bluuebunny/crossref_metadata_2025 huggingface dataset:
Host cell-mediated selection of a mutant influenza A virus that has lost a complex oligosaccharide from the tip of the hemagglutinin.
Authors: Deom C M , Caton A J , Schulze I T
Published: May 2006

During serial passage in Madin-Darby bovine kidney (MDBK) cells, a substrain of influenza virus A/WSN is lost from the population and is replaced by a mutant virus with altered host cell binding properties. This selection does not occur during growth in chicken embryo fibroblasts (CEF). It occurs during growth in MDBK cells because the parental virus produced by these cells has a dramatically reduced affinity for cellular receptors [Crecelius, D.M., Deom, C. M. & Schulze, I. T. (1984) Virology 139, 164-177]. We have now compared the hemagglutinin (HA) subunits, HA1 and HA2, of the parent and mutant viruses by NaDodSO4/PAGE and have found that when the viruses are grown in either host cell the HA1 subunit of the mutant is smaller than that of the parent virus. The nonglycosylated HAs, made in the presence of tunicamycin, have the same apparent molecular weight, indicating that the HA1 subunit of the mutant virus contains less carbohydrate than that of the parent. This reduction in carbohydrate content was observed with 11 independently derived mutants that had been selected by growth in MDBK cells. The nucleotide sequence of the HA gene of the parent and mutant viruses indicates that there are five potential glycosylation sites on the parent HA1 subunit and four on the mutant and that the mutation responsible for this difference is a single base change that eliminates the glycosylation site at amino acid 125 of the parent HA1 subunit. Treatment of the parent and mutant HAs from both cell sources with endo-beta-N-acetylglucosaminidases F and H showed that the HA1 of the parent virus has four complex and one high-mannose oligosaccharides, whereas that of the mutant virus has three complex and one high-mannose oligosaccharides. Thus, all of the potential sites on both HA1 subunits are glycosylated. We conclude that the oligosaccharide attached to amino acid 125 of the parent HA by MDBK cells can reduce the affinity of the virus for cellular receptors and that the mutant virus has a higher affinity than the parent because the mutant HA is not glycosylated at that site. Since amino acid 125 of the parent HA is glycosylated by both CEF and MDBK cells, we further conclude that the host-determined structure of the oligosaccharide at that site affects the affinity of the parent virus for cellular receptors and, thereby, determines whether the mutant virus will have a growth advantage.
Survival of UV-irradiated mammalian cells correlates with efficient DNA repair in an essential gene.
Authors: Bohr V A , Okumoto D S , Hanawalt P C
Published: May 2006

The survival of UV-irradiated mammalian cells is not necessarily correlated with their overall capacity to carry out DNA repair. Human cells typically remove 80% of the pyrimidine dimers produced by a UV dose of 5 J/m2 within 24 hr. In contrast, a Chinese hamster ovary (CHO) cell line survives UV irradiation equally well while removing only 15% of the dimers. Using a newly developed technique to measure dimer frequencies in single-copy specific sequences, we find that the CHO cells remove 70% of the dimers from the essential dihydrofolate reductase (DHFR) gene but only 20% from sequences located 30 kilobases or more upstream from the 5' end of the gene in a 24-hr period. Repair-deficient human cells from xeroderma pigmentosum complementation group C (XPC) are similar to the CHO cells in overall repair levels, but they are extremely sensitive to killing by UV irradiation. In the XPC cells, we find little or no repair in the DHFR gene; in contrast, in normal human fibroblasts and epidermal keratinocytes, greater than 80% of the dimers induced in the gene by 20 J/m2 are removed in 24 hr. Since the CHO and normal human cells exhibit similar UV resistance, much higher than that of XPC cells, our findings suggest a correlation between efficient repair of essential genes and resistance to DNA-damaging agents such as UV light.
Insertion and/or deletion of many repeated DNA sequences in human and higher ape evolution.
Authors: Hwu H R , Roberts J W , Davidson E H , Britten R J
Published: May 2006

The total numbers of copies of two repeat families, L1 (Kpn I) and Alu, have been measured in the DNA of four higher apes by an accurate titration method. The number of members of the Alu family repeats in the four genomes are as follows: human, 910,000; chimpanzee, 330,000; gorilla, 410,000; orangutan, 580,000. For the Kpn I family (3'-ward higher frequency region) the number of copies in these genomes are as follows: human, 107,000; chimpanzee, 51,000; gorilla, 64,000; orangutan, 84,000. Thermal stability measurements show that, although the families of repeats are moderately divergent in sequence, little net sequence change has occurred during the evolution of the higher apes. Most or all of the members of these families of repeats are interspersed throughout the genome. Therefore, a large number of events of insertion and/or deletion of these DNA sequences has occurred during higher primate evolution.
Piliation control mechanisms in Neisseria gonorrhoeae.
Authors: Bergström S , Robbins K , Koomey J M , Swanson J
Published: May 2006

Gonococci (Gc) undergo pilus+ to pilus- "phase transitions" readily in vitro. In the present study we sequenced pilin mRNA from reverting, pilus- Gc by oligonucleotide primer extension and compared these pilin mRNA sequences with those expressed by their pilus+ predecessors and pilus+ revertants. The results suggest that genetic rearrangement within the pilin structural gene can generate defective pilin gene products, resulting in a pilus- phenotype. These pilus- Gc give rise to pilus+ revertants upon reconstitution of their modified pilin gene.
Mitosis in sand dollar embryos is inhibited by antibodies directed against the calcium transport enzyme of muscle.
Authors: Silver R B
Published: May 2006

Monospecific antibodies to the calcium transport enzyme (alpha-Ca pump) inhibit mitosis when microinjected into sand dollar embryos. Immunoglobulins were raised against the calcium transport enzyme (Ca pump) of sarcoplasmic reticulum (SR) from rat skeletal muscle and guinea pig ileum smooth muscle. Specific antibodies were further isolated from IgG fractions by using electrophoretically purified SR Ca-pump protein as the immobilized ligand for immunoaffinity chromatography. ELISA demonstrated that common antigenic determinants are shared by SR, SR Ca pump (of rat skeletal and guinea pig ileum smooth muscle), and isolated membrane containing "native" mitotic apparatus (MA). Preimmune sera gave negative results in identical control assays. Triton X-100 extraction of MA removes the Ca-pump antigen. SR Ca pump and the MA Ca pump have nearly identical molecular masses as determined by NaDodSO4/PAGE. These alpha-SR Ca-pump IgGs inhibit ATP-dependent Ca2+ sequestration by purified SR and MA membranes. Indirect immunofluorescence of isolated native MA demonstrated coincident localization of the MA Ca pump, sequestered calcium, and membrane vesicles. Fluorescent foci were regionally concentrated within the volumes of the asters and spindle. Microinjection of the anti-Ca-pump IgGs into one of two sister blastomeres at second metaphase resulted in mitotic arrest of the injected cell accompanied by a rapid loss of spindle birefringence. Karyomeres formed and fused to form nuclei either at the site of the metaphase plate or at the position the chromosomes occupied during anaphase A. The cleavage furrow did not develop in the injected cell, while the sister and neighbor cells continued normal mitotic cycling. Injection later in mitosis yielded cells with two nuclei whose cleavage furrow relaxed completely. Routine control injections of boiled immune IgG, preimmune IgG, Wesson oil, buffer, or goat anti-rabbit IgG did not affect mitosis, birefringence of the MA, or cleavage furrow activity.
Identification of conserved isotype-defining variable region sequences for four vertebrate beta tubulin polypeptide classes.
Authors: Sullivan K F , Cleveland D W
Published: May 2006

We report the determination of the complete sequences for two chicken beta tubulin genes, beta 3 and beta 5. Taken with the previously published efforts, we have determined the primary structures of five of the seven beta tubulin genes in this vertebrate species. A comparison of these sequences unambiguously reveals that amino acid sequence variations among different beta tubulin gene products are distinctly clustered within an otherwise highly conserved framework of the beta tubulin molecule. To determine the extent to which this pattern of structural heterogeneity is conserved among vertebrates, we have isolated novel beta tubulin sequences from human and mouse cDNA libraries and compared these and all other known vertebrate beta tubulin sequences with the family of chicken polypeptide sequences. What emerges from such comparison is the recognition of distinct, evolutionarily conserved isotypes of beta tubulin that are distinguished primarily by their characteristic carboxyl-terminal variable region sequence and, to a lesser extent, by sequence in an amino-terminal variable domain as well. These correlations represent a convincing demonstration that multiple beta tubulin genes in vertebrates encode a family of closely related but structurally distinct beta tubulin isotypes and further serve to define the sequences of four classes of polypeptide isotypes that constitute that family.
Locus specificity in the mutability of L5178Y mouse lymphoma cells: the role of multilocus lesions.
Authors: Evans H H , Mencl J , Horng M F , Ricanati M , Sanchez C , Hozier J
Published: May 2006

Mouse L5178Y strain LY-S and its parental strain LY-R differ in their comparative sensitivities to the cytotoxic effects of various mutagenic agents--i.e., strain LY-S has been found to be more sensitive, less sensitive, or similarly sensitive to individual agents in comparison to strain LY-R. Nevertheless, strain LY-S has been found to be uniformly less mutable than strain LY-R at the hypoxanthine (guanine) phosphoribosyltransferase (Hprt) locus following treatment with x-radiation, UV radiation, or alkylating agents. In the present work we have isolated subclones of strains LY-R and LY-S that are heterozygous at the thymidine kinase (Tk) locus (chromosome 11). We have found that a heterozygous LY-S Tk+/Tk- strain shows as high or higher mutability at the Tk locus than do heterozygous LY-R strains following treatment with x-radiation, UV radiation, or ethyl methanesulfonate. Mutability of all heterozygous strains at the Tk locus is much higher than at the Hprt locus following treatment with these mutagenic agents, with the exception of one heterozygous LY-R strain that possesses only one chromosome 11 and that is poorly mutable at the Tk locus by x-radiation. On the basis of these results, we have suggested that because of a repair deficiency, multilocus lesions are formed in the DNA of LY-S strains following treatment with radiation or alkylating agents; multilocus lesions lead to poor recovery of viable mutants when the target locus is closely linked to essential genes and situated on a hemizygous chromosomal region (e.g., the Hprt locus on the X chromosome or the Tk locus in strains monosomic for chromosome 11); and x-radiation is a relatively poor mutagen at loci situated on hemizygous chromosomal regions, in repair-efficient and repair-deficient cells, because of its tendency to form multilocus lesions.
Expression of cellular oncogenes in primary cells from human acute leukemias.
Authors: Mavilio F , Sposi N M , Petrini M , Bottero L , Marinucci M , De Rossi G , Amadori S , Mandelli F , Peschle C
Published: May 2006

The structure and the expression of 11 cellular oncogenes (protooncogenes) were analyzed in primary cells from 20 acute lymphocytic (ALL) and 31 acute myelogenous (AML) leukemia patients. Neoplastic cells, obtained prior to initiation of therapy, were purified and classified, on the basis of both surface antigen pattern and morphology, into pre-B, B, and T ALL and M1-M5 AML. RNA was extracted and analyzed for expression of cellular oncogenes coding for nuclear proteins (c-myc, c-myb, c-fos), the beta-chain of platelet-derived growth factor (c-sis), growth factor receptors or related proteins (c-src, c-abl, c-fes, c-erbB), or putative intermediate transducers of mitogenic signals (c-Ha-ras, c-Ki-ras, c-N-ras). Quantitative analysis of total RNA was carried out by dot blot hybridization to specific cDNA or genomic probes. Number and size of transcripts were evaluated by blot hybridization of electrophoretically fractionated poly(A)+ RNA. Expression of c-myc and c-myb was detected in all leukemic cells at variable levels and was characterized by well-defined patterns within ALL subtypes. Conversely, significant levels of c-fos transcripts were detected only in myelomonocytic (M4) and monocytic (M5) leukemias. Among the "src-family," c-fes was expressed more in AML than ALL, and c-abl was expressed at variable but not elevated levels in all leukemia types. c-Ha-ras was uniformly expressed at low levels, as in non-neoplastic cells. c-Ki-ras transcription was detected only in T ALL; N-ras expression was barely demonstrable. The structure of these protooncogenes was not grossly modified, as evaluated by Southern analysis, except for c-myc rearrangement in B ALL. These studies indicate that cellular oncogene expression in specific subtypes of leukemic cells may relate to either the proliferative activity (c-myc, c-myb) or the differentiation state (c-fos) of the cells, or possibly to expression of receptors for putative hemopoiesis-related growth factors (c-fes, c-abl). Our data provide a basis for in-depth analysis of protooncogene expression in normal and neoplastic hemopoiesis.
Single amino acid substitutions in the enzyme acetolactate synthase confer resistance to the herbicide sulfometuron methyl
Authors: Yadav Narendra , McDevitt Raymond E. , Benard Susan , Falco S. Carl
Published: May 2006

Sulfometuron methyl, a sulfonylurea herbicide, blocks growth of bacteria, yeast, and higher plants by inhibition of acetolactate synthase (EC 4.1.3.18), the first common enzyme in the biosynthesis of branched-chain amino acids. Spontaneous mutations that confer increased resistance to the herbicide were obtained in cloned genes for acetolactate synthase from
Escherichia coli
and
Saccharomyces cerevisiae
. The DNA sequence of a bacterial mutant gene and a yeast mutant gene revealed single nucleotide differences from their respective wild-type genes. The mutations result in single amino acid substitutions in the structurally homologous aminoterminal regions of the two proteins, but at different positions. The bacterial mutation results in reduced levels of acetolactate synthase activity, reduced sensitivity to sulfometuron methyl, and unaltered resistance to feedback inhibition by valine. The yeast mutation results in unaltered levels of acetolactate synthase activity, greatly reduced sensitivity to sulfometuron methyl, and slightly reduced sensitivity to valine.
Host participation in plasmid maintenance: dependence upon dnaA of replicons derived from P1 and F.
Authors: Hansen E B , Yarmolinsky M B
Published: May 2006

Nonparticipation of the bacterial dnaA gene in plasmid replication has been assumed to be the general rule. In conditional dnaA mutants of Escherichia coli, only plasmid pSC101 has been shown to have a dnaA requirement. Experiments with dnaA null mutants of E. coli, presented here, show that dnaA plays a critical and direct role in the replication of miniplasmids derived from P1 and F as it does in the initiation of bacterial replication. Evidence is also presented for the existence of a dnaA-independent secondary replicon of P1 that is able to drive bacterial chromosome replication but is inadequate to support the maintenance of P1 as a plasmid in E. coli.
Generation of diversity of the beta chain of the human T-lymphocyte receptor for antigen.
Authors: Leiden J M , Strominger J L
Published: May 2006

Human T-cell receptor beta-chain (Ti beta) genes are formed by the rearrangement of variable (V), diversity (D), and joining (J) gene segments. A comparison of the nucleotide and deduced amino acid sequences of the variable regions of six human Ti beta cDNAs reveals that they display a level of homology similar to that shared by human immunoglobulin heavy chain V genes. In contrast to immunoglobulin V regions, which contain three discrete regions of hypervariability, the Ti beta V regions display a more widely distributed pattern of variability. Southern blot analyses show that most human Ti V beta gene families contain one to three members. However, a single family containing at least eight members is identified. This analysis allows the identification of at least 15 human Ti V beta germ-line genes. The sequence data show that at least one germ-line Ti beta J gene is used preferentially in Ti beta cDNAs. Moreover, they suggest the presence of at least four human germ-line Ti beta D genes. At least three mechanisms are involved in generating the diversity of human Ti beta genes: (i) the combinatorial rearrangement of different V, D, and J genes; (ii) imprecise V-D-J joining, including V-D joining in any of three translational reading frames; and (iii) the addition of extra nucleotides at the V-D-J joints (N-region diversity).
Lack of class I H-2 antigens in cells transformed by radiation leukemia virus is associated with methylation and rearrangement of H-2 DNA.
Authors: Meruelo D , Kornreich R , Rossomando A , Pampeno C , Boral A , Silver J L , Buxbaum J , Weiss E H , Devlin J J , Mellor A L
Published: May 2006

Transformation of murine thymocytes by radiation leukemia virus is associated with reduced expression of the class I antigens encoded in the major histocompatibility complex (MHC) and increased methylation and altered restriction enzyme patterns of MHC DNA. These changes may play a role in host susceptibility to virus-induced leukemogenesis and accord with the notion that viral genomes play a regulatory function when they integrate adjacent to histocompatibility genes.
Phototoxicity of the tetracyclines: photosensitized emission of singlet delta dioxygen.
Authors: Hasan T , Khan A U
Published: May 2006

The spectroscopic observation of 1268-nm emission of singlet oxygen photosensitized by tetracyclines in oxygenated solutions at room temperature is reported. In the series demeclocycline, tetracycline, and minocycline, the efficiency of singlet oxygen generation is found to parallel the clinical observation of relative frequency of phototoxicity of these antibiotics, suggesting singlet oxygen generation as the origin of their phototoxicity.
DNA-binding site of major regulatory protein alpha 4 specifically associated with promoter-regulatory domains of alpha genes of herpes simplex virus type 1.
Authors: Kristie T M , Roizman B
Published: May 2006

Herpes simplex virus type 1 genes form at least five groups (alpha, beta 1, beta 2, gamma 1, and gamma 2) whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Previous studies have shown that functional alpha 4 gene product is essential for the transition from alpha to beta protein synthesis and have suggested that alpha 4 gene expression is autoregulatory. We have previously reported that labeled DNA fragments containing promoter-regulatory domains of three alpha (alpha 0, alpha 4, and alpha 27) and a gamma 2 gene form stable complexes with proteins from lysates of infected cells as detected by a gel electrophoresis binding assay and that monoclonal antibody to alpha 4 protein reduced the electrophoretic mobility of the complex of labeled DNA and protein from infected cells. In this study we identified one monoclonal antibody, H950, from a panel of monoclonal antibodies to the alpha 4 protein that blocks the formation of specific infected cell complexes with labeled DNA fragments containing promoter and regulatory domains of alpha genes. We also report the nucleotide sequence of the alpha 0 promoter domain protected from exonuclease III digestion by alpha 4 protein in the absence of H950 monoclonal antibody but not in its presence. In addition, we identified a 59-base-pair sequence from the regulatory domain of the alpha 4 gene that binds alpha 4 protein. Deletion clones of this fragment localize sequence elements required for formation of the alpha 4 protein-DNA complex. Furthermore, deletion of the in vitro binding site of the SP1 transcription factor from the 59-base-pair fragment did not affect the formation of the alpha 4 protein-DNA complex.
Cerebellar granule cells in culture: monosynaptic connections with Purkinje cells and ionic currents.
Authors: Hirano T , Kubo Y , Wu M M
Published: May 2006

Electrophysiological properties of cerebellar granule cells and synapses between granule and Purkinje cells were studied in dissociated cultures. Electrophysiological properties of neurons and synapses in the mammalian central nervous system are best studied in dissociated cell cultures because of good target cell visibility, control over the contents of the extracellular solution, and the feasibility of whole-cell patch electrode recording, which has been a powerful technique in analyzing biophysical properties of ionic channels in small cells. We have applied this whole-cell recording technique to cultured cerebellar granule cells whose electrophysiological properties have been almost unknown because of their small cell size. In this study, simultaneous intracellular recordings from presynaptic granule and postsynaptic Purkinje cells demonstrated that granule cells made functional monosynaptic connections with Purkinje cells in dissociated cell cultures. Further, the existence of Na, Ca, and K channels in granule cells is demonstrated by external ion substitutions.
Molecular cloning of an oncogene from a human hepatocellular carcinoma.
Authors: Ochiya T , Fujiyama A , Fukushige S , Hatada I , Matsubara K
Published: May 2006

A transforming DNA, named lca (for liver cancer), was obtained from a primary human hepatocellular carcinoma (HCC) in transformation assays using NIH 3T3 cells and a calcium phosphate coprecipitation method. High molecular weight DNA obtained from the HCC tissue was employed for this purpose. This transforming DNA had a linkage to the Alu sequence and was cloned in lambda phage for further studies. Restriction enzyme analyses showed that the minimal size of the lca transforming DNA is about 10 kilobase pairs and that its cleavage profiles are different from those of any one of the previously reported human transforming genes or retroviral oncogenes. No cross-hybridization was observed between these genes and the lca DNA. Southern blot analyses of DNAs from flow-sorted human chromosomes and human-mouse somatic cell hybrids indicated that the lca DNA is located on human chromosome 2. An independently obtained transforming DNA from another HCC exhibited identical restriction enzyme cleavage profiles. Thus, lca DNA is likely to represent a commonly encountered transforming DNA in HCC.
Evidence and characterization of the receptor to eye-derived growth factor I, the retinal form of basic fibroblast growth factor, on bovine epithelial lens cells.
Authors: Moenner M , Chevallier B , Badet J , Barritault D
Published: May 2006

Eye-derived growth factor I (EDGF-I), the retinal form of the basic fibroblast growth factor, has been purified to homogeneity from bovine retina by heparin-Sepharose chromatography. The radioiodinated EDGF-I retained full mitogenic activity and was used to study the interaction of the growth factor with bovine epithelial lens cells. We showed that 125I-labeled EDGF-I bound in a saturable and reversible manner to a specific cellular receptor. Scatchard analysis of the equilibrium binding gave a Kd of 53 X 10(-12) M with approximately equal to 20,000 binding sites per cell. Crosslinking experiments using two homobifunctional reagents induced the formation of a specific major complex with a Mr of approximately equal to 145,000, as determined by NaDodSO4/PAGE, and independent of reducing conditions. These data establish the existence of a receptor for the basic growth factor derived from neural tissues and give an estimation of the size of this receptor at Mr 130,000.
Analysis of the human alpha-globin gene cluster reveals a highly informative genetic locus.
Authors: Higgs D R , Wainscoat J S , Flint J , Hill A V , Thein S L , Nicholls R D , Teal H , Ayyub H , Peto T E , Falusi A G
Published: May 2006

Extensive molecular studies have characterized 15 dimorphic and 2 multiallelic genetic markers within the human alpha-globin gene cluster. Analysis of these markers in 9 populations has shown that the alpha-globin locus is remarkably polymorphic and is therefore an ideal marker on chromosome 16 for the construction of a human genetic linkage map. The combined analysis of 9 polymorphic markers has established alpha-globin haplotypes that provide the means to study the molecular genetics and common mutants of this cluster. The novel association of a conventional restriction fragment length polymorphism haplotype and linked, hypervariable regions of DNA should allow a comparison of the rate of change of such markers.
Analysis of the structure, transcripts, and protein products of bcl-2, the gene involved in human follicular lymphoma.
Authors: Tsujimoto Y , Croce C M
Published: May 2006

We have determined that the bcl-2 (B-cell leukemia/lymphoma 2) gene is transcribed into three overlapping mRNAs, and we have cloned bcl-2 cDNA sequences. Sequence analysis of the bcl-2 cDNA clones and comparison of their sequences to their genomic counterparts indicate that the bcl-2 gene contains at least two exons. The three bcl-2 transcripts, which are 8.5, 5.5, and 3.5 kilobases (kb) long, overlap within the first exon, but only the 8.5-kb and 5.5-kb transcripts contain sequences of the second exon. The 8.5-kb and 5.5-kb transcripts seem to use different polyadenylylation sites. Sequence analysis of the cDNA clones corresponding to the 5.5-kb and 3.5-kb mRNAs indicates that the two bcl-2 transcripts carry two overlapping open reading frames, one of which is 717 nucleotides long and codes for a protein (bcl-2 alpha) of 239 amino acids and a molecular mass of 26 kDa, while the other codes for a protein of 205 amino acids (bcl-2 beta, molecular mass 22 kDa) that is identical to bcl-2 alpha except at the carboxyl terminus. The bcl-2 protein products in follicular lymphomas with or without bcl-2 rearrangements are identical to the normal bcl-2 products.
Hebbian synapses in hippocampus.
Authors: Kelso S R , Ganong A H , Brown T H
Published: May 2006

A combination of current- and voltage-clamp techniques applied to hippocampal brain slices was used to evaluate the role of postsynaptic electrogenesis in the induction of associative synaptic enhancement. In accordance with Hebb's postulate for learning, repetitive postsynaptic spiking enabled enhancement in just those synapses that were eligible to change by virtue of concurrent presynaptic activity. However, the essential postsynaptic electrogenic event that controlled the enhancement was shown to involve biophysical processes that were unknown when Hebb formulated his neurophysiological postulate. The demonstrated spatiotemporal specificity of this pseudo-Hebbian conjunctive mechanism can account qualitatively for the known neurophysiological properties of associative long-term potentiation in these synapses, which in turn can explain the "cooperativity" requirement for long-term potentiation.
Effect of errors, redundancy, and solvent content in the molecular replacement procedure for the structure determination of biological macromolecules.
Authors: Arnold E , Rossmann M G
Published: May 2006

The power of molecular replacement as a tool for analyzing macromolecular structures such as viruses has been demonstrated by an increasing number of successful determinations. We examine here the effects of noncrystallographic redundancy, N; the fraction of solvent volume (1- U/V); error in structure amplitude measurements, R; the fraction, f, of the unique data that were measured; error in the description of the noncrystallographic symmetry; and definition of the molecular envelope. The formula P = (Nf)1/2/R(U/V) has been derived and represents the inherent phasing power (P) for a given problem. The ability of "solvent flattening" procedures to determine phases was analyzed and found to be analogous to the effect of noncrystallographic redundancy. However, in the limiting case, the effect of solvent flattening approaches the power of Sayre's equations.
Spatial pattern of catalase (Cat2) gene activation in scutella during postgerminative development in maize.
Authors: Tsaftaris A S , Scandalios J G
Published: May 2006

The scutellum of maize is a fully differentiated, nondividing, diploid embryonic tissue. Two distinct structural genes (Cat1 and Cat2) encoding the enzyme catalase (CAT) are differentially expressed in this tissue during postgerminative development. As development proceeds, the expression of Cat1 diminishes, while that of Cat2 is enhanced, leading to the disappearance of the CAT-1 protein and the gradual accumulation of the CAT-2 protein. The present investigation was undertaken to determine whether all scutellar cells may be genetically programmed to activate expression of Cat2 synchronously or whether there is an asynchronous spatial gradient of Cat2 activation. By using immunofluorescence microscopy and anti-CAT-2 IgG, we have found that a gradient of Cat2 activation occurs within the scutellar cell mass during postgerminative development. The gradient of Cat2 activation occurs from the outer perimeter of the tissue inward toward the embryonic axis. To determine a potential site of origin for any putative "triggering signal" for Cat2 activation, we demonstrated that Cat2 is expressed in the single layer of aleurone cells prior to its expression in any other tissue during kernel development. To our knowledge, this is the first observation of a gradient-type spatial pattern of a eukaryote gene activation occurring in a stable, virtually nondividing tissue such as the maize scutellum. The significance of these results with respect to developmental gene regulation is discussed.
Isolation of a rat liver delta-aminolevulinate dehydrase (ALAD) cDNA clone: evidence for unequal ALAD gene dosage among inbred mouse strains.
Authors: Bishop T R , Cohen P J , Boyer S H , Noyes A N , Frelin L P
Published: May 2006

We have isolated several cDNA clones encoding delta-aminolevulinate dehydrase [ALAD; porphobilinogen synthase; 5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing), EC 4.2.1.24], the second enzyme in the heme biosynthetic pathway. We used a rabbit polyclonal antibody developed against the purified 35-kDa subunit of rat liver ALAD to screen a lambda gt11 cDNA expression library constructed from rat liver mRNA. A prototype clone (ALAD-1) contained a 680-base-pair insert and expressed a 140-kDa beta-galactosidase gene cDNA insert fusion protein immunoreactive with both polyclonal and monoclonal anti-ALAD. Identity of ALAD-1 was verified by hydridization to ALAD mRNA that had been enriched via immunopurification of rat liver polysomes with anti-ALAD. Intensity of such hybridization to a 1500-nucleotide-long mRNA was approximately equal to 200-fold greater than that realized with whole liver mRNA, a result consistent with the degree of immunoenrichment of ALAD mRNA found independently by analysis of cell-free translation products. A second ALAD cDNA clone (ALAD-3) was isolated when the rat liver cDNA expression library was rescreened with ALAD-1. The identity of both ALAD cDNA clones was established by correspondence between their nucleotide sequence and the reported amino-terminal protein sequence of bovine ALAD. Hybridization of ALAD cDNA to mouse genomic DNA indicates that the previously unexplained incremental differences in ALAD enzymatic activity among inbred mouse strains has arisen through alterations in ALAD gene dose.
Detection of restriction fragment length polymorphisms at the centromeres of human chromosomes by using chromosome-specific alpha satellite DNA probes: implications for development of centromere-based genetic linkage maps.
Authors: Willard H F , Waye J S , Skolnick M H , Schwartz C E , Powers V E , England S B
Published: May 2006

We describe a general strategy for the detection of high-frequency restriction fragment length polymorphisms in the centromeric regions of human chromosomes by molecular analysis of alpha satellite DNA, a diverse family of tandemly repeated DNA located near the centromeres of all human chromosomes. To illustrate this strategy, cloned alpha satellite repeats isolated from two human chromosomes, 17 and X, have been used under high-stringency conditions that take advantage of the chromosome-specific organization of this divergent repeated DNA family. Multiple high-frequency restriction fragment length polymorphisms are described for the centromeric region of both chromosome 17 and X chromosome. Mendelian inheritance of the variants is demonstrated. The X-linked alpha satellite polymorphisms in particular are highly informative and constitute a virtually unique centromeric DNA marker for each X chromosome examined. Since the strategy we describe is a general one, the alpha satellite family of DNA should provide a rich source of molecular variation in the human genome and should contribute to the development of centromere-based genetic linkage maps of human chromosomes.
Ribonuclease H(70) from Saccharomyces cerevisiae possesses cryptic reverse transcriptase activity.
Authors: Karwan R , Kühne C , Wintersberger U
Published: May 2006

Yeast cells contain a protein of molecular size 70 kDa that possesses RNase H activity. A polyclonal antibody against it reacts in addition with proteins of molecular sizes 160 kDa from yeast extracts. All these immunologically related proteins exhibit reverse transcriptase activity and in this respect they resemble the products of retroviral pol genes, relatives of which reside in Ty elements and mitochondrial introns of yeast. Experimental evidence, however, indicates that the protein described here that combines RNase H and reverse transcriptase activity is not coded for by a known element of the retrotransposon family. It may originate from a cellular gene distantly related to retrotransposon sequences.
Gene encoding the sigma 37 species of RNA polymerase sigma factor from Bacillus subtilis.
Authors: Binnie C , Lampe M , Losick R
Published: May 2006

sigma 37 is a minor species of RNA polymerase sigma factor found in the Gram-positive bacterium Bacillus subtilis. sigma 37 governs the transcription in vitro of genes that are turned on at an early stage in spore formation, as well as other genes that are switched on at the end of the exponential phase of growth but that are not under sporulation control. To study the role of sigma 37 in B. subtilis gene expression, we have cloned the gene for this minor species of sigma factor in Escherichia coli by using as a hybridization probe a synthetic oligonucleotide that was designed on the basis of the NH2-terminal amino acid sequence of sigma 37 protein. We determined the nucleotide sequence of the entire sigma 37 gene, which was found to encode a 262-amino acid residue polypeptide of 29.9 kDa. The predicted amino acid sequence of sigma 37 showed significant homology to that of other sigma proteins in a region that has been proposed to be the site of binding of these factors to core RNA polymerase. Genetic mapping experiments placed the gene for sigma 37, herein designated sigB, at 40 degrees on the genetic map of Piggot and Hoch [Piggot, P. & Hoch, J. A. (1985) Microbiol. Rev. 49, 158-179]. An insertion mutation was constructed in sigB and found not to impair growth or sporulation.
Replication forks are underrepresented in chromosomal DNA of Xenopus laevis embryos.
Authors: Gaudette M F , Benbow R M
Published: May 2006

Chromosomal DNA was isolated from rapidly dividing cells of Xenopus laevis embryos at blastulation, at gastrulation, and at the beginning of hatching. Few, if any, replication forks were seen by electron microscopy in DNA isolated at any stage of embryogenesis. Instead, unbranched DNA, which appeared to be single-stranded, was abundant at all stages. The percentage of chromosomal DNA that was single-stranded was quantitated by electron microscopy and by monitoring the release of acid-soluble radioactivity during digestion of labeled chromosomal DNA with nucleases specific for single-stranded DNA. The amount of single-stranded DNA was inversely correlated with the length of S phase during embryogenesis. We postulate that chromosomal DNA replication in X. laevis embryos takes place by a mechanism in which strand separation is uncoupled from DNA synthesis.
Enhanced delivery to target cells by heat-sensitive immunoliposomes.
Authors: Sullivan S M , Huang L
Published: May 2006

Heat-sensitive immunoliposomes are capable of releasing the entrapped content at the target cell surface upon a brief heating to the phase transition temperature of the liposome membrane. In this study we have examined the delivery efficiency of drugs entrapped in heat-sensitive immunoliposomes. Immunoliposomes composed of dipalmitoyl phosphatidylcholine with entrapped [3H]uridine were incubated with target cells at 4 degrees C. The cell-liposome mixture was then heated to 41 degrees C and the uptake of [3H]uridine into the intracellular pool of phosphorylated uridine-containing molecules was measured. The immunoliposomes showed maximal release of the uridine at 41 degrees C, the phase transition temperature of dipalmitoyl phosphatidylcholine liposomes. The largest accumulation of [3H]uridine in the target cells also took place at 41 degrees C. The initial level of uptake of [3H]uridine released from immunoliposomes by heating was greatly enhanced over that observed for free [3H]uridine and [3H]uridine released from liposomes without attached antibody. The nucleoside uptake inhibitors nitrothiobenzylinosine, dipyridamole, and unlabeled uridine were able to inhibit uptake of [3H]uridine released from immunoliposomes. This supports the hypothesis that the enhanced uptake is due to a heat-induced release of [3H]uridine at the cell surface followed by transport and phosphorylation of [3H]uridine by the target cells. These results indicate the feasibility of using the heat-sensitive immunoliposomes as a target-specific drug delivery system.
Hyperpolarization of the membrane potential caused by somatostatin in dissociated human pituitary adenoma cells that secrete growth hormone.
Authors: Yamashita N , Shibuya N , Ogata E
Published: May 2006

Membrane electrical properties and the response to somatostatin were examined in dissociated human pituitary adenoma cells that secrete growth hormone (GH). Under current clamp condition with a patch electrode, the resting potential was -52.4 +/- 8.0 mV, and spontaneous action potentials were observed in 58% of the cells. Under voltage clamp condition an outward K+ current, a tetrodotoxin-sensitive Na+ current, and a Ca2+ current were observed. Cobalt ions suppressed the Ca2+ current. The threshold of Ca2+ current activation was about -60 mV. Somatostatin elicited a membrane hyperpolarization associated with increased membrane permeability in these cells. The reversal potential of somatostatin-induced hyperpolarization was -78.4 +/- 4.3 mV in 6 mM K+ medium and -97.2 +/- 6.4 mV in 3 mM K+ medium. These reversal potential values and a shift with the external K+ concentration indicated that membrane hyperpolarization was caused by increased permeability to K+. The hyperpolarized membrane potential induced by somatostatin was -63.6 +/- 5.9 mV in the standard medium. This level was subthreshold for Ca2+ and Na+ currents and was sufficient to inhibit spontaneous action potentials. Hormone secretion was significantly suppressed by somatostatin and cobalt ions. Therefore, we suggest that Ca2+ entering the cell through voltage-dependent channels are playing an important role for GH secretion and that somatostatin suppresses GH secretion by blocking Ca2+ currents. Finally, we discuss other possibilities for the inhibitory effect of somatostatin on GH secretion.
Dopaminergic control of 125I-labeled neurotensin binding site density in corticolimbic structures of the rat brain.
Authors: Herve D , Tassin J P , Studler J M , Dana C , Kitabgi P , Vincent J P , Glowinski J , Rostene W
Published: May 2006

In the rat brain, destruction of dopaminergic cell groups by injections of 6-hydroxydopamine into the ventral mesencephalic tegmentum results in large decreases in the number of neurotensin binding sites in the mesencephalon and the striatum. In contrast, these lesions produce an increase in the number of 125I-labeled neurotensin binding sites in the lateral part of the prefrontal cortex despite a large decrease in cortical dopamine levels. Increases in the number of 125I-labeled neurotensin binding sites in this cortical area as well as in the entorhinal cortex, the nucleus accumbens, and the central part of the striatum were also obtained after chronic blockade of dopamine neurotransmission by a long-acting neuroleptic pipotiazine palmitic ester. We propose that dopamine inputs regulate the density of postsynaptic neurotensin binding sites through cortical and subcortical dopamine receptors. Therefore, some of the clinical effects of neuroleptics in schizophrenic patients could be partly related to changes in neurotensin neurotransmission.
Viroids and virusoids are related to group I introns.
Authors: Dinter-Gottlieb G
Published: May 2006

Group I introns are found in nuclear rRNA genes, mitochondrial mRNA and rRNA genes, and chloroplast tRNA genes. The hallmarks of this intron class are a 16-nucleotide consensus sequence and three sets of complementary sequences. The viroids (circular pathogenic plant RNAs) and the virusoids (plant satellite RNAs) also contain the consensus sequence and the three sets of complementary bases. Pairing of the complementary bases would generate a viroid structure resembling a group I intron, which might be stabilized in vivo through interactions with proteins. The Tetrahymena self-splicing rRNA intron further has sequences homologous with regions of potato spindle tuber viroid associated with the severity of viroid symptoms.
Cyclosporin A prevents induction of the interleukin 2 receptor gene in cultured murine thymocytes.
Authors: Gauchat J F , Khandjian E W , Weil R
Published: May 2006

Blast formation and mitotic activation of G0-arrested mouse thymocytes were triggered by the addition of concanavalin A plus interleukin 2 (IL-2) to the culture medium. When added alone, Con A induces within 6 hr a complex reprogramming ("priming") that comprises the activation of the IL-2 receptor gene. The primed thymocytes are competent to interact with IL-2 and to respond to its growth-promoting effect, which corresponds to blast formation and mitotic activation. Cyclosporin A, an immunosuppressive cyclic peptide of fungal origin, prevents in T lymphocytes the activation of a set(s) of genes encoding lymphokines and the IL-2 receptor but does not affect their expression once they have been activated. The biomedical implications of these observations are discussed.
Gene-dosage compensation of endosperm proteins in hexaploid wheat
Triticum aestivum
Authors: Galili Gad , Levy Avraham A. , Feldman Moshe
Published: May 2006

Several aneuploid lines and one intervarietal substitution line of the hexaploid wheat
Triticum aestivum
(2
n
= 6
x
= 42; genomes AABBDD) cv. Chinese Spring were used to study the effects of different doses of chromosomes 1B, 1D, or 1A on the amount of the high molecular weight (“HMW”) glutenins and gliadins of endosperm. These homeologous chromosomes carry HMW glutenin and gliadin gene clusters on their long and short arms, respectively. Increasing the dosage of chromosome 1B of Chinese Spring in plants having in their 3
n
endosperm zero or the normal three doses of the homeologue 1D, as well as in plants carrying in their endosperm one dose of 1B of the cultivar Timstein, had a dual effect: on one hand, a nonlinear increase in the amount of each subunit encoded by the chromosome whose dosage was elevated and, on the other hand, a compensating nonspecific decrease in the amount of other HMW glutenin and gliadin subunits encoded either by the homeoalleles on 1D or by the homoalleles on 1B of Timstein, respectively. Deletion of chromosome arm 1BL, which carries only a few HMW glutenin genes, had no significant effect on the amount of HMW glutenins encoded by 1DL and HMW gliadins encoded by 1DS and 1BS. However, deletion of 1BS or 1DS, each carrying many gliadin genes, caused a significant but nonspecific increase in the HMW glutenins and gliadins encoded by the remaining arms of 1B and 1D. The possible mechanism and evolutionary implications of gene-dosage compensation in polyploid wheat are discussed.
Base substitutions in the tRNA anticodon arm do not degrade the accuracy of reading frame maintenance.
Authors: Curran J F , Yarus M
Published: May 2006

We have examined the activities of a set of 34 site-directed mutants of tRNA Su7 for their ability to shift reading frame during translation of amber codons in vivo. The set includes variants at every position in the distal three base pairs of the anticodon stem and saturates the anticodon loop, with the exception of the anticodon itself. Most anticodon-stem mutations were made pairwise to preserve the secondary structure of that region. Variants of the Hirsh (A24) coding alteration were also tested. The mutations have varied and often dramatic effects on the ability of Su7 to act in translation, which indicates that they cause distortions of the codon-anticodon complex. However, none of the tested mutations affects the intrinsic accuracy of translocation, which we show to be very high. These results suggest that translocation must be independent of the conformational detail of the codon-anticodon complex and stand in contrast to frameshifts that occur when tRNAs misread codons. We suggest that when the tRNA is properly paired to the codon, translocation proceeds normally. Thus, we conclude that selection of a cognate tRNA ensures highly accurate reading frame maintenance. As a corollary, inefficient amber suppressors are not inefficient because they frameshift. Instead, they are likely to fail because a release factor translates the amber codon.
Cloning of the esterase D gene: a polymorphic gene probe closely linked to the retinoblastoma locus on chromosome 13.
Authors: Squire J , Dryja T P , Dunn J , Goddard A , Hofmann T , Musarella M , Willard H F , Becker A J , Gallie B L , Phillips R A
Published: May 2006

The study of recessive oncogenes such as those responsible for retinoblastoma and Wilms tumor is difficult because the gene products involved are unknown and because the diseases are not associated with unique cellular or molecular phenotypes suitable for genetic manipulation. Since the gene for esterase D (ESD) is known to be tightly linked to the retinoblastoma locus (RB1) in the q14.1 band of chromosome 13, we have cloned the ESD gene from a human cDNA library by using oligonucleotides specific for a partial amino acid sequence of the purified enzyme to provide a genetic marker for further studies on retinoblastoma. The putative ESD gene codes for a message of 1.2 kilobases, which is present in all cell types examined, and maps to 13q14.1, thus confirming that it is the ESD gene. Restriction enzyme analysis reveals a restriction fragment length polymorphism with Apa I; this polymorphism results from the heterozygosity of 32% of the individuals tested and is shown to be useful in identifying carriers of the mutation responsible for retinoblastoma. A preliminary screen of 24 retinoblastoma tumors by Southern blot did not reveal any homologous deletions or rearrangements of the ESD locus.