bluuebunny/crossref_metadata_2025 huggingface dataset:
Exonuclease III and endonuclease IV remove 3' blocks from DNA synthesis primers in H2O2-damaged Escherichia coli.
Authors: Demple B , Johnson A , Fung D
Published: May 2006

Escherichia coli deficient in exonuclease III (xth gene mutants) are known to be hypersensitive to hydrogen peroxide. We now show that such mutants accumulate many more DNA single-strand breaks than do wild-type bacteria upon exposure to H2O2. DNA isolated from H2O2-treated xth- cells contains strand breaks that do not efficiently support synthesis by E. coli DNA polymerase I, indicating the presence of blocking groups at the DNA 3' termini. Purified E. coli exonuclease III activates this blocked DNA to allow substantial synthesis by polymerase I in vitro. Another E. coli enzyme, endonuclease IV, also activates primers for DNA polymerase. Exonuclease III accounts for greater than 95% of the total activity in E. coli crude extracts for removal of 3'-terminal phosphoglycolaldehyde esters from model DNA substrates. Purified exonuclease III and endonuclease IV can each efficiently remove 3'-terminal phosphoglycolaldehyde in vitro. An important physiological function for exonuclease III is thus the activation of blocked 3' ends for DNA repair synthesis. Endonuclease IV can also initiate the repair of ruptured 3'-deoxyribose in DNA.
Functional complementation between mutations in a yeast suppressor tRNA gene reveals potential for evolution of tRNA sequences.
Authors: Willis I , Nichols M , Chisholm V , Söll D , Heyer W D , Szankasi P , Amstutz H , Munz P , Kohli J
Published: May 2006

Successive rounds of mutagenesis of a Schizosaccharomyces pombe strain bearing the UGA-reading sup3 tRNASer suppressor have been carried out for two cycles of inactivation and reactivation of the suppressor. The suppressor phenotype at each stage was found to involve different combinations of three mutations, A30, A53, and A67, in the sup3-UGA gene. Single mutations A30 and A53 inactivate the suppressor as does the presence of all three mutations. A67 by itself is phenotypically neutral, but in combination with either A30 or A53 suppressor function is restored. The frequency with which these and other complementation events occur in S. pombe demonstrates a significant potential for nucleotide sequence evolution in tRNA. Differential expression of the S. pombe genes in Saccharomyces cerevisiae suggests that the two yeasts have diverged at the transcriptional and RNA processing level. Processing of the mutant tRNA precursors in S. cerevisiae reveals a hierarchy of structural domains within the tRNA that vary in their importance for RNase P cleavage.
Rapid degradation restricts measles virus matrix protein expression in a subacute sclerosing panencephalitis cell line.
Authors: Sheppard R D , Raine C S , Bornstein M B , Udem S A
Published: May 2006

Measles virus matrix protein expression is restricted in the persistently infected brain cells of patients with the chronic neurological disease subacute sclerosing panencephalitis (SSPE). Prior studies of the nature of this restriction have identified polyadenylylated matrix gene-encoded RNA transcripts unable to direct effective translation. The defective nature of these mRNAs readily accounted for the inability to detect matrix protein in these persistently infected cells and suggested that in SSPE the restriction of matrix protein expression is achieved by preventing its synthesis. Recently, however, we reported evidence that matrix protein is synthesized in at least one example of this persistent infection, the SSPE cell line IP-3-Ca. In this case, failure of matrix protein to accumulate normally accounted for its restricted expression [Sheppard, R. D., Raine, C. S., Bornstein, M. B. & Udem, S. A. (1985) Science 228, 1219-1221]. To clarify the nature of the restriction displayed by IP-3-Ca cells, the synthesis and fate of the matrix protein of this SSPE cell line were examined in detail. No evidence of constraints on the efficiency of matrix protein mRNA transcription or translation was found. Instead, the restricted expression proved to be the result of rapid posttranslational degradation of matrix protein. We suggest that matrix protein gene mutations incurred in the course of genome replication are likely to be responsible for the diversity of observed mechanisms restricting matrix protein expression. In that event, the nature and position of the nucleotide substitution(s) would be the determinants of the level at which restricted expression is achieved.
Light paired with serotonin mimics the effect of conditioning on phototactic behavior of Hermissenda.
Authors: Crow T , Forrester J
Published: May 2006

A conditioning procedure consisting of pairing-specific stimulation of the eyes and gravity-detecting statocysts in Hermissenda results in a long-term modification of normal positive phototactic behavior. The learning is expressed by a significant suppression of the initiation of locomotion in the presence of light. We now report that an analogue of the classical conditioning procedure, consisting of light paired with serotonin (5-HT) applied directly to the exposed circumesophageal nervous system of otherwise intact animals, mimics the effect of conditioning on long-term changes in phototactic behavior. The effect of the conditioning analogue on behavior shows some specificity with 5-HT since light paired with dopamine or octopamine does not significantly affect phototactic behavior. The conditioning analogue exhibits pairing specificity since unpaired light and 5-HT and 5-HT applied in the dark do not produce behavioral suppression. Animals that initially received unpaired light and 5-HT do show behavioral suppression after receiving paired light and 5-HT. These results indicate that light (the conditioned stimulus) paired with the putative transmitter of the unconditioned stimulus pathway (5-HT) is sufficient to produce long-term phototactic suppression.
Location of regulatory elements responsible for drug induction in the rat cytochrome P-450c gene.
Authors: Sogawa K , Fujisawa-Sehara A , Yamane M , Fujii-Kuriyama Y
Published: May 2006

The synthesis of cytochrome P-450c is induced remarkably in cultured cells as well as animal tissues in response to added chemicals such as 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzodioxin. To study this mechanism, we joined the sequence of 5'-flanking and upstream regions of the P-450c gene to the structural gene for chloramphenicol acetyltransferase. The fusion gene was introduced into Hepa-1 cells for the assay of the expressed acetyltransferase activity. At least three cis-acting regulatory regions that are responsible for the inductive expression were determined in the sequences from nucleotide -3674 to -3067, from -1682 to -1429, and from -1139 to -1029, relative to the transcription start site, by external deletion analysis. Further detailed analysis of the region (nucleotides -1139 to -1029) most influential on the inducibility revealed that a regulatory element consisting of 10 base pairs termed a drug regulatory element (DRE) and its homologues were tandemly arranged in this region. The consensus sequence deduced from DREs is 5'-GCNTGAGGCTGGG-3'. The regulatory sequence from nucleotide -1140 to -844 is capable of conferring inducibility on a heterologous promoter in a manner independent of its orientation and distance from the subordinate promoter.
Action mechanism of ABC excision nuclease on a DNA substrate containing a psoralen crosslink at a defined position.
Authors: Van Houten B , Gamper H , Holbrook S R , Hearst J E , Sancar A
Published: May 2006

Many carcinogenic as well as chemotherapeutic agents cause covalent linkages between complementary strands of DNA. If unrepaired, DNA crosslinks are blocks to DNA replication and transcription and therefore represent potentially lethal lesions to the cell. Genetic studies of Escherichia coli have demonstrated that the repair enzyme ABC excision nuclease, coded for by the three unlinked genes, uvrA, uvrB, and uvrC, plays a crucial role in DNA crosslink repair. To study the molecular events of ABC excision nuclease-mediated crosslink repair, we have engineered a DNA fragment with a psoralen-DNA interstrand crosslink at a defined position, digested this substrate with pure enzyme, and analyzed the reaction products on DNA sequencing gels. We find that the excision nuclease cuts only one of the two strands involved in the crosslink, incises the crosslink by hydrolyzing the ninth phosphodiester bond 5' and the third phosphodiester bond 3' to the furan-side thymine of the crosslink, and does not produce double-strand breaks at any significant level. Based on these data, we present a model by which ABC excision nuclease initiates crosslink repair in vivo.
Site-specific mutagenesis of the calcium-binding photoprotein aequorin
Authors: Tsuji Frederick I. , Inouye Satoshi , Goto Toshio , Sakaki Yoshiyuki
Published: May 2006

The luminescent protein aequorin from the jellyfish
Aequoria victoria
emits light by an intramolecular reaction in the presence of a trace amount of Ca
2+
. In order to understand the mechanism of the reaction, a study of structure-function relationships was undertaken with respect to modifying certain of its amino acid residues. This was done by carrying out oligonucleotide-directed site-specific mutagenesis of apoaequorin cDNA and expressing the mutagenized cDNA in
Escherichia coli
. Amino acid substitutions were made at the three Ca
2+
-binding sites, the three cysteines, and a histidine in one of the hydrophobic regions. Subsequent assay of the modified aequorin showed that the Ca
2+
-binding sites, the cysteines, and probably the histidine all play a role in the bioluminescence reaction of aequorin.
Liver-specific expression of the mouse alpha-fetoprotein gene is mediated by cis-acting DNA elements.
Authors: Widen S G , Papaconstantinou J
Published: May 2006

We have identified cis-acting regulatory elements in the 5' flanking region of the mouse alpha-fetoprotein (Afp) gene, using the expression of the bacterial gene for chloramphenicol acetyltransferase (CAT) in a transient expression assay. Tissue-specific enhancer activity was determined by transfection of mouse hepatoma (BWTG3) and fibroblast cells (C127, NIH 3T3) with various DNA fragments linked to the CAT gene. A 5.4-kilobase restriction fragment was shown to have characteristics typical of enhancers, including the ability to function independent of orientation and position and the ability to enhance transcription from a heterologous promoter. The enhancer activity was greatest in the hepatoma cells, which express Afp. By deletion analysis, it was demonstrated that enhancer activity is present in several subfragments, indicating the presence of more than one element in this fragment. An additional regulatory element within 950 base pairs of the Afp transcription initiation site has been identified and shown to confer tissue-specific expression on the CAT gene. This fragment, which lacks enhancer activity, contains the Afp promoter region and mediates the tissue-specific expression of the CAT gene when driven by nonspecific viral enhancers. We conclude from our studies that there are several types of regulatory elements in the 5' flanking region of the Afp gene that help mediate tissue-specific expression.
Antibodies to liver cell adhesion molecule perturb inductive interactions and alter feather pattern and structure.
Authors: Gallin W J , Chuong C M , Finkel L H , Edelman G M
Published: May 2006

Cell adhesion molecules (CAMs) may act as regulators of morphogenesis by constraining cell motion, forming borders, and controlling intercellular communications that lead to embryonic induction. This postulated causal role of CAMs in inductive events was tested here in an in vitro system of feather induction. In the developing chicken skin, an ectodermal sheet of epithelium interacts with mesodermal cell collectives to form more or less circular feather germs arranged in a hexagonal pattern. Cells of the epidermal epithelium are linked by liver CAM (L-CAM) and mesodermal cells in dermal condensations are linked by neural CAM (N-CAM); neither of these CAMs links cells in one tissue of this inductive couple to cells in the other. After perturbation of the L-CAM linkage in epidermis by antibodies to L-CAM, nonhexagonal striped patterns of dermal condensations were observed in culture. The stripes did not follow straight lines but meandered in lateral and oblique directions. Histological examination of the perturbed tissues showed extensive changes in dermal cell density distributions. After 10 days of culture, the perturbed tissues developed a cobbled or plaque-like morphology resembling scales rather than the feather-like filamentous structures that formed in unperturbed skin cultures. The results indicate that perturbation of CAM binding in tissues linked by one CAM can alter fates and interactions of cells linked by another, presumably by altering the amount or effect of inductive signals crossing the border between the inducing cell collectives. A computer model based on the notion that the response of L-CAM-linked epidermal cells to signals from N-CAM-linked dermal cells depends cooperatively on the degree of L-CAM linkage was found to generate hexagonal patterns for the unperturbed case and stripes after perturbation of L-CAM bonds.
Trophic molecules and evolution of the nervous system.
Authors: Black I B
Published: May 2006

Although recent work has reemphasized the general importance of ontogeny in evolution, underlying developmental molecular mechanisms are largely undefined. What heritable ontogenetic mechanisms result in the evolution of new morphologies and functions? Such questions are particularly difficult in the nervous system, in which each of 10(11) neurons forms approximately equal to 10(4) specific interconnections. I propose that specific heritable, trophic interactions during development, which determine cell survival and pathway size, form a substrate for neural evolution. This model is based on the observation that neurons are vastly overproduced during ontogeny; neurons, their pathways and connections are dependent on target-derived trophic factors for developmental survival; and co-innervating, functionally and anatomically distinct neural populations compete for common trophic factors for survival. Focusing on sympathetic and sensory neurons, which require the target-derived, trophic protein nerve growth factor at different times for developmental survival, and which innervate common targets, different classes of ontogenetic evolutionary mechanisms may be characterized. Evolution may occur from heritable changes in the structure of trophic gene products or altered timing of expression. Molecular mechanisms underlying heterochrony are thereby described. The model is directly applicable to evolution of the brain and is testable in a variety of situations.
Repression mediates cell-type-specific expression of the rat growth hormone gene.
Authors: Larsen P R , Harney J W , Moore D D
Published: May 2006

A plasmid containing 1.8 kilobase pairs of rat growth hormone (rGH) promoter and upstream flanking sequences fused to the bacterial gene for chloramphenicol acetyltransferase (CAT) was transiently introduced into pituitary, fibroblast, and kidney cell lines. Significant CAT activity was detectable only in the pituitary cell lines, demonstrating that this relatively large fragment directs strongly cell-type-specific expression. However, plasmids containing only 200-300 bases of rGH promoter and flanking sequences directed expression of CAT in all three cell types, suggesting that upstream sequences directly repress the activity of a minimal rGH promoter in nonpituitary cell types. S1 nuclease analysis showed that the RNA synthesis directed by one of the short rGH promoter fragments in fibroblasts initiated from the site used by the natural promoter in pituitary cells. Insertion of rGH upstream sequences in their natural orientation upstream of the mouse metallothionein I promoter caused a decrease in its activity in fibroblasts by a factor of 4, but there was a 2.5-fold increase in its activity in pituitary cells. Insertion of the rGH fragment upstream of the thymidine kinase promoter in either orientation lowered its activity in both fibroblasts and pituitary cells. Thus, the negatively acting rGH flanking sequences can act on a heterologous promoter and have at least some of the properties of positively acting enhancers.
Immunization with synthetic peptides of a Plasmodium falciparum surface antigen induces antimerozoite antibodies.
Authors: Cheung A , Leban J , Shaw A R , Merkli B , Stocker J , Chizzolini C , Sander C , Perrin L H
Published: May 2006

Polypeptides expressed on the surface of merozoites, the invasive stage of the asexual blood cycle, are good candidates for the development of malaria vaccines. Five synthetic peptides with predetermined specificity deduced from a genomic DNA clone coding for the NH2-terminal portion of the main merozoite surface polypeptide of Plasmodium falciparum were evaluated for their capability to raise antibodies that react with the P. falciparum merozoites. Antibodies induced by two of the peptides (3 and 5) reacted with the membrane surfaces of seven of seven isolates of P. falciparum from different geographic areas. Antibodies against peptide 4, which contains a repeated amino acid sequence (Gly-Gly-Ser and Val-Ala-Ser), reacted with six of seven isolates. Structural analysis of the deduced polypeptides suggests that peptide 3 is exposed at the surface of merozoites. When it was used to immunize monkeys, three of the four animals were partially protected from a challenge infection that induced a fulminant infection in control animals.
Sequence and expression of transcripts of the T-cell antigen receptor alpha-chain gene in a functional, antigen-specific suppressor-T-cell hybridoma.
Authors: Imai K , Kanno M , Kimoto H , Shigemoto K , Yamamoto S , Taniguchi M
Published: May 2006

We have detected rearrangement and expression of a gene encoding a T-cell antigen receptor alpha chain in the keyhole limpet hemocyanin (KLH)-specific, inducible suppressor-T-cell (Ts) hybridoma 34S-281 (BW5147 lymphoma-C57BL/6Ts) by using alpha-chain cDNA clones isolated from this Ts hybridoma. The cDNA clones have a restriction length polymorphism in the constant region that identifies them as being of C57BL/6 origin. The cDNA sequence has an ATG start codon for an open reading frame including variable, joining, and constant gene segments. Furthermore, the Ts alpha-chain gene transcripts were detected on membrane-bound polysomes by RNA blot analysis using a variable-region fragment from one of the alpha-chain cDNA clones as probe, suggesting that they are actively translated in the Ts hybridoma. As the beta-chain gene is deleted in all Ts hybridomas analyzed, and since a disulfide-linked dimer is detectable by two-dimensional NaDodSO4/PAGE of lysates of surface-radioiodinated Ts, we suggest that Ts antigen receptors are either alpha-chain homodimers or heterodimers composed of the alpha chain in association with an undefined chain.
A repetitive antigen of Plasmodium falciparum that is homologous to heat shock protein 70 of Drosophila melanogaster.
Authors: Bianco A E , Favaloro J M , Burkot T R , Culvenor J G , Crewther P E , Brown G V , Anders R F , Coppel R L , Kemp D J
Published: May 2006

We describe an antigen of Plasmodium falciparum, defined by a cDNA clone designated Ag63. The antigen is an abundant, soluble cytoplasmic polypeptide of Mr 75,000 present in all stages of asexual development in the blood and in gametocytes, but not in sporozoites. The sequence of the cDNA clone revealed that, like many other antigens of P. falciparum, it contains tandemly repeated amino acid sequences, in this case Gly-Gly-Met-Pro. However, the rest of the sequence is 70% homologous at the amino acid level to the heat shock protein hsp70 of Drosophila melanogaster.
The role of the T-cell receptor in thymocyte maturation: effects in vivo of anti-receptor antibody.
Authors: McDuffie M , Born W , Marrack P , Kappler J
Published: May 2006

The T-cell receptor, which recognizes antigen plus a product of the major histocompatibility complex, has been postulated to drive T-cell maturation in the thymus by engaging major histocompatibility complex proteins expressed on thymic stromal cells. We tested this idea by injecting neonatal animals with an anti-receptor antibody, KJ16, that binds to about 20% of T cells and is capable of blocking receptor function. In the presence of this antibody, mature T cells bearing the KJ16 epitope failed to develop. On the other hand, although the antibody could be shown to bind to receptors on cortical thymocytes, it did not prevent the rapid expansion or survival of the bulk of the KJ16+ cells in this population. These results are consistent with the hypothesis that most cortical thymocytes arise by a receptor-independent mechanism and that only a small proportion of these cells mature by a process dependent on receptor-major histocompatibility complex interactions.
Antibody-targeted photolysis: selective photodestruction of human T-cell leukemia cells using monoclonal antibody-chlorin e6 conjugates.
Authors: Oseroff A R , Ohuoha D , Hasan T , Bommer J C , Yarmush M L
Published: May 2006

Selective in vitro photodestruction of HPB-ALL human T-cell leukemia cells was accomplished using the photosensitizer chlorin e6 coupled through dextran molecules to an anti-T-cell monoclonal antibody (mAb), anti-Leu-1. Conjugates with mAb/chlorin molar ratios as high as 1:36 retained mAb binding activity, and the absorption spectrum and quantum efficiency for singlet oxygen production of bound chlorin (0.7 +/- 0.2) were unchanged from that of the free photosensitizer. Phototoxicity, as measured by a clonogenic assay and by uptake of ethidium bromide, was dependent on the doses of both mAb-chlorin and 630- to 670-nm light, was enhanced by 2H2O, and was observed only in target populations that bound the mAb. Similarly, free chlorin e6 in solution had no photodynamic effect in amounts 100 times more than that carried by the mAb. For this antibody-targeted system, approximately 10(10) molecules of singlet oxygen were necessary to kill a cell.
Monoclonal antibodies to glutamic acid decarboxylase.
Authors: Gottlieb D I , Chang Y C , Schwob J E
Published: May 2006

Five monoclonal antibodies that recognize chicken brain glutamic acid decarboxylase (GAD) have been selected and designated GAD-1 to -5. GAD-1 to -5 were selected on the basis of their ability to immunoprecipitate active GAD from crude brain extracts. GAD-1 recognizes an epitope that is conserved in many vertebrates; the epitope recognized by GAD-5 is restricted to the chicken. Radioimmunoassays with GAD-1 indicate that GAD is highly enriched in brain relative to other tissues. GAD was localized immunocytochemically with GAD-1 and GAD-2 in rat cerebellum, spinal cord, and retina. The staining pattern is in agreement with that obtained previously with polyclonal antisera to GAD. GAD from the chicken brain was purified by chromatography on an immunoaffinity column made of GAD-1. NaDodSO4/PAGE analysis of the immunoaffinity-purified GAD fractions shows a major band of 59 kDa and minor bands at 63 and 54 kDa.
Topoisomerase I mutants: the gene on pBR322 that encodes resistance to tetracycline affects plasmid DNA supercoiling.
Authors: Pruss G J , Drlica K
Published: May 2006

Plasmid pBR322 DNA isolated from topoisomerase I mutants of Escherichia coli and Salmonella typhimurium exhibits a distinctive supercoiling distribution characterized by an extremely heterogeneous distribution of linking numbers that contains highly negatively supercoiled topoisomers. Analysis of the supercoiling distributions of deletion and insertion derivatives of pBR322 shows that the presence of the gene on pBR322 encoding resistance to tetracycline is responsible for the unusual supercoiling distribution. Both an intact promoter and a portion of the remainder of the gene, but not the gene product, are required. However, no particular section of the gene outside the promoter appears to be necessary; only the size of the section remaining appears to be important. These observations suggest that transcription of this gene may be responsible for its effect on DNA supercoiling.
Disulfide bonds and the translocation of proteins across membranes.
Authors: Maher P A , Singer S J
Published: May 2006

We are concerned with the mechanisms whereby hydrophilic proteins synthesized in the cytoplasm are translocated across one or two membranes into different cellular organelles. On the basis of a model of the translocation process to be described elsewhere, we propose an explanation of previous findings that the in vitro translocation across the endoplasmic reticulum of secretory proteins of higher eukaryotic cells appears to be obligatorily co-translational (i.e., occurs only while the polypeptide chain is being synthesized on the ribosome). We suggest that in vitro the intrachain disulfide bonds of the polypeptide rapidly form after it is released from the ribosome; the three-dimensional conformation of the chain is thereby stabilized and cannot undergo the unfolding that is required for post-translational translocation. In accord with this proposal, we show that the secretory preprotein human preprolactin, after translation and release from the ribosome, can indeed undergo translocation across endoplasmic reticulum membranes in vitro if the medium is sufficiently reducing. Those polypeptides that, in the absence of reducing agents, can be post-translationally translocated in vitro across bacterial, mitochondrial, and other types of membranes may generally lack intrachain disulfide bonds.
Cognate homeo-box loci mapped on homologous human and mouse chromosomes.
Authors: Rabin M , Ferguson-Smith A , Hart C P , Ruddle F H
Published: May 2006

The homeotic genes of Drosophila, which regulate pattern formation during larval development, contain a 180-base-pair DNA sequence termed the "homeo-box." Nucleotide sequence comparisons indicate that the homeo-box motif is highly conserved in a variety of motazoan species. As in Drosophila, homeo-box sequences of mammalian species are expressed in a temporal and tissue-specific pattern during embryogenesis. These observations suggest functional homologies between dipteran and mammalian homeo-box gene products. To identify possible relationships between homeo-box genes of mice and humans, we have compared the chromosomal location of homeo-box genes in these species. Using in situ hybridization and somatic cell genetic techniques, we have mapped the chromosome 6-specific murine Hox-1 homolog to the region p14-p21 on human chromosome 7. We have also regionally mapped the murine Hox-3 locus to 15F1-3 and its human cognate to 12q11-q21. These comparative mapping data indicate that a syntenic relationship in mice and humans is maintained for all homeo-box loci examined to date. We suggest these regions represent evolutionarily conserved genomic domains encoding homologous protein products that function in regulating patterns of mammalian development.
Structural properties of double-stranded RNAs associated with biological control of chestnut blight fungus
Authors: Tartaglia James , Paul Cynthia P. , Fulbright Dennis W. , Nuss Donald L.
Published: May 2006

Double-stranded RNAs (ds RNAs) are thought to be the cytoplasmic determinants responsible for the phenomenon of transmissible hypovirulence in the chestnut blight fungus
Endothia parasitica
[Murr.] Anderson. The three major ds RNA components associated with the North American hypovirulent strain, Grand Haven 2, were characterized with respect to molecular-hybridization specificity and RNase T1-digestion patterns. The large (L-RNA; ≈9 kilobase pairs) and middle-sized (M-RNA; ≈3.5 kilobase pairs) ds RNA components cross-hybridized under stringent conditions and exhibited indistinguishable partial and complete RNase T1 digestion patterns relative to their 5′ and 3′ termini. These results suggest that M-RNA was derived from L-RNA by an internal deletion event. The small (S-RNA; ≈1 kilobase pair) RNA was unrelated to L- and M-RNA by these criteria. However, all three ds RNA components contained RNase T1-resistant oligonucleotides at one 5′ terminus and at the corresponding 3′ terminus of the complementary strand. These RNase T1-resistant species exhibited properties consistent with stretches of poly(uridylic acid) and poly(adenylic acid), respectively. The combined results are discussed in terms of the structural organization of hypovirulence-associated ds RNA molecules and their similarities to “double-stranded” RNA molecules observed in plant and animal cells infected with single-stranded RNA viruses.
Regulation of a transfected human class II major histocompatibility complex gene in human fibroblasts.
Authors: Boss J M , Strominger J L
Published: May 2006

To investigate the cis-acting DNA elements that are involved in regulation of class II major histocompatibility complex genes, including gamma-interferon (gamma-IFN) induction, 5' flanking DNA deletions of a DQ beta "minigene" were analyzed in stable transfected cell lines. At least four elements 5' to the gene were found to be involved in DQ beta regulation. Deletion of sequences from -2500 to -159 base pairs (bp) resulted in increased transcription, suggesting that negative regulatory elements resided in the deleted region. These clones were all capable of responding to gamma-IFN. Further deletion of sequences from -159 to -128 bp resulted in constitutive high level transcription and the inability of these constructions to respond to gamma-IFN. A deletion to -107 bp resulted in a decrease in the basal level of expression that was restored by removal of the 5' DNA sequence to -82 bp, suggesting the presence of a second negative element. Finally, deletion to -64 bp caused a marked decrease in expression, suggesting the loss of an element necessary for high levels of transcription. The gamma-IFN control and the transcription control elements contain the conserved upstream sequences found in all class II genes, suggesting a role for these sequences.
A synthetic peptide vaccine involving the product of the pre-S(2) region of hepatitis B virus DNA: protective efficacy in chimpanzees.
Authors: Itoh Y , Takai E , Ohnuma H , Kitajima K , Tsuda F , Machida A , Mishiro S , Nakamura T , Miyakawa Y , Mayumi M
Published: May 2006

The S gene encoding the major surface polypeptide of hepatitis B virus is preceded by the region pre-S(2) with a capacity to code for 55 amino acid residues. In the product of region pre-S(2), the sequence of 19 amino acid residues (amino acids 14-32 from the N terminus) representing an area of high local hydrophilicity is shared by viral strains of subtypes adr, ayw, and ayr; residue 22, phenylalanine, is replaced by leucine in a strain of the other subtype, adw. A synthetic peptide vaccine involving these 19 amino acid residues, when given to two chimpanzees, raised antibodies that bound to viral particles and protected the animals from challenge with 10(6) chimpanzee infectious doses of hepatitis B virus.
Current issues in developing a strategy for dealing with the acquired immunodeficiency syndrome.
Authors: Fauci A S
Published: May 2006

A number of current issues exist that must be taken into account in the development of a strategy for dealing with the problem of the acquired immunodeficiency syndrome (AIDS) in the United States and throughout the world. Given the complexity of the problem and the fact that the epidemic is still in its evolving stages, such issues must be considered individually and as a group. The present discussion focuses on several of these critical issues and outlines approaches that might be useful in the formulation of basic scientific and public health strategies for dealing with the currently appreciated and projected problems in AIDS.
Regulation of insulin-like growth factor I gene expression by growth hormone.
Authors: Mathews L S , Norstedt G , Palmiter R D
Published: May 2006

Recombinant clones containing exon 3 of the insulin-like growth factor I (IGF-I) gene were isolated from a mouse genomic library. These sequences were used to generate an RNA probe, which was used in a solution hybridization assay to quantitate IGF-I mRNA in various murine tissues as a function of growth hormone status. The liver is the major site of IGF-I synthesis and the level of IGF-I mRNA is regulated about 10-fold by growth hormone in the growth hormone-deficient lit/lit mouse. Nuclear run-on assays were used to show that growth hormone regulation is manifested in part at the transcriptional level. Growth hormone also affects the size distribution of hepatic IGF-I mRNAs. Pancreas showed the highest non-hepatic expression, but every tissue analyzed contained some IGF-I mRNA. Expression was not growth hormone-dependent in most non-hepatic tissues.
Conserved protein domains in a myosin heavy chain gene from Dictyostelium discoideum.
Authors: Warrick H M , De Lozanne A , Leinwand L A , Spudich J A
Published: May 2006

The 2116-amino acid myosin heavy chain sequence from Dictyostelium discoideum was determined from DNA sequence analysis of the cloned gene. The gene product can be divided into two distinct regions, a globular head region and a long alpha-helical, rod-like tail. In comparisons with nematode and mammalian muscle myosins, specific areas of the head region are highly conserved. These areas presumably reflect conserved functional and structural domains. Certain features that are present in the head region of nematode and mammalian muscle myosins, and that have been assumed to be important for myosin function, are missing in the Dictyostelium myosin sequence. The protein sequence of the Dictyostelium tail region is very poorly conserved with respect to the other myosins but displays the periodicities similar to those of muscle myosins. These periodicities are believed to play a role in filament formation. The 196-residue repeating unit that determines the 14.3-nm repeat seen in muscle thick filaments, the 28-residue charge repeating unit, and the 1,4 hydrophobic repeat previously described for the nematode myosin are all present in the Dictyostelium myosin rod sequence, suggesting that the filament structures of muscle and Dictyostelium myosins must be similar.
Tissue-specific transcription enhancement of the fibroin gene characterized by cell-free systems.
Authors: Suzuki Y , Tsuda M , Takiya S , Hirose S , Suzuki E , Kameda M , Ninaki O
Published: May 2006

Six cell-free extracts have been used to characterize the nature of DNA signals and trans-acting factors responsible for the transcription enhancement of the Bombyx mori fibroin gene. The upstream element of the fibroin gene involved in the enhancement can be divided into two regions. The proximal region, -72 to -32, is recognized as a common enhancing signal by all B. mori extracts from the posterior silk gland, the middle silk gland, the ovarian tissue, and an embryonic cell line. It is weakly recognized by an Antheraea silkworm cell line extract but not by a HeLa cell extract. The distal region, -238 to -73, appears to be a tissue-specific enhancing signal that is recognized more effectively by the posterior silk gland extract than by the middle silk gland extract. These observations suggest that the use of these cell-free systems can offer a means for the biochemical characterization of the trans-acting factors involved in the tissue-specific regulation of the fibroin gene.
Molecular cloning, encoding sequence, and expression of vaccinia virus nucleic acid-dependent nucleoside triphosphatase gene.
Authors: Rodriguez J F , Kahn J S , Esteban M
Published: May 2006

A rabbit poxvirus genomic library contained within the expression vector lambda gt11 was screened with polyclonal antiserum prepared against vaccinia virus nucleic acid-dependent nucleoside triphosphatase (NTPase)-I enzyme. Five positive phage clones containing from 0.72- to 2.5-kilobase-pair (kbp) inserts expressed a beta-galactosidase fusion protein that was reactive by immunoblotting with the NTPase-I antibody. Hybridization analysis allowed the location of this gene within the vaccinia HindIIID restriction fragment. From the known nucleotide sequence of the 16-kbp vaccinia HindIIID fragment, we identified a region that contains a 1896-base open reading frame coding for a 631-amino acid protein. Analysis of the complete sequence revealed a highly basic protein, with hydrophilic COOH and NH2 termini, various hydrophobic domains, and no significant homology to other known proteins. Translational studies demonstrate that NTPase-I belongs to a late class of viral genes. This protein is highly conserved among Orthopoxviruses.
Nucleotide sequence analysis of murine 21-hydroxylase genes: mutations affecting gene expression.
Authors: Chaplin D D , Galbraith L J , Seidman J G , White P C , Parker K L
Published: May 2006

Steroid 21-hydroxylase [21-OHase; steroid 21-monooxygenase; steroid, hydrogen-donor:oxygen oxidoreductase (21-hydroxylating); EC 1.14.99.10] is a cytochrome P-450 enzyme required for the adrenal synthesis of mineralocorticoids and glucocorticoids. The gene encoding this protein is present in two copies (21-OHase A and B) in the S region of the murine major histocompatibility complex. Previous studies utilizing gene-specific oligonucleotide probes and gene transfer showed that only the 21-OHase A gene is expressed in the BALB/c mouse. Here, we present the complete primary structures of both BALB/c 21-OHase encoding genes. Comparison of the nucleotide sequences defines a deletion of 215 nucleotides spanning the second exon of the 21-OHase B gene; other nucleotide changes in the 21-OHase B gene introduce frame shifts and premature termination codons. Southern blot analysis of C57BL/6 and DBA/2J mice indicates that a similar deletion is present in these strains; however the C3H/HeJ strain is a structural variant. A hybrid gene composed of the 21-OHase B promoter placed 5' of the 21-OHase A structural sequences was efficiently transcribed following transfection into Y1 adrenocortical tumor cells. These findings demonstrate that the 21-OHase B gene promoter is functional and suggest that mutations within the 21-OHase B structural gene are responsible for its lack of expression.
Mutational analysis of the immunoglobulin heavy chain promoter region.
Authors: Ballard D W , Bothwell A
Published: May 2006

Complete immunoglobulin heavy chain (IgH) genes (gamma and mu) containing the intronic IgH enhancer and mutations in the upstream promoter region were constructed in vitro and introduced into murine J558L myeloma cells by protoplast fusion. S1-nuclease mapping experiments demonstrated that IgH gene expression was extremely sensitive to mutation in an upstream region containing the octanucleotide sequence ATGCAAAT. Significant IgH mRNA levels were detected in RNA from cells transfected with IgH gene constructs in which all upstream sequences on the 5' proximal side of this element were deleted. Similar results were obtained using the precise inverse of the IgH octamer, which is found in the upstream promoter region of immunoglobulin light chain genes. Deletion of the IgH octamer, or point mutation of adenine to guanine at position 6, resulted in the loss of correctly initiated IgH mRNA. A DNA binding factor from J558L nuclear extracts was identified that appeared to recognize the octamer on the basis of differential binding to homologous restriction fragments containing the various mutations and that bound preferentially with octamer DNA fragments derived from functional relative to nonfunctional IgH constructs. Collectively, these data suggest that the octamer element contains residues that are critical to accurate immunoglobulin gene transcription and that may serve as part of a recognition locus for nuclear factors important to B-cell-specific immunoglobulin expression.
Evidence on precise time-coded symbols and memory of patterns in monkey cortical neuronal spike trains.
Authors: Strehler B L , Lestienne R
Published: May 2006

High-resolution examination of pulse sequences generated by single visual cortex cells of the rhesus monkey in response to precisely controlled visual stimuli has disclosed (i) that the outputs of such neurons contain highly improbable (P less than 10(-7) numbers of identical triplets of precisely repeating pulse patterns; (ii) that the precision of such matches is better than 1/6000th of a second; (iii) that there is a similarly improbable high number of precisely matching pairs of triplets and anti-triplets, about half of which are present in symmetrical quadruplets of the form A-B-A, and that precisely replicating quadruplets and quintuplets are similarly generated in improbably large numbers; (iv) that identical triplets occur highly preferentially during immediately succeeding presentation of the same stimulus to the eye; and (v) that identical triplet (and doublet) patterns occur much more frequently in the responses of the same nerve when the eye receives identical or similar stimuli in different experiments than when dissimilar stimuli are applied. From these findings it is concluded (i) that the high precision of pattern replication required for triplets of pulses in time to serve to encode specific inputs and to permit their decoding through spatial summation is met (observations i-iii); (ii) that stimulus-specific triplets symbolize components of responses to specific stimuli (observation iv); (iii) that a temporary memory store of previous responses exists (observations iv and v); and (iv) that the mammalian brain uses precise patterns of discharges in time to represent and store specific data, rather than statistical qualities associated with pulse trains to symbolize qualitative stimulus components.
Mesencephalic dopamine neurons regulate the expression of neuropeptide mRNAs in the rat forebrain.
Authors: Young W S , Bonner T I , Brann M R
Published: May 2006

We used in situ hybridization histochemistry with synthetic oligodeoxyribonucleotide probes to identify cells that synthesize mRNAs encoding tyrosine hydroxylase in the mesencephalon and substance P, enkephalin, and dynorphin in the rat forebrain. Dopaminergic cells in the mesencephalon project to the forebrain and influence neuropeptide levels. We examined the effect of unilateral 6-hydroxydopamine lesions (which eliminated tyrosine hydroxylase mRNA-containing cells in the mesencephalon) on substance P, enkephalin, and dynorphin mRNA levels. Substance P mRNA levels were depressed, whereas enkephalin mRNA levels were elevated in consecutive sections from striatal areas in all animals. The effects of the lesions on dynorphin mRNA levels were less robust, and considerable variation between animals was observed. Changes were evident in the levels of message in individual cells but not in the numbers of labeled cells. These effects were not uniform throughout the dopamine-innervated areas, suggesting degrees of control not apparent with RNA blot-hybridization or dot-blot analyses.
Deficiency of adenosine deaminase in the wasted mouse.
Authors: Abbott C M , Skidmore C J , Searle A G , Peters J
Published: May 2006

Wasted (wst) is a spontaneous mutation with autosomal recessive inheritance. Abnormally low levels of adenosine deaminase have been found in erythrocytes from the wasted mouse. Enzyme activity in wst/wst mice is reduced to 38% of that found in the erythrocytes from control mice, and the apparent Km for adenosine is reduced to 51% of control. These changes imply an alteration in the catalytic properties of the enzyme arising from a change in the primary structure of the protein. We postulate that wasted is a mutation in the structural gene for adenosine deaminase. In man, the autosomal recessive form of severe combined immunodeficiency is associated, in about one-third of cases, with a deficiency of adenosine deaminase. Wasted mice are immunodeficient, develop neurological abnormalities, and die soon after weaning. These features are shared with the human syndrome. We therefore further suggest that the wasted mouse is an animal model for this form of severe combined immunodeficiency that will have potential use in gene-therapy studies.
Amplification units containing human N-myc and c-myc genes.
Authors: Kinzler K W , Zehnbauer B A , Brodeur G M , Seeger R C , Trent J M , Meltzer P S , Vogelstein B
Published: May 2006

The amplification units in human tumors containing amplified myc genes were examined. The amplification unit in all cases consisted of a large genomic region coamplified with the coding region of the myc genes themselves. In eight independent neuroblastomas containing N-myc amplifications, the amplification unit was estimated to be 290 to 430 kilobases. This amplification unit was highly conserved among the different neuroblastomas, with some neuroblastomas containing almost identical units. In contrast, five tumor cell lines containing c-myc amplifications exhibited amplification units that were more variable in size (90 to 300 kilobases) and sequence content; at least three different patterns of c-myc amplification units could be discerned.
Rabbit and human antibodies to a repeated amino acid sequence of a Plasmodium falciparum antigen, Pf 155, react with the native protein and inhibit merozoite invasion.
Authors: Berzins K , Perlmann H , Wåhlin B , Carlsson J , Wahlgren M , Udomsangpetch R , Björkman A , Patarroyo M E , Perlmann P
Published: May 2006

The Plasmodium falciparum-derived antigen of Mr 155,000 designated Pf 155, deposited in the membrane of infected erythrocytes, contains at least two blocks of tandemly repeated amino acid sequences. The peptide Glu-Glu-Asn-Val-Glu-His-Asp-Ala, which corresponds to a subunit of a C-terminally located repeat, was synthesized. Rabbits immunized with the octapeptide conjugated with either keyhole limpet hemocyanine or tetanus toxoid formed antibodies against the octapeptide. These antibodies reacted with Pf 155 as detected by immunoblotting or a modified immunofluorescence assay. Sera from humans exposed to P. falciparum also contained antibodies binding to the octapeptide in a dot-blot immunoassay. Their anti-octapeptide titers were correlated with their immunofluorescence titers in the assay detecting Pf 155 and other parasite antigens in the membrane of infected erythrocytes. Human octapeptide-reactive antibodies were isolated on an affinity column with the octapeptide conjugated to bovine serum albumin as ligand. These human antibodies reacted with Pf 155 in immunoblotting and strongly stained the surface of infected erythrocytes in the modified immunofluorescence assay. Approximately 20% of this immunofluorescence activity in a high-titered human serum could be recovered from the octapeptide column, indicating that a significant fraction of these anti-parasite antibodies react with epitopes associated with the octapeptide. Furthermore, the human octapeptide-reactive antibodies very efficiently inhibited merozoite reinvasion into erythrocytes in vitro. Similarly purified rabbit antibodies also significantly inhibited reinvasion. Our results suggest that the C-terminal segment of repeated peptides in Pf 155 is a major antigenic region of the molecule and may contain target sites for protective immunity in P. falciparum malaria.