bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for specific DNA sequences in the nuclear acceptor sites of the avian oviduct progesterone receptor.
Authors: Toyoda H , Seelke R W , Littlefield B A , Spelsberg T C
Published: May 2006

Recent studies have shown that saturable high-capacity nuclear binding sites (termed acceptor sites) for the avian oviduct progesterone receptor can be reconstituted by rehybridizing a specific oviduct chromatin protein fraction (CP-3) to pure hen DNA to generate a reconstituted nucleoacidic protein (NAP). Only a limited number of acceptor sites can be generated on hen DNA even at high protein/DNA ratios. This suggests the existence of a limited number of specific sequences in the avian genome that can participate in the acceptor sites. The studies presented in this paper show a specificity as to the source of DNA that can generate acceptor sites using hen oviduct CP-3 protein. The acceptor protein binds to all DNAs but generates acceptor sites only on DNAs from certain animals. The acceptor sites for the progesterone receptor, generated with heterologous mammalian DNAs and the avian oviduct CP-3 fraction, show saturation not only in number of acceptor sites generated on the DNAs but also in progesterone receptor binding. Binding to these sites is also receptor dependent. Using oviduct receptors from particular physiological states of the birds wherein the receptors do not bind to nuclear sites in vivo, it was found that the cell-free binding to these heterologous complexes of hen CP-3 protein and DNA from another species, termed heterologous NAP, is similarly absent. Thus, the cell-free binding to the native oviduct NAP and the heterologous NAP markedly resembles the nuclear binding in vivo. Interestingly, synthetic DNAs rich in adenine and thymine, but not those rich in guanine and cytosine, are capable of generating acceptor sites. Species-specific DNA sequences, as well as specific chromatin proteins, therefore, appear to be involved in the nuclear acceptor sites for the avian oviduct progesterone receptor. The DNA sequences appear to be conserved throughout most of the vertebrates but not among nonvertebrates as are the steroid hormones and their receptors. The exact numbers and distributions of these sequences in the avian genome are not known.
A predicted structure of calmodulin suggests an electrostatic basis for its function.
Authors: O'Neil K T , DeGrado W F
Published: May 2006

By using interactive computer graphics, two models for calmodulin have been constructed based on the structures of two functionally and structurally related proteins, intestinal calcium-binding protein and carp parvalbumin. The two models have been compared and contrasted to the parent proteins with respect to proportion of solvent-exposed hydrophobic residues, solvent-accessible surface area, and side-chain packing. Electrostatic potential surfaces generated for the models suggest a probable binding site for basic amphiphilic alpha-helical peptides located between the last E and F helices in the second domain of calmodulin. Both electrostatic and hydrophobic complementarity can contribute to stabilization of a peptide-protein complex in this region.
Production and characterization of a monoclonal antibody to a human interferon-induced double-stranded RNA-binding Mr 68,000 protein kinase.
Authors: Penn L J , Williams B R
Published: May 2006

One of the interferon-induced proteins thought to be involved in the antiviral effects of interferon is a double-stranded RNA-dependent protein kinase. This paper reports the development of a monoclonal antibody, 10A5, that recognizes a protein that co-migrates with the double-stranded RNA-dependent protein kinase at an approximate molecular weight of 68,000. Levels of this protein and of the protein kinase activity increase 3-fold on interferon treatment of T98G cells. The specificity of the monoclonal antibody was determined by ELISA, immunoblotting, and immunoprecipitation procedures. Furthermore, immunoaffinity chromatography of an interferon-induced T98G cell extract previously phosphorylated in the presence of double-stranded RNA and radiolabeled ATP resulted in the specific elution of a phosphorylated Mr 68,000 protein from the monoclonal antibody 10A5-Sepharose column. Monoclonal antibody 10A5 recognizes both native and denatured protein kinase, independent of double-stranded RNA binding or phosphorylation, and should therefore serve as a useful tool in analyzing the role of the double-stranded RNA-dependent protein kinase in the mechanism of interferon action.
Expression of autophosphorylating protein kinase 500 in normal and neoplastic rat cells.
Authors: Majumdar G , Cashel M , Sharma R K
Published: May 2006

Autophosphorylating protein kinase 500 (AUT-PK 500) is a unique serine protein kinase that was originally purified and characterized from the rat adrenocortical carcinoma. A specific RIA with an assay sensitivity of 10 ng (0.02 pmol) was developed for AUT-PK 500 and applied to normal, embryonic, fetal, neonatal, immortal, and neoplastic tissues and cultured cells. As compared to normal rat tissues, the expression of AUT-PK 500 is elevated 100-fold in spontaneously occurring adrenocortical carcinoma 494, 50- to 60-fold in four chemically induced, rapidly growing hepatomas, 30-fold in the chemically induced mammary carcinoma, 20-fold in the cultured hepatoma cell line, and 4-fold in the Rat I and Rat II established tissue culture cell lines. There was also a 5-fold increase in the enzyme when freshly cultured rat skin epithelial-like cells were established. Furthermore, in vivo studies showed that when the rat liver was chemically transformed into its premalignant altered foci, there was a 7-fold elevation of AUT-PK 500. Embryonic cells and fetal and neonatal tissues contained barely detectable (less than 0.22 micrograms/mg of protein) amounts of the protein kinase. These results suggest that AUT-PK 500 is not involved in the differentiation process during fetal development but may be elevated during early steps of carcinogenesis and is further elevated during later stages.
Cell-type-specific pattern of myc protooncogene expression in developing human embryos.
Authors: Pfeifer-Ohlsson S , Rydnert J , Goustin A S , Larsson E , Betsholtz C , Ohlsson R
Published: May 2006

The expression of viral oncogenes in cells transformed by acutely transforming retroviruses profoundly alters proliferation and differentiation in the target cell, suggesting that the cellular homologues of the viral oncogenes, the protooncogenes, have a role in normal cell proliferation and differentiation. To investigate the possible developmental role of protooncogenes in human embryogenesis, we have determined the spatial distribution of myc gene transcripts in early human embryos by using in situ hybridization of a labeled myc exon to thin sections. The results indicate a stage- and cell-type-specific regulation of c-myc gene expression in primarily epithelial cells of late first trimester embryos. Furthermore, the data suggest that the linkage between c-myc gene expression and cellular proliferation holds for only a restricted set of embryonic cells.
Linkage map of the short arm of human chromosome 11: location of the genes for catalase, calcitonin, and insulin-like growth factor II.
Authors: Kittur S D , Hoppener J W , Antonarakis S E , Daniels J D , Meyers D A , Maestri N E , Jansen M , Korneluk R G , Nelkin B D , Kazazian H H
Published: May 2006

The following order of genes on the short arm of human chromosome 11 (11p) was determined previously: parathyroid hormone (PTH)-the beta-globin gene cluster (HBBC)-HRAS1/insulin. Although it is generally agreed that HRAS1 (formerly termed c-Ha-ras-1) and the insulin gene are close to each other [1-4 centimorgans (cM)], their order on chromosome 11p is still in question. We have now added three other genes, those for catalase, calcitonin, and insulin-like growth factor II (IGF-II), to this map of chromosome 11p by use of restriction site polymorphisms adjacent to these genes in classical linkage analysis. Most importantly, we find no evidence of linkage between the catalase and HBBC loci. In addition, our data indicate that the calcitonin gene is located between the catalase gene and the PTH gene. Our best estimate of the distance between the catalase and calcitonin gene is approximately 16 cM, while that between the calcitonin and PTH genes is approximately equal to 8 cM. In agreement, very loose linkage was found between the catalase and PTH loci (approximately 26 cM). Since the catalase locus has been mapped to 11p13, these data support the view that the PTH, HBBC, HRAS1, and insulin loci are located on the distal short arm of chromosome 11. The IGF-II gene is tightly linked to both the HRAS1 oncogene and the insulin gene since no recombinants were observed between the IGF-II and the HRAS1/insulin loci. Thus, based on our linkage analysis we propose that the most likely gene order for the short arm of chromosome 11 is centromere-catalase-calcitonin-PTH-HBBC-HRAS1/insulin-tel ome re and that the IGF-II gene is very close to both the HRAS1 and the insulin genes.
Duck hepatitis B virus DNA in liver and serum of Chinese ducks: integration of viral DNA in a hepatocellular carcinoma.
Authors: Yokosuka O , Omata M , Zhou Y Z , Imazeki F , Okuda K
Published: May 2006

The presence of duck hepatitis B virus (DHBV) DNA in liver and serum and its state (integrated vs. free) were studied in 23 ducks from Chi-tung county in China by spot hybridization and Southern blot hybridization, respectively. In 16 of 23 (70%), DHBV DNA was detected in serum and/or in liver tissue. These infected ducks showed a variety of pathological changes including advanced chronic disease in the liver. In contrast, none of the virus-negative ducks had advanced hepatic changes. One DHBV DNA-seropositive duck had a large hepatocellular carcinoma. Southern blot analysis demonstrated integrated DHBV DNA in neoplastic tissue and abundant episomal DHBV DNA in non-neoplastic tissue of the liver. In one noninfected duck with a small adenoma, no viral DNA was detected in tumor or non-neoplastic tissue. The detection of integrated DHBV DNA in hepatocellular carcinoma suggests that DHBV behaves like human and woodchuck hepatitis viruses in relation to chronic liver disease and hepatocarcinogenesis.
Antibodies specific for amino acid 12 of the ras oncogene product inhibit GTP binding.
Authors: Clark R , Wong G , Arnheim N , Nitecki D , McCormick F
Published: May 2006

An antibody (anti-p21ser) was raised against a ras p21-related synthetic peptide and was able to recognize specifically the substitution of serine for glycine at amino acid 12 of p21. This substitution causes oncogenic activation of p21. Anti-p21ser was found to immunoprecipitate v-Ki-ras p21 and to strongly inhibit its ability to autophosphorylate and to bind GTP in an immunoabsorption assay. Furthermore, binding of the antibody to p21 was specifically inhibited by GTP or GDP, suggesting that amino acids around position 12 are part of the GTP/GDP binding site. These results, taken together with the observation that the microinjection of anti-p21ser into cells transformed by v-Ki-ras p21 causes a transient reversion of the cells to a normal phenotype [Feramisco, J. R., Clark, R., Wong, G., Arnheim, N., Milley, R. & McCormick, F. (1985) Nature (London) 314, 639-642], support the idea that interaction of p21 with guanine nucleotides is crucial to the transforming function of this protein.
Cloning and characterization of cDNAs encoding 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible rabbit mRNAs for cytochrome P-450 isozymes 4 and 6.
Authors: Okino S T , Quattrochi L C , Barnes H J , Osanto S , Griffin K J , Johnson E F , Tukey R H
Published: May 2006

Monoclonal antibodies toward rabbit liver cytochrome P-450 isozyme 6 (P-450 6) were used to identify several recombinant clones from a pBR322 cDNA library that express beta-lactamase-P-450 6 hybrid proteins. The nucleic acid sequence and predicted amino acid sequence of a rabbit P-450 6 cDNA shows a high degree of homology with rat P-450c and mouse P1-450. When used as a probe to rescreen the library, the P-450 6 cDNA hybridized to several heterologous classes of cDNAs. One such class was shown to encode P-450 4 by comparison of its predicted amino acid sequence to amino acid sequences of cysteine-containing tryptic peptides derived from P-450 4. DNA sequence analysis of a cDNA clone belonging to a third class demonstrated that it contained a 131-base-pair intervening sequencing when compared to the cDNA coding for P-450 6. This sequence corresponds in location to intron E of the rat P-450c gene. Blot-hybridization analysis demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dramatically induced P-450 4 and 6 mRNAs, which differ in size. The sizes of these mRNAs differ from their analogs in the mouse as a result of divergence in the 3' untranslated portions of the mRNAs.
Characterization of a pigment-dispersing hormone in eyestalks of the fiddler crab
Uca pugilator
Authors: Rao K. Ranga , Riehm John P. , Zahnow Cynthia A. , Kleinholz Lewis H. , Tarr George E. , Johnson Linda , Norton Sheila , Landau Matthew , Semmes Oliver J. , Sattelberg R. Mark , Jorenby William H. , Hintz Mary F.
Published: May 2006

A pigment-dispersing hormone (PDH) from eyestalks of the fiddler crab
Uca pugilator
has been purified by gel filtration, ion-exchange chromatography, partition chromatography, and reversed-phase liquid chromatography. Based on automated gas-phase sequencing and subsequent identification of carboxyl-terminal amide, we have assigned the primary structure of this peptide as Asn-Ser-Glu-Leu-Ile-Asn-Ser-Ile-Leu-Gly-Leu-Pro-Lys-Val-Met-Asn-Asp-Ala-NH
2
. We have confirmed the sequence by synthesizing this peptide and demonstrating that the synthetic PDH and the native PDH display identical chromatographic behavior and biological activity. This hormone is a member of a family of invertebrate neuropeptides that includes a light-adapting/pigment-dispersing octadecapeptide hormone from the prawn
Pandalus borealis
. In assays for melanophore pigment dispersion in destalked fiddler crabs,
Uca
PDH was 21-fold more potent than
Pandalus
PDH. These two hormones share a hexapeptide core sequence (residues 5-10: -Ile-Asn-Ser-Ile-Leu-Gly-) as well as the amino- and carboxyl-terminal residues but differ at positions 3, 4, 11, 13, 16, and 17. These results point to speciesrelated or group-specific structural differences among crustacean PDHs.
c-myc mRNA levels in the cell cycle change in mouse erythroleukemia cells following inducer treatment.
Authors: Lachman H M , Hatton K S , Skoultchi A I , Schildkraut C L
Published: May 2006

Several lines of evidence suggest that the c-myc protooncogene is involved in some aspect of cell division in mammalian cells. We have been investigating changes in the expression of c-myc mRNA in mouse erythroleukemia cells during chemically induced terminal erythroid differentiation. In vitro induction of erythroleukemia cell differentiation results in a switch from cells with unlimited proliferative capacity to cells that undergo a small number of terminal cell divisions. The level of c-myc mRNA changes rapidly following treatment with inducing agents. After a very rapid decline the mRNA is restored to pretreatment levels and then declines again. We have now measured the level of c-myc mRNA with respect to position in the cell cycle. Prior to inducer treatment the level of c-myc mRNA is relatively constant throughout the cell cycle. However, when the mRNA is restored following treatment with hypoxanthine or hexamethylenebisacetamide, it is found primarily in cells in the G1 phase. Thus, treatment with inducers of differentiation leads to a change in the cell cycle regulation of c-myc mRNA. This change may be involved in the altered proliferative capacity of the cells that occurs during terminal differentiation.
The 90-kDa component of reticulocyte heme-regulated eIF-2 alpha (initiation factor 2 alpha-subunit) kinase is derived from the beta subunit of spectrin.
Authors: Kudlicki W , Fullilove S , Kramer G , Hardesty B
Published: May 2006

Antibodies from three different lines of monoclonal hybridomas crossreact with both the beta subunit of spectrin and the 90-kDa peptide present in highly purified preparations of the heme-controlled eIF-2 alpha (initiation factor 2 alpha-subunit) kinase from rabbit reticulocytes. Antibodies from two of the three lines enhance the enzymatic activity of the kinase preparation for phosphorylation of the alpha subunit of eukaryotic translational initiation factor 2 (eIF-2) and for phosphorylation of the 100-kDa peptide thought to be a peptide of the kinase that is phosphorylated during its activation. Also, it is shown that both the beta subunit of spectrin and the 90-kDa peptide can be phosphorylated by two protein kinases from reticulocytes, the catalytic subunit of cAMP-dependent protein kinase and a cAMP-independent protein kinase similar to casein kinase II. Furthermore, a phosphorylated 90-kDa peptide can be derived from phosphorylated beta subunit of spectrin by tryptic proteolysis. We conclude that the 90-kDa peptide is derived by proteolysis from the beta subunit of spectrin, probably from its carboxyl terminus, and suggest that the heme-sensitive eIF-2 alpha kinase, like the 56-kDa phosphatase [Wollny, E., Watkins, K., Kramer, G. & Hardesty, B. (1984) J. Biol. Chem. 259, 2484-2492], is associated with an element of the membrane skeleton in intact reticulocytes.
Detection of a fundamental modular format common to transfer and ribosomal RNAs: second-order spectral analysis.
Authors: Nazarea A D , Bloch D P , Semrau A C
Published: May 2006

Second-order spectral analysis is used to detect rigorously and to characterize the principal periodicities in the positions of conserved sequences common to tRNAs and rRNAs. It is shown that the shared periodicity having the largest spectral amplitude is 9, followed by 8 and 10, thus forming a closed triad of significant multiplets centered at 9 bases. This conclusion is proposed to reflect a closed triadic set of fundamental tandem repeat lengths in a class of ancestral macromolecules possessing a restricted sequence symmetry. The terms "remanent" and "archeomodular" are used to describe a relic modular format, traces of which are shown here to persist despite the changes that have occurred in the primary structures of ribonucleic acids during the course of their evolution.
Kinetic analysis of regulatory events in G1 leading to proliferation or quiescence of Swiss 3T3 cells.
Authors: Zetterberg A , Larsson O
Published: May 2006

Kinetic analysis of cellular response to serum deprivation or inhibition of protein synthesis was performed on Swiss 3T3 cells. Time-lapse cinematographic analysis of individual cells transiently exposed to serum-free medium (with or without the addition of purified growth factors) or cycloheximide enabled a detailed mapping of the magnitude and variability of cellular response in different parts of the cell cycle. In all cells, in all stages of the cell cycle, serum deprivation resulted in inhibition of protein synthesis, but only in postmitotic cells in the first 3-4 hr of G1 (here denoted the G1pm phase) did it produce cell-cycle arrest. During G1pm, the cells are highly dependent on the continuous presence of serum growth factors and a high level of protein synthesis in order to progress toward mitosis. A 1-hr exposure to serum-free medium or to cycloheximide was sufficient to force most G1pm cells into a state of quiescence (G0), from which the cells required 8 hr to return to G1pm. During G1pm the cells complete the growth factor-dependent processes leading to commitment for proliferation. Thereafter they enter the growth factor-independent pre-DNA-synthetic part of G1 (here denoted G1ps). The commitment process in G1pm could be successfully completed in the presence of platelet-derived growth factor as the only supplied growth factor. Epidermal growth factor and insulin were insufficient for the completion of the commitment processes in G1pm, although they were able to temporarily prevent the G1pm cells from entering G0 during serum starvation. Under conditions optimal for proliferation, the cells complete the commitment processes in G1pm within a remarkably constant time period. Almost all cells in the population left G1pm and entered G1ps between the third and fourth hour after mitosis. The duration of G1ps, on the other hand, showed a large intercellular variability consistent with a transition-probability event. In fact, G1ps accounts for most of the variability in G1 and cell cycle time.
Cell size as a determinant of the clone-forming ability of human keratinocytes.
Authors: Barrandon Y , Green H
Published: May 2006

Keratinocytes isolated from human epidermis and subsequently cultured may form clones if they are 11 micron or less in diameter but are irreversibly committed to further enlargement and terminal differentiation if they are 12 micron or more in diameter. When a founding cell of 11 micron or less forms a small rapidly growing clone in culture, the cells of that clone are able to found new colonies even when their diameter is as great as 20 micron. As the clone becomes larger and grows more slowly, the maximal size of its clonogenic cells is reduced toward that of the epidermis. A cultured cell of up to 20 micron in diameter can, when it divides, give rise to clonogenic progeny smaller than itself, thus reversing the process of enlargement. Cells larger than 20 micron cannot divide and therefore cannot be rescued from terminal differentiation. It is concluded that when keratinocytes multiply rapidly, they extend reversibly the maximal size at which they are capable of generating clones into the range usually characteristic of terminally differentiating cells. It is proposed that this mechanism enables the keratinocyte to accommodate an increased rate of multiplication to its need to attain a large size during terminal differentiation.
Papillomavirus genomes in human cervical tumors: analysis of their transcriptional activity.
Authors: Lehn H , Krieg P , Sauer G
Published: May 2006

Four of six human cervical carcinoma biopsies were shown to contain the DNA of the human papillomavirus type 16 (HPV-16) covalently linked with the tumor cell DNA. HPV-16-specific mRNA species were observed in only one of the four tumors. No such sequences were found in the three other specimens in conditions that permitted the detection of less than one mRNA molecule per cell. It is concluded that maintenance of the malignant nature of these cervical tumors does not depend on the continuous transcriptional activity of HPV-16.
Antiserum to lens antigens immunostains Müller glia cells in the neural retina.
Authors: Moscona A A , Fox L , Smith J , Degenstein L
Published: May 2006

Antiserum to a lens fraction enriched for alpha-crystallin selectively immunostains Müller glia cells in the neural retina of several vertebrate species. Also, in embryonic retina (chicken), this antiserum reacts with Müller cells and, at early stages of development, with their apparent precursors. Thus, antibodies to a lens product(s) detect a Müller glia cell marker that begins to be expressed very early in their ontogeny and can be useful in studies on differentiation, function, and pathologies of this cell type.
Quantitative structural analysis of eukaryotic ribosomal RNA by scanning transmission electron microscopy.
Authors: Oostergetel G T , Wall J S , Hainfeld J F , Boublik M
Published: May 2006

The conformation of 28S ribosomal RNA isolated from baby hamster kidney cells was studied by scanning transmission electron microscopy (STEM) and circular dichroic spectroscopy to establish the conditions under which STEM images of unstained freeze-dried rRNA are a meaningful representation of the conformation of rRNA in solution. We have determined the conformation of 28S rRNA under various buffer conditions, the molecular mass, the mass distribution, and the number of polynucleotide strands within the individual molecules, and the apparent radii of gyration. The 28S rRNA molecule is highly extended in water and becomes compact with increasing ionic strength. However, even in the "reconstitution buffer" (30 mM Tris/HCl/20 mM MgCl2/360 mM KCl, pH 7.6) the compactness does not reach a state in which the rRNA molecule appears structurally similar to the 60S ribosomal subunit. Our approach has a broad application in high-resolution structural studies of nucleic acids and nucleic acid-protein interactions.
Cloning, expression in Escherichia coli, and reconstitution of human myoglobin.
Authors: Varadarajan R , Szabo A , Boxer S G
Published: May 2006

A full-length cDNA clone for human myoglobin has been isolated from a human skeletal muscle cDNA library. The clone as isolated has a cDNA insert approximately one kilobase long and has 5' and 3' untranslated regions of approximately 80 and 530 base pairs, respectively. The sequence of the translated region corresponds exactly to that predicted for human myoglobin. The cDNA was expressed in high yield in Escherichia coli as a fusion protein consisting of the first 31 amino acids of the phage lambda cII gene, the tetrapeptide Ile-Glu-Gly-Arg, and the myoglobin sequence by following the approach of Nagai and Thogersen [Nagai, K. & Thogersen, M. C. (1984) Nature (London) 309, 810-812]. The fusion product was isolated, reconstituted with heme, cleaved with trypsin, and purified to generate a protein whose properties are indistinguishable from those for authentic human myoglobin. Myoglobin can be readily prepared on a gram scale by using these methods.
F1 and F2: Two similar genes regulated differently during development of Drosophila melanogaster
Authors: Walldorf Uwe , Hovemann Bernd , Bautz Ekkehard K. F.
Published: May 2006

Screening a cDNA library of 2- to 3-day-old flies with poly(A)
+
RNA from male and female flies, we were able to isolate a small number of clones hybridizing preferentially with RNA from female flies. Four cDNA clones, derived from a single mRNA species, proved to be highly abundant in female flies. When we screened a genomic library with the longest cDNA, we obtained two genomic clones,
F1
and
F2
;
F1
was a direct copy of the cDNA and
F2
was obtained by cross-hybridization. A detailed analysis of these genomic clones revealed two independent genes coding for proteins of 50 kDa that are >90% homologous. RNA analysis with gene-specific probes from the 3′ untranslated region showed an expression of
F1
in all stages of development with a 5- to 10-fold overexpression of this RNA in female flies compared with males. In contrast to
F1, F2
is mainly expressed in late pupae and is expressed only at low levels in adult flies.
Color categorization: a possible concordance between genes and culture.
Authors: Lumsden C J
Published: May 2006

A marked correspondence is found to relate the categories of infant wavelength discrimination and the ethnographic distribution of adult color terms. The structure of the infant category system accounts for at least 75% of the variance in the world ethnographic categories (P less than 0.001). Such a correspondence is predicted to be favored by natural selection when perceptual mechanisms interact with their associated cultural traits.
Bone marrow-derived thymic antigen-presenting cells determine self-recognition of Ia-restricted T lymphocytes.
Authors: Longo D L , Kruisbeek A M , Davis M L , Matis L A
Published: May 2006

We previously have demonstrated that in radiation-induced bone marrow chimeras, T-cell self-Ia restriction specificity appeared to correlate with the phenotype of the bone marrow-derived antigen-presenting (or dendritic) cell in the thymus during T-cell development. However, these correlations were necessarily indirect because of the difficulty in assaying thymic function directly by adult thymus transplant, which has in the past been uniformly unsuccessful. We now report success in obtaining functional T cells from nude mice grafted with adult thymuses reduced in size by treatment of the thymus donor with anti-thymocyte globulin and cortisone. When (B10 Scn X B10.D2)F1 nude mice (I-Ab,d) are given parental B10.D2 (I-Ad) thymus grafts subcutaneously, their T cells are restricted to antigen recognition in association with I-Ad gene products but not I-Ab gene products. Furthermore, thymuses from (B10 X B10.D2)F1 (I-Ab,d)----B10 (I-Ab) chimeras transplanted 6 months or longer after radiation (a time at which antigen-presenting cell function is of donor bone marrow phenotype) into (B10 X B10.D2)F1 nude mice generate T cells restricted to antigen recognition in association with both I-Ad and I-Ab gene products. Thymuses from totally allogeneic bone marrow chimeras appear to generate T cells of bone marrow donor and thymic host restriction specificity. Thus, when thymus donors are radiation-induced bone marrow chimeras, the T-cell I-region restriction of the nude mice recipients is determined at least in part by the phenotype of the bone marrow-derived thymic antigen presenting cells or dendritic cells in the chimeric thymus.
Secondary structure of the immunoglobulin J chain.
Authors: Zikan J , Novotny J , Trapane T L , Koshland M E , Urry D W , Bennett J C , Mestecky J
Published: May 2006

J chain is a 137-residue polypeptide that is covalently linked to polymeric immunoglobulins and participates in their synthesis and transport to external secretions. To clarify these roles, the secondary structure of J chain was characterized by computer-assisted analyses of human and mouse sequences and by circular dichroism measurements of the isolated J chain. The secondary-structure profiles obtained were very similar to those of superoxide dismutase or immunoglobulin light chain variable domains, suggesting that the J chain folds into an eight-stranded antiparallel beta-barrel and should contain approximately 37% beta-sheet conformation, with the rest of the structure existing as reverse turns (random coil). The circular dichroism measurements indicated that the conformation of denatured, S-carboxymethylated or S-sulfonated J chain consists of 75% random coil and 25% beta-structure. Upon reformation of disulfide bonds the percentage of beta-structure in the air-oxidized J chain increased to 34%, a value that is in good agreement with the secondary-structure analysis. Two alternative models of J-chain structure, a two-domain model [Cann, G., Zaritsky, A. & Koshland, M.E. (1982) Proc. Natl. Acad. Sci. USA 79, 6656-6660] and a single-domain antiparallel beta-sheet bilayer model (proposed in this paper), are compared.
Partial purification and characterization of a pp60v-src-related tyrosine kinase from bovine brain.
Authors: Neer E J , Lok J M
Published: May 2006

We have identified and substantially purified a tyrosine protein kinase from normal bovine brain that is immunologically related to the product of the Rous sarcoma virus oncogene (pp60v-src). The enzyme, a 61-kDa protein (p61), is solubilized with detergent from bovine cerebral cortical membranes and purified by column chromatography. In the purest preparations, this protein is phosphorylated only on tyrosine, but it can also be a substrate for serine- and threonine-specific protein kinases. The p61 protein phosphorylates the heavy chain of immunoglobulins from rabbits bearing Rous sarcoma virus-induced tumors (TBR IgG) but not normal IgG. TBR IgG precipitates the 61-kDa phosphoprotein and protein kinase activity from purified preparations. The activity of the purified brain tyrosine kinase is 10 times higher in the presence of 7-10 mM Mn2+ and 6 mM Mg2+ than it is with 6 mM Mg2+ alone. With Mn2+, the p61 enzyme has a Km for ATP of 2 microM. All preparations of p61 also contain a 64-kDa protein (p64) that is phosphorylated on tyrosine. Measurement of the Stokes radius of p61 and p64 by gel filtration shows that they are not physically associated in buffer containing the nonionic detergent Lubrol 12A9. The p64 protein is not precipitated by TBR IgG. We do not know whether p64 is only a substrate for the p61 tyrosine kinase or is itself a kinase.
Ren-1 and Ren-2 loci are expressed in mouse kidney.
Authors: Field L J , Gross K W
Published: May 2006

Inbred strains of mice can be categorized into two groups based on the absence or presence of a duplicated copy of the renin structural gene; one-gene strains carry a single renin gene (Ren-1), whereas two-gene strains carry two renin genes (Ren-1 and Ren-2). To investigate the contribution that each locus makes to the composite levels of renin mRNA observed to accumulate in different tissues of two-gene strains, we have developed two assays capable of distinguishing the highly homologous Ren-1 and Ren-2 transcripts. Both methods take advantage of established base sequence differences between Ren-1 and Ren-2 coding regions by using reverse transcriptase-mediated primer extension of oligonucleotide primer/mRNA hybrids in the presence of appropriate deoxy-and dideoxynucleotide phosphates. Using these techniques we found that Ren-1 and Ren-2 mRNAs accumulate in the kidney of two-gene strains to approximately equal levels. These observations are discussed in light of potential mechanisms regulating the tissue-specific expression of the Ren-1 and Ren-2 loci.
Evidence that major histocompatibility complex restriction of foreign transplantation antigens occurs when tolerance is induced in neonatal mice and rats.
Authors: Desquenne-Clark L , Kimura H , Silvers W K
Published: May 2006

Studies on the survival of skin-specific antigen (Skn)-incompatible skin grafts in mice rendered tolerant at birth with major histocompatibility complex (MHC)-incompatible lymph node and spleen or bone marrow cells, as well as studies concerned with the survival of third-party skin grafts in rats rendered tolerant at birth with MHC-incompatible bone marrow cells, indicate that MHC restriction of foreign transplantation antigens occurs when tolerance is induced. Thus, evidence is presented that animals rendered tolerant with MHC-incompatible bone marrow cells depleted of mature T lymphocytes will accept any graft that is homozygous for the bone marrow donor's foreign MHC. Evidence has also been obtained that continuous exposure to foreign transplantation antigens in association with an MHC different from that of the graft may induce unresponsiveness to the same antigens in association with the MHC of the graft.
Monkey-derived monoclonal antibodies against Plasmodium falciparum.
Authors: Stanley H A , Reese R T
Published: May 2006

A system has been developed that allows efficient production of monkey monoclonal antibodies from owl monkeys. Splenocytes or peripheral blood lymphocytes from monkeys immune to the human malarial parasite, Plasmodium falciparum, were fused with P3X63 Ag8.653 mouse myelomas. The resulting hybridomas were screened by an indirect fluorescent antibody test for the production of monkey monoclonal antibodies (mAb) reactive with P. falciparum. Most of the mAb reacted with the P. falciparum merozoites and immunoprecipitated a parasite-derived glycoprotein having a relative molecular weight of 185,000. These mAb gave a minimum of five different immunoprecipitation patterns, thus demonstrating that a large number of polypeptides obtained when parasitized erythrocytes are solubilized share epitopes with this large glycoprotein. In addition, mAb were obtained that reacted with antigens associated with the infected erythrocyte membrane. One of these mAb bound a Mr 95,000 antigen.
Replication of phage phi 29 DNA with purified terminal protein and DNA polymerase: synthesis of full-length phi 29 DNA.
Authors: Blanco L , Salas M
Published: May 2006

A system that replicates bacteriophage phi 29 DNA with protein p3 covalently attached to the two 5' ends, using as the only proteins the phi 29 DNA polymerase and the terminal protein, is described. Restriction analysis of the 32P-labeled DNA synthesized in vitro showed that all phi 29 DNA fragments were labeled. Analysis by alkaline sucrose gradient centrifugation of the DNA labeled during a 10-min pulse showed that, after a 20-min chase, about half of the DNA molecules had reached apparently full-length phi 29 DNA (approximately equal to 18,000 nucleotides). Ammonium ions strongly stimulated phi 29 DNA-protein p3 replication, the effect being due to stimulation of the initiation reaction. ATP was not required for phi 29 DNA-protein p3 replication, either in the initiation or elongation steps. The results show that the phi 29 DNA polymerase functions, not only in the formation of the p3-dAMP covalent initiation complex but also in the elongation of the latter, as the only DNA polymerase to produce full-length phi 29 DNA.
Conserved features of eukaryotic hsp70 genes revealed by comparison with the nucleotide sequence of human hsp70.
Authors: Hunt C , Morimoto R I
Published: May 2006

We have determined the nucleotide sequence of the human hsp70 gene and 5' flanking region. The hsp70 gene is transcribed as an uninterrupted primary transcript of 2440 nucleotides composed of a 5' noncoding leader sequence of 212 nucleotides, a 3' noncoding region of 242 nucleotides, and a continuous open reading frame of 1986 nucleotides that encodes a protein with predicted molecular mass of 69,800 daltons. Upstream of the 5' terminus are the canonical TATAAA box, the sequence ATTGG that corresponds in the inverted orientation to the CCAAT motif, and the dyad sequence CTGGAAT/ATTCCCG that shares homology in 12 of 14 positions with the consensus transcription regulatory sequence common to Drosophila heat shock genes. Comparison of the predicted amino acid sequences of human hsp70 with the published sequences of Drosophila hsp70 and Escherichia coli dnaK reveals that human hsp70 is 73% identical to Drosophila hsp70 and 47% identical to E. coli dnaK. Surprisingly, the nucleotide sequences of the human and Drosophila genes are 72% identical and human and E. coli genes are 50% identical, which is more highly conserved than necessary given the degeneracy of the genetic code. The lack of accumulated silent nucleotide substitutions leads us to propose that there may be additional information in the nucleotide sequence of the hsp70 gene or the corresponding mRNA that precludes the maximum divergence allowed in the silent codon positions.
Molecular packing and area compressibility of lipid bilayers.
Authors: White S H , King G I
Published: May 2006

Knowledge of the molecular packing of lipids and water in lipid bilayers is important for understanding bilayer mechanics and thermodynamics. Information on packing is most often obtained from x-ray or neutron diffraction measurements. Given the d spacing, composition, and partial specific volumes of the lipid and water, it is a simple matter to calculate the area per lipid molecule, bilayer thickness, and bilayer mass density. The partial specific volumes are commonly assumed to be those of bulk water and of lipid in excess water regardless of the degree of bilayer hydration. We present evidence here that these assumptions should be seriously questioned. At low hydrations, we find the head groups of egg and dioleoyl lecithin to be much less tightly packed than previously thought and the partial specific volume of water to be considerably smaller than 1 ml/g. Because the molecular packing affects the mechanical properties of bilayers, we use the results to reevaluate published experiments concerning the elastic area compressibility modulus of egg lecithin bilayers and the repulsive hydration force between bilayers.
Induction of Rhizobium meliloti nodC expression by plant exudate requires nodD.
Authors: Mulligan J T , Long S R
Published: May 2006

The soil bacterium Rhizobium meliloti invades and establishes a symbiosis with host plants such as alfalfa. Bacterial nodulation (nod) genes are required for this invasion, but their mechanism of action and the timing of their expression are not known. We have used translational lacZ fusions to monitor expression of nodD and nodC, which are located in the cluster of four nod genes on the R. meliloti megaplasmid (pSym). nodD is expressed at comparable levels by broth-grown bacterial cells and by cells exposed to exudates from aseptically grown plants. Activity of the nodC-lacZ protein fusion in broth-grown bacterial cells is very low. nodC-lacZ activity is increased approximately equal to 30-fold by plant exudate when nodD is expressed at a high level but not when nodD expression is low. Both fusions show differences in expression when borne on inc-P vectors as compared to when located on the pSym megaplasmid. nodD expression from vector-borne copies of the nod segment and response of nodC to plant exudate appear to require additional loci on the megaplasmid. Our results suggest that regulation of bacterial nod gene expression is an important control mechanism early in the symbiosis, and that the biochemical nature of some nod gene products may be cryptic except in cells grown in the presence of plant exudate.
Structure of the chromosomal gene for murine interleukin 3.
Authors: Miyatake S , Yokota T , Lee F , Arai K
Published: May 2006

We have isolated the mouse interleukin 3 (IL-3) gene from a mouse sperm DNA library based on homology with the mouse mast-cell growth-factor (MCGF) cDNA sequence. The nucleotide sequence determined for the gene and its flanking regions reveals that the IL-3 gene is composed of four introns and five exons. The nucleotide sequence of the exons agrees with that determined for the MCGF cDNA. A "TATA"-like sequence preceded by a G + C-rich region is found in the 5' flanking region. At the 3' region of the second intron are nine repeats of a closely related 14-base-pair (bp) sequence. These repeated sequences share extensive homology with a 20-bp repeated sequence found in the human genome, which was shown to have enhancer activity. Eight out of nine repeats form a 73-bp duplicated sequence and each 73-bp repeat contains sequences homologous to the core sequence suggested for enhancer elements. These sequences may play a role in the expression of the IL-3 gene in concanavalin A- or antigen-stimulated T lymphocytes. Stable transformants of L cells generated by co-transformation of the IL-3 gene with pSV2neo constitutively express MCGF activity indicating that the isolated gene is functional in vivo.
A selenium-containing nucleoside at the first position of the anticodon in seleno-tRNAGlu from Clostridium sticklandii.
Authors: Ching W M , Alzner-DeWeerd B , Stadtman T C
Published: May 2006

In previous studies, the single selenonucleoside component of a selenium-containing tRNAGlu isolated from Clostridium sticklandii has been shown to be 5-methyl-aminomethyl-2-selenouridine. Here, we show that this selenonucleoside is most likely located at the "wobble" position of the anticodon of the clostridial seleno-tRNAGlu. Nuclease T1 digestion of this seleno-tRNAGlu generated one major selenium-containing oligonucleotide (25 bases long). The selenium-containing residue within this oligonucleotide was located by sequence analysis of the oligonucleotide before and after removal of selenium by treatment with cyanogen bromide. The sequence of this oligonucleotide, A-A-C-C-G-C-C-C-U-U+-U-C-A+C-G-G-C-G-G-U-A-A-C-A-G, is homologous to that of the Escherichia coli tRNAGlu2 from residues 27 to 50, including the anticodon region and the variable loop, except that the E. coli tRNA has 5-methylaminomethyl-2-thiouridine instead of the selenonucleoside.
Highly structured sequence homology between an insertion element and the gene in which it resides
Authors: Rhodes Patsy R. , Vodkin Lila O.
Published: May 2006

The recessive allele for soybean seed lectin results from the insertion of a DNA segment (designated Tgm1) into the coding region of the gene. The termini of Tgm1 display structural features characteristic of a transposable element. The complete sequence of Tgm1 contains 3550 base pairs (bp) and can be divided into three regions (left arm, mid-section, and right arm). No large open reading frames were found, but an extensive, highly structured border with homology to the lectin gene was revealed. The left border (726 bp) comprising most of the left arm and extreme right border (144 bp) of the right arm consist of various forms of a basic 54-bp repeating unit. This 54-bp unit is comprised of a stem-loop structure and interhairpin sequence that occurs 13 times in the left arm and 2 times in the right arm of Tgm1. Progressively degenerate forms of this repeating unit appear toward the termini of Tgm1, but the dyad symmetry remains highly conserved. Seven nucleotides (A-C-A-T-C-G-G and its complement) maintained within the stem also appear as a subset of inverted repeats found at nearly equal distances from the target site in the lectin gene. Together with the inverted repeat termini and a duplication in the left arm, this 7-bp sequence occurs a total of 33 times in Tgm1. We infer that the dyad symmetries containing this sequence are involved in target gene selection. The repeating unit format of Tgm1 describes a distinct class of eukaryotic elements that includes representatives known to be mobile in snapdragon and maize.
Mapping of steroid 21-hydroxylase genes adjacent to complement component C4 genes in HLA, the major histocompatibility complex in man.
Authors: Carroll M C , Campbell R D , Porter R R
Published: May 2006

The genes for four components (C) of complement in the human major histocompatibility complex (HLA) have been aligned previously in a series of overlapping cosmid cloned inserts. Those inserts, which contained the two C4 genes C4A and C4B, hybridized with human adrenal mRNA, indicating that they contain a gene expressed in the adrenal. The mRNA fraction of 2.4 kilobases (kb) hybridizes with genomic DNA of 4.5 kb, which is duplicated and lies about 1.5 kb 3' of both the C4A and the C4B complement genes. Sequencing of a 430-base section and comparison with the published cDNA sequence of bovine cytochrome P-450 21-hydroxylase, peptide sequences of porcine 21-hydroxylase, and a cDNA sequence of a rat liver cytochrome P-450 identified the gene as coding for human steroid 21-hydroxylase [steroid,hydrogen-donor:oxygen oxidoreductase (21-hydroxylating), EC 1.14.99.10]. Mapping of the gene was helped by use of a synthetic oligonucleotide based on the bovine cDNA sequence.
uvrA and recA mutations inhibit a site-specific transition produced by a single O6-methylguanine in gene G of bacteriophage phi X174.
Authors: Chambers R W , Sledziewska-Gojska E , Hirani-Hojatti S , Borowy-Borowski H
Published: May 2006

Using site-specific mutagenesis, we have examined the mutagenic activity in vivo of O6-methylguanine or O6-n-butylguanine located at a preselected site in gene G of bacteriophage phi X174. The experiments were designed so that the phage mutant produced by a targeted transition from either of these alkylated derivatives would be recognizable by a simple plaque assay. Spheroplasts derived from normal and repair-deficient cells were transfected, and the lysates were screened for mutant virus. In cells with normal repair, DNA carrying the methylguanine produced the expected transition in 15% of the total phage; DNA carrying the butylguanine produced the same mutation in 0.3% of the phage. In cells deficient in excision repair (uvrA) the transition frequency went up by a factor of 8 for O6-butylguanine and down by a factor of 40 for O6-methylguanine. In cells deficient in recombination (recA), the transition frequency increased 1.5-fold for butylguanine and decreased by a factor of 8 for methylguanine. The data show that both methyl- and butylguanine produce site-directed transitions in phi X174; the transition occurs in recA cells; the frequency of the transition is influenced by both recA and uvrA mutations; the recA and uvrA mutations alter the transition frequency for methylguanine and butylguanine in opposite directions.