasmid gene cat-86 specifies chloramphenicol-inducible chloramphenicol acetyltransferase in Bacillus subtilis. Induction by the antibiotic is primarily due to activation of the translation of cat-86-encoded mRNA. It has been suggested that the inducer stalls ribosomes at a discrete location in the leader region of cat-86 mRNA, which causes the destabilization of a downstream RNA secondary structure that normally sequesters the cat-86 ribosome binding site. It is the destabilization of this RNA secondary structure that permits translation of the cat-86 coding sequence. In the present report, we show that ribosomes that were stalled in the cat-86 leader by starvation of host cells for the amino acid specified by leader codon 6 induced gene expression to a level above that detected when cells were starved for the amino acids specified by leader codons 7 and 8. Starvation for amino acids specified by leader codons 3, 4, or 5 failed to activate cat-86 expression. These results indicate that the stalled ribosome that is most active in cat-86 induction has its aminoacyl site occupied by leader codon 6. To determine if chloramphenicol also stalled ribosomes in the cat-86 regulatory leader such that the aminoacyl site was occupied by codon 6, we separately changed leader codons 3, 4, 5, and 6 to the translation termination (ochre) codon TAA. Each of the mutated genes was tested for its ability to be induced by chloramphenicol. The results show that replacement of leader codons 3, 4, or 5 by the ochre codon blocked induction, whereas replacement of leader codon 6 by the ochre codon permitted induction. Collectively, these observations lead to the conclusion that cat-86 induction requires ribosome stalling in leader mRNA, and they identify leader codon 6 as the codon most likely to be occupied by the aminoacyl site of a stalled ribosome that is active in the induction.
Cell-surface changes associated with transformation of human hepatocytes to the malignant phenotype.
Authors: Wilson B , Ozturk M , Takahashi H , Motté P , Kew M , Isselbacher K J , Wands J R
Published: May 2006

Hepatocellular carcinoma is one of the leading causes of cancer death in the world. To understand the cellular changes associated with transformation of hepatocytes to the malignant state, we have made several libraries of monoclonal antibodies against the hepatocellular carcinoma cell line FOCUS and have found six antibodies (AF-20, SF-25, SF-31, SF-90, XF-4, and XF-8) that recognize antigens expressed at consistently higher levels on hepatoma cells. We have studied malignant and nontransformed liver tissue from the same individual by using direct 125I-labeled antibody binding and immunoperoxidase staining techniques. For each of these antibodies, we found striking increases in antigen expression on the transformed tissues. These antigens were found to be expressed throughout the tumor and on distant metastases, with little, if any, expression on the nontransformed adjacent liver. These antibodies demonstrate that hepatic transformation may be accompanied by stereotyped and predictable antigenic changes. The uniformity of such antigenic changes suggests an association between these cell-surface alterations and the malignant transformation process.
A translational enhancer derived from tobacco mosaic virus is functionally equivalent to a Shine-Dalgarno sequence.
Authors: Gallie D R , Kado C I
Published: May 2006

When present at the 5' end of mRNAs, the untranslated leader sequence (omega) of tobacco mosaic virus RNA significantly enhances translation in eukaryotes and prokaryotes. We have tested a deletion derivative of the omega sequence, omega delta 3, for its enhancing ability on gene constructs in which the ribosomal binding site was either present or deleted, in several Gram-negative bacterial species including Escherichia coli, Agrobacterium tumefaciens, Xanthomonas campestris pv. vitians, Erwinia amylovora, and Salmonella typhimurium. In vivo production of chloramphenicol acetyltransferase from a gene construct lacking its native ribosomal binding site was enhanced 40- to 120-fold by the presence of omega delta 3. Similar levels of enhancement (30- to 240-fold) were observed when the gene encoding beta-glucuronidase was tested. With a chloramphenicol acetyltransferase construct containing a ribosomal binding site, enhancement was markedly less, between 1- and 3.8-fold. Omega delta 3 appeared to enhance translation independent of its position upstream of the AUG codon used for initiation.
Chromatin architecture and nuclear RNA.
Authors: Nickerson J A , Krochmalnic G , Wan K M , Penman S
Published: May 2006

The maintenance of normal chromatin morphology requires ongoing RNA synthesis. We have examined the role of RNA in chromatin organization, using selective detergent extraction of cells, RNA synthesis inhibitors, and enzymatic digestion of nuclear RNA. Comparison of extracted and unextracted cells showed that the important features of chromatin architecture were largely unchanged by the extraction procedure. Normally, chromatin was distributed in small heterochromatic regions and dispersed euchromatic strands. Ribonucleoprotein granules were dispersed throughout the euchromatic regions. Exposure to actinomycin led to the redistribution of chromatin into large clumps, leaving large empty spaces and a dense clustering of the remaining ribonucleoprotein granules. When the nuclei of extracted cells were digested with RNase A, there was a rearrangement of chromatin similar to but more pronounced than that seen in cells exposed to actinomycin. The inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole also inhibits RNA synthesis but by a different mechanism that leaves no nascent RNA chains. The drug had little effect on chromatin after brief exposure but resembled actinomycin in its effect at longer times. We also examined the structure of the nuclear matrix to which most heteronuclear RNA remains associated. Pretreatment of cells with actinomycin or digestion of the nuclear matrix with RNase A caused the matrix fibers to collapse and aggregate. The experiments show a parallel decay of chromatin and of nuclear matrix organization with the depletion of nuclear RNA and suggest that RNA is a structural component of the nuclear matrix, which in turn may organize the higher order structure of chromatin.
Expression and replication of hepatitis B virus genome in transgenic mice.
Authors: Araki K , Miyazaki J , Hino O , Tomita N , Chisaka O , Matsubara K , Yamamura K
Published: May 2006

We produced transgenic mice by microinjecting a partial tandem duplication of the complete hepatitis B virus (HBV) genome into fertilized eggs of C57BL/6 mice. One of eight transgenic mice was a high producer for HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) in the serum. The HBV genomes were transmitted to the next generation and these F1 mice also produced HBsAg and HBeAg. mRNAs of 3.5, 2.1, and 0.8 kilobases were detected in the livers and the kidneys of these mice. In addition, a 0.8-kilobase RNA was detected in the testis. Single-stranded and partially double-stranded HBV DNAs were shown to be produced in the cytoplasm of the liver and kidneys. These HBV DNAs were associated with the core particles, indistinguishable from nucleocapsid produced in an infected human liver. Viral genome DNA was detected in the serum. These results demonstrate that the HBV genome integrated into the mouse chromosome acted as a template for viral gene expression, allowing viral replication. Thus, these transgenic mice should be useful for detailed studies of the replication and expression of HBV and for pathological studies of hepatitis, including the development of hepatocellular carcinoma.
Isolation and characterization of a cDNA and gene coding for a fourth CD1 molecule.
Authors: Balk S P , Bleicher P A , Terhorst C
Published: May 2006

The CD1 locus encodes a family of major histocompatibility complex (MHC) antigen-like glycoproteins which associate with beta 2-microglobulin and are expressed on immature thymocytes and Langerhans cells. Three CD1 molecules have been identified by monoclonal antibodies and molecular cloning: CD1a, -b, and -c. We have isolated a cDNA coding for a fourth CD1 molecule from a human thymocyte library and termed this molecule CD1d. Reported here are the complete nucleotide sequence and genomic organization of CD1d. They predict that this molecule is related to the previously identified CD1a, -b, and -c molecules and to MHC class I molecules, with three external domains, a transmembrane domain, and a short cytoplasmic tail. The sequence of CD1d is the most divergent among the CD1 molecules in the membrane-distal alpha 1 and alpha 2 domains and in the 5' untranslated region. In contrast, all four CD1 molecules are highly homologous in the membrane-proximal alpha 3 domain, which is likely involved in beta 2-microglobulin binding. A comparison of CD1 and MHC class I sequences suggests that these molecules each evolved to interact with a distinct set of cell surface proteins.
Rearrangement by inversion of a T-cell receptor delta variable region gene located 3' of the delta constant region gene.
Authors: Korman A J , Maruyama J , Raulet D H
Published: May 2006

We have located a T-cell receptor variable (V) delta gene segment immediately 3' of the delta constant (C) region gene and 5' to the known joining (J) alpha gene segments. This V delta gene is in the opposite transcriptional polarity to C delta and has rearranged to C delta by inversion in a gamma/delta-expressing hybridoma, DN7.3. This V delta gene is commonly rearranged in adult but not fetal gamma/delta-expressing thymocytes and has not been observed among alpha gene rearrangements reported to date. The reciprocal joining sequence isolated from this cell line contains N region nucleotides between the recombination signal sequences, in contrast to previously analyzed reciprocal joints. The results are discussed in the context of models accounting for ordered V gene usage during lymphocyte development.
B-lymphoma cells process and present their endogenous immunoglobulin to major histocompatibility complex-restricted T cells.
Authors: Weiss S , Bogen B
Published: May 2006

Antigen-presenting B-lymphoma cells were transfected with the gene encoding the immunoglobulin lambda 2 light chain of MOPC315 cells (lambda 2(315). The lambda 2 chain is expressed on the cell surface of the transfectants together with the endogenous heavy chain. The transfectants present an idiotope of the lambda 2(315) light chain to class II-restricted T-cell clones. Recognition by the T cells requires processing of the lambda 2(315) light chain. From these data we conclude that B-lymphoma cells constitutively process and present their immunoglobulins. Secretion and reuptake of the light chain was not necessary for the presentation. Thus, B cells bear two types of idiotypes on their membrane, a native form as surface immunoglobulin and a processed form in the context of products of the major histocompatibility complex.
Structure of the NGFI-A gene and detection of upstream sequences responsible for its transcriptional induction by nerve growth factor.
Authors: Changelian P S , Feng P , King T C , Milbrandt J
Published: May 2006

The NGFI-A gene encodes a "zinc-finger" protein that is rapidly induced by nerve growth factor (NGF) in PC12 rat pheochromocytoma cells. The complete exon/intron organization and nucleotide sequence of the rat NGFI-A gene have been determined. The gene spans 3789 nucleotides (nt) and is interrupted by a single intron at nt 588. All three zinc-finger DNA-binding domains are contiguously coded for within the 3' exon; this is in contrast to the structure described by others for the Xenopus laevis transcription factor TFIIIA gene. To analyze the transcription of this gene, we have determined the transcription start site and nucleotide sequence of the 5' flanking region. Transfection of PC12 cells with a fragment from the 5' flanking region linked to the chloramphenicol acetyltransferase (CAT) gene revealed that it contains an element which imparts an NGF-inducible phenotype to the normally silent CAT gene. Several regions with homologies to recognizable sequence elements are present in this fragment, including a TATA box at nt -27, serum response elements at nt -84, -106, -370, and -408, a cAMP-responsive element at nt -140, and a transcription factor Sp1-binding site at nt -286. These results establish the genomic structure of this mammalian multifinger protein and demonstrate that an NGF-responsive element lies upstream of the NGFI-A gene.
Peptide sequencing and site-directed mutagenesis identify tyrosine-727 as the active site tyrosine of Saccharomyces cerevisiae DNA topoisomerase I.
Authors: Lynn R M , Bjornsti M A , Caron P R , Wang J C
Published: May 2006

Extensive digestion of the covalent intermediate between DNA and Saccharomyces cerevisiae DNA topoisomerase I with trypsin yields a 7-amino acid peptide covalently linked to DNA. Direct sequencing of the DNA-linked peptide identifies Tyr-727 as the active site tyrosine that forms an O4-phosphotyrosine bond with DNA when the enzyme cleaves a DNA phosphodiester bond. Site-directed mutagenesis of the cloned yeast TOP1 gene encoding the enzyme confirms the essentiality of Tyr-727 for the relaxation of supercoiled DNA by the enzyme. From amino acid sequence homology, Tyr-771 and -773 are readily identified as the active site tyrosines of Schizosaccharomyces pombe and human DNA topoisomerase I, respectively. Sequence comparison and site-directed mutagenesis also implicate Tyr-274 of vaccinia virus DNA topoisomerase as the active site residue. There appears to be a 70-amino acid domain near the carboxyl terminus of eukaryotic DNA topoisomerase I and vaccinia topoisomerase, within which the active site tyrosine resides.
Cloning and characterization of mammalian homologs of the Drosophila dunce+ gene.
Authors: Davis R L , Takayasu H , Eberwine M , Myres J
Published: May 2006

A probe representing the Drosophila dunce+ (dnc+) gene, the structural gene for a cAMP phosphodiesterase (PDEase), detects homologous sequences in many different organisms, including mouse, rat, and human. Genomic and cDNA clones representing a homolog of the Drosophila dnc+ gene were isolated from rat libraries and characterized. This gene has been named ratdnc-1. One cDNA clone defines a large open reading frame of approximately 1.8 kilobases (kb), predicting a protein sequence of 610 amino acids with significant homology to a conserved domain of approximately 275 residues found in most other PDEases. The amino acid identity value to the Drosophila cAMP PDEase within this domain is a striking 75%. Other cDNA clones show blocks of sequence divergence from this cDNA clone close to the predicted N terminus, indicating the potential existence of a family of related enzymes encoded by alternatively spliced messenger RNAs from ratdnc-1. Genomic blotting experiments suggest the existence of at least one other rat gene with homology to ratdnc-1. RNAs homologous to ratdnc-1 are heterogeneous in size between tissues, with heart containing a major transcript of 4.4 kb and brain one of 4.0 kb. The potential identity of the product of the ratdnc-1 gene with known PDEases is discussed.
Lithium evokes expression of vegetal-specific molecules in the animal blastomeres of sea urchin embryos.
Authors: Livingston B T , Wilt F H
Published: May 2006

The mechanism of determination of early embryonic cells has been investigated using sea urchin embryos. An efficacious method of isolating blastomere pairs from the animal or vegetal half of sea urchin embryos was developed. The overt differentiation of separated animal and vegetal blastomere pairs resembles that of separated animal and vegetal hemispheres isolated by manual dissection. Treatment of animal blastomeres with LiCl caused them to display a morphology resembling that of isolated vegetal blastomeres. The effects of separation of animal and vegetal blastomeres and of treatment of animal blastomeres with LiCl were examined at the molecular level using gut alkaline phosphatase and a spicule matrix protein RNA as markers of differentiation. Histochemical staining and in situ hybridization studies showed that these markers are normally only expressed in vegetal blastomeres but that their expression can be evoked in animal blastomeres by treatment with LiCl.
Nonrandom utilization of codon pairs in Escherichia coli.
Authors: Gutman G A , Hatfield G W
Published: May 2006

We have analyzed protein-coding sequences of Escherichia coli and find that codon-pair utilization is highly biased, reflecting overrepresentation or underrepresentation of many pairs compared with their random expectations. This effect is over and above that contributed by nonrandomness in the use of amino acid pairs, which itself is highly evident; it is much weaker when nonadjacent codon pairs are examined and virtually disappears when pairs separated by two or three intervening codons are evaluated. There appears to be a high degree of directionality in this bias: any codon that participates in many nonrandom pairs tends to make both over- and underrepresented pairs, but preferentially as a left- or right-hand member. We show a relationship between codon-pair utilization patterns and levels of gene expression: genes encoding proteins expressed at high levels tend to contain more abundant, but more highly underrepresented, codon pairs, relative to genes expressed at low levels. The nonrandom utilization of codon pairs may be a consequence of their effects on translational efficiency, which in turn may be related to the compatibility of adjacent aminoacyl-tRNA isoacceptors at the A and P sites of a translating ribosome.
Genetic characterization of clones of the bacterium Listeria monocytogenes causing epidemic disease.
Authors: Piffaretti J C , Kressebuch H , Aeschbacher M , Bille J , Bannerman E , Musser J M , Selander R K , Rocourt J
Published: May 2006

One hundred and seventy-five isolates of the pathogenic bacterium Listeria monocytogenes recovered from human clinical (blood and cerebrospinal fluid), animal, and environmental sources in Europe, North America, and elsewhere were analyzed electrophoretically for allelic variation at 16 genetic loci encoding metabolic enzymes. Forty-five distinctive allele profiles (electrophoretic types, ETs) were distinguished, among which mean genetic diversity per locus (H) was 0.424. Cluster analysis of a matrix of genetic distances between paired ETs revealed two primary phylogenetic divisions of the species separated at a distance of 0.54. ETs in division I were presented by strains of serotypes 4b, 1/2b, and 4a, whereas strains of ETs in division II were of serotypes 1/2a and 1/2c. Human and animal isolates did not represent distinctive subsets of ETs. The occurrence of linkage disequilibrium between enzyme loci and the widespread distribution of certain ETs indicate that the genetic structure of L. monocytogenes is clonal. One clone, marked by ET1, caused major epidemics of human disease in western Switzerland in the period 1983-1987 and in Los Angeles County, California, in 1985, both of which were attributed to contamination of soft cheese. ET 1 is closely related to the clone (ET7) that caused two large outbreaks of listeriosis in Massachusetts in 1979 and 1983.
Dipyridamole potentiates the inhibition by 3'-azido-3'-deoxythymidine and other dideoxynucleosides of human immunodeficiency virus replication in monocyte-macrophages.
Authors: Szebeni J , Wahl S M , Popovic M , Wahl L M , Gartner S , Fine R L , Skaleric U , Friedmann R M , Weinstein J N
Published: May 2006

Dipyridamole (DPM) is commonly used as a coronary vasodilator and inhibitor of platelet aggregation in the treatment of cardiovascular diseases. We report here that DPM potentiates the inhibitory effects of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine against human immunodeficiency virus type 1 (HIV-1) in human monocyte-macrophages. At the same concentrations, DPM does not potentiate the toxic effects of AZT on these cells or on human bone marrow (granulocyte-monocyte) progenitor cells. Since monocyte-macrophage lineage cells appear to be the major reservoir for HIV-1 in vivo, these findings suggest the possibility of using DPM or its analogues in combination chemotherapy of HIV infections.
Identification of icsA, a plasmid locus of Shigella flexneri that governs bacterial intra- and intercellular spread through interaction with F-actin.
Authors: Bernardini M L , Mounier J , d'Hauteville H , Coquis-Rondon M , Sansonetti P J
Published: May 2006

The capacity of Shigella to spread within the cytosol of infected epithelial cells and to infect adjacent cells is critical for the development of infection foci, which lead to mucosal abscesses. Shigella is a nonmotile microorganism that appears to utilize host cell microfilaments to generate intra- as well as intercellular movements, since this movement was inhibited by cytochalasin D and involvement of F-actin was demonstrated by direct labeling of infected cells with the specific dye N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin. Such movements led to the formation of extracellular protrusions, which may explain cell to cell spread. icsA, a locus necessary for intra- and intercellular spread, was identified on the Shigella flexneri virulence plasmid pWR100. This locus was cloned and shown to express a 120-kDa outer membrane protein, which plays an important role in the interactions established between host cell microfilaments and the bacterial surface, thus leading to intracellular movement.
Purification and characterization of glia maturation factor beta: a growth regulator for neurons and glia.
Authors: Lim R , Miller J F , Zaheer A
Published: May 2006

A protein has been isolated from bovine brains by using a modification of the procedure used to purify glia maturation factor. The method consists of ammonium sulfate precipitation, chromatography with DEAE-Sephacel, Sephadex G-75, and hydroxylapatite columns, passage through a heparin-Sepharose column, and finally fractionation by reverse-phase HPLC with a C4 column. The isolated protein reacts strongly with the mouse monoclonal antibody G2-09 and has a molecular weight of approximately 17,000 and an isoelectric point of pH 4.9. The N terminus is blocked, but tryptic digestion releases 28 peptides, 8 of which have been sequenced. The total known residues add up to more than two-thirds of the entire 140-residue protein, estimated from amino acid composition, and show no sequence homology with any known protein. Reversible thermal renaturation greatly enhances its biological activity. The purified protein stimulates differentiation of normal neurons as well as glial cells. It inhibits the proliferation of the N-18 neuroblastoma line and the C6 glioma line while promoting their phenotypic expression. We designate this protein glia maturation factor beta.
Correlation between preferred sugar ring conformation and activity of nucleoside analogues against human immunodeficiency virus.
Authors: Van Roey P , Salerno J M , Chu C K , Schinazi R F
Published: May 2006

Analysis of the solid-state conformations of six active and two inactive anti-human immunodeficiency virus nucleoside analogues is used to correlate conformational features with the relative activities of the compounds. Ten of the 11 observations of the active compounds (3'-azido-3'-deoxythymidine, 3'-azido-2',3'-dideoxyuridine, 3'-azido-2',3'-dideoxy-5-ethyluridine, 2',3'-dideoxyadenosine, 2',3'-dideoxycytidine, and 3'-deoxythymidine) have C3'-exo sugar ring conformations. The only exception is one of the two molecules of 3'-deoxythymidine. Four have unusual low anti glycosyl link conformations coupled with unusual C3'-exo/C4'-endo sugar ring conformations. The inactive compounds, 2',3'-dideoxyuridine and 3'-(propyl-2-ene)-2',3'-dideoxyuridine, have C3'-endo conformations. The C3'-exo and C3'-endo conformations place C5' in axial and equatorial positions, respectively. This affects the location of the 5'-hydroxyl group in relation to the location of the base. The 5'-hydroxyl group is the site of phosphorylation of the nucleoside and the observation of this conformational preference of the active compounds may be of importance for the development of new nucleosides with activity against human immunodeficiency virus.
Chemical synthesis of echistatin, a potent inhibitor of platelet aggregation from Echis carinatus: synthesis and biological activity of selected analogs.
Authors: Garsky V M , Lumma P K , Freidinger R M , Pitzenberger S M , Randall W C , Veber D F , Gould R J , Friedman P A
Published: May 2006

Echistatin, a polypeptide from the venom of the saw-scaled viper, Echis carinatus, containing 49 amino acids and 4 cystine bridges was synthesized by solid-phase methodology in 4% yield. In the final step, air oxidation of the octahydroderivative was found to be optimal at pH 8. The synthetic product was shown to be physically and biologically indistinguishable from native material. It inhibits fibrinogen-dependent platelet aggregation stimulated by ADP with IC50 = 3.3 x 10(-8) M and also prevents aggregation initiated by thrombin, epinephrine, collagen, or platelet-activating factor. Reduction of purified synthetic echistatin to octahydroechistatin with dithiothreitol followed by air oxidation regenerated homogeneous echistatin in quantitative yield. This highly specific refolding strongly suggests that the linear sequence of octahydroechistatin contains all of the information that is required for the proper folding of the peptide. The sequence Arg24-Gly-Asp of echistatin occurs also in adhesive glycoproteins that bind to the platelet fibrinogen receptor--a heterodimeric complex composed of glycoproteins IIb and IIIa. In an effort to evaluate the role of this putative binding site we have synthesized analogs of echistatin with substitution of Arg-24. Replacement with ornithine-24 (Orn-24) resulted in an analog having a platelet aggregation inhibitory activity with IC50 = 1.05 x 10(-7) M. Substitution with Ala-24 gave IC50 = 6.1 x 10(-7) M. The inhibitory activity of the corresponding short sequence analogs Arg-Gly-Asp-Phe (IC50 = 6 x 10(-6) M), Orn-Gly-Asp-Phe (IC50 = 1.3 x 10(-4) M), and Ala-Gly-Asp-Phe (IC50 = 5.0 x 10(-4) M) was also determined. These results suggest that arginine plays a more important role in the binding of the tetrapeptide than in that of echistatin.