bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional expression in yeast of the Escherichia coli plasmid gene coding for chloramphenicol acetyltransferase.
Authors: Cohen J D , Eccleshall T R , Needleman R B , Federoff H , Buchferer B A , Marmur J
Published: May 2006

The Escherichia coli R factor-derived chloramphenicol resistance (camr) gene is functionally expressed in the yeast Saccharomyces cerevisiae. the gene was introduced by transformation into yeast cells as part of a chimeric plasmid, pYT11-LEU2, constructed in vitro. The plasmide vector consists of the E. coli plasmid pBR325 (carrying the camr gene), the yeast 2-micron DNA plasmid, and the yeast LEU2 structural gene. Yeast cells harboring pYT11-LEU2 acquire resistance to chloramphenicol and cell-free extracts prepared from such cells contain chloramphenicol acetyltransferase (acetyl-CoA: chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28), the enzyme specified by the camr gene in E. coli. Resistance to chloramphenicol and the presence of chloramphenicol acetyltransferase activity segregate with the yeast marker LEU2, carried by the transforming plasmid, during both mitotic growth and meiotic division.
Organization of kappa light chain genes in germ-line and somatic tissue.
Authors: Joho R , Weissman I L , Early P , Cole J , Hood L
Published: May 2006

We studied the organization of the kappa light chain genes in germ-line (sperm) and somatic (embryo) tissues. We constructed a plasmid containing a DNA insert coding for the kappa chain MOPC 167 and used the Southern blotting technique to determine the organization of kappa variable and constant region genes. In the haploid genome of the mouse there is only one constant region gene detectable and it has the same organization in sperm and embryo DNAs. There are several variable region genes in sperm and embryo that are related to the Vk167 gene. The organization of the V genes in sperm and embryo DNAs is identical. These results show that there is no rearrangement of variable region genes (or "minigenes") during early embryogenesis.
Hydroboration kinetics: Unusual kinetics for the reaction of 9-borabicyclo[3.3.1]nonane with representative alkenes
Authors: Brown Herbert C. , Wang Kung K. , Scouten Charles G.
Published: May 2006

The kinetics of hydroboration of alkenes with 9-borabicyclo[3.3.1]nonane dimer, (9-BBN)
2
, exhibit interesting characteristics. With more reactive alkenes, such as 1-hexene, 2-methyl-1-pentene, 3,3-dimethyl-1-butene, and cyclopentene, the reaction exhibits first-order kinetics, first order in (9-BBN)
2
and zero order in alkene. On the other hand, with less reactive alkenes, such as cyclohexene, 1-methylcyclohexene, and 2,3-dimethyl-2-butene, the reaction exhibits three-halves-order kinetics, first order in alkene and one-half order in (9-BBN)
2
. These kinetics can be accounted for in terms of the following mechanism:

(9-BBN)
2
⇄ 2 9-BBN

9-BBN + alkene →
B
-alkyl-9-BBN

In the case of the more reactive alkenes, the rate-determining step is the dissociation of the dimer. With less reactive alkenes, the reaction of the alkene with the monomer becomes the rate-determining step. For certain alkenes, such as 2-methyl-2-butene and
cis
-3-hexene, neither of the two steps is a decisive rate-determining step. Therefore, the reaction exhibits kinetic behavior between that of first- and three-halves-order kinetics.
Cloning of sea urchin actin gene sequences for use in studying the regulation of actin gene transcription
Authors: Merlino Glenn T. , Water Robert D. , Chamberlain John P. , Jackson David A. , El-Gewely M. Raafat , Kleinsmith Lewis J.
Published: May 2006

In order to investigate the regulation of actin gene transcription during early sea urchin development, a specific hybridization probe for actin sequences is required. Such a probe was produced by cloning cDNA transcribed from a sea urchin poly(A)-containing mRNA preparation enriched for actin message. Double-stranded DNA was ligated into the
Bam
HI restriction site of plasmid pBR322, and the resulting hybrid molecules were used to transform the
Escherichia coli
strain ML100. After preliminary screening of bacterial colonies by antibiotic sensitivity and hybridization back to the original cDNA, clones containing sea urchin DNA were further characterized by a positive translation assay in which total sea urchin mRNA was hybridized to plasmid, and the hybridized message then was eluted and translated in a reticulocyte cell-free protein-synthesizing system. In this way, one clone (pSA38) was found to hybridize selectively to sea urchin mRNA coding for a protein of 43,000 daltons. This protein was identified as actin by three criteria: electrophoretic migration in two-dimensional polyacrylamide gels, affinity for DNase I, and peptide mapping. Restriction endonuclease and heteroduplex mapping of pSA38 indicate that it contains a 1.5-kilobase-pair insert and is therefore likely to contain a large portion of the actin coding sequence. By using pSA38 as a hybridization probe, it has been found that the level of actin-specific RNA sequences increases dramatically during early sea urchin development.
Inhibition of RNA polymerase activity by the Escherichia coli protein biosynthesis elongation factor Ts.
Authors: Biebricher C K , Druminski M
Published: May 2006

The transcribing activity of DNA-dependent RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from Escherichia coli is inhibited in vitro by addition of preparations of elongation factor Ts purified to homogeneity. The inhibitory activity of elongation factor Ts on the RNA polymerase activity and the enzymatic activity of elongation factor Ts show the same temperature sensitivity. The extent of inhibition is strongly dependent on the template used for transcription. A mechanism for the control of RNA synthesis in vivo based on this inhibition found in vitro is proposed.
Vero cells injected with adenovirus type 2 mRNA produce authentic viral polypeptide patterns: early mRNA promotes growth of adenovirus-associated virus.
Authors: Richardson W D , Carter B J , Westphal H
Published: May 2006

Adenovirus type 2 mRNAs were injected via glass capillaries into Vero cells, a line of African green monkey kidney cells permissive for adenovirus growth. Polypeptides synthesized after injection were labeled with 35S-labeled amino acids, precipitated with antiviral sera, and analyzed by polyacrylamide gel electrophoresis. Both early and late viral mRNAs give rise to authentic polypeptides. The in vivo translation of mRNAs can be measured as late as 24 hr after injection. The ability to analyze the protein products of microinjected mRNAs directly should greatly extend the applications of the procedure. Vero cells injected with early mRNA from adenovirus type 2 support the growth of adenovirus-associated virus, a defective virus that is dependent on adenovirus helper functions. This result demonstrates that a measurable biological activity, the ability to overcome the defectiveness of adenovirus-associated virus, resides in early adenovirus mRNA.
X-ray scattering by cybotactic nematic mesophases
Authors: Azároff Leonid V.
Published: May 2006

A structural model is proposed to account fully for observed x-ray scattering by certain nematic mesophases above their transition to smectic-C phases. In an external magnetic field, the constituent molecules tend to align into nearly periodic parallel chains that give rise to disc-like intensity distributions along the meridian in reciprocal space. Close to the transition temperature, the chains aggregate into parallel sheets which cause a ring-shaped intensity rise in these dises. The planar array in the cybotactic nematic bears a close similarity to that in cholesteric mesophases.
Copper electron-nuclear double resonance of cytochrome c oxidase.
Authors: Hoffman B M , Roberts J E , Swanson M , Speck S H , Margoliash E
Published: May 2006

Electron-nuclear double resonance of copper was observed while monitoring the "intrinsic copper" electron paramagnetic resonance signal of cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) near g = 2. This unambiguously establishes the presence of the metal (Cua) in the redox center responsible for this signal. The hyperfine couplings to copper are largely istropic and the maximum value is about half that seen in type I blue copper proteins. The magnetic properties of this oxidized copper center are not consistent with those of a thiyl radical (R-S) coordinated to Cu(I), and thus favor the identification of this redox center as a Cu(II) ion in a unique environment.
Different half-lives of the carbohydrate and protein moieties of a 110,000-dalton glycoprotein isolated from plasma membranes of rat liver.
Authors: Kreisel W , Volk B A , Büchsel R , Reutter W
Published: May 2006

By using a four-step procedure (i, solubilization with Triton X-100; ii, affinity chromatography on concanavalin A-Sepharose; iii, affinity chromatography on wheat germ lectin-Sepharose; iv, preparative sodium dodecyl sulfate gel electrophoresis) a glycoprotein was isolated from rat liver plasma membrane. The molecular weight is 110,000 and the isoelectric point is 5.8. It contains L-fucose, N-acetylneuraminic acid, D-galactose, D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and considerable quantities of aspartate, threonine, serine, and leucine. In pulse-chase experiments the half-lives of methionine and arginine, representing the half-life of the protein, were determined as 70 hr and 78 hr, respectively. The half-lives of the terminal carbohydrates L-fucose and N-acetylneuraminic acid were 12.5 and 33 hr, respectively. The galactose half-life was 20 hr. From this it is concluded that terminal sugars turn over several times in the life-span of this protein molecule. This process may be operative during membrane recycling mechanisms.
Multivalent translational control of transcription termination at attenuator of ilvGEDA operon of Escherichia coli K-12.
Authors: Lawther R P , Hatfield G W
Published: May 2006

The regulatory region for the ilvGEDA operon of Escherichia coli K-12 has been located and characterized. ilv leader RNA transcribed from this region is described, and the DNA sequence of the region is presented. This DNA sequence contains a transcription promoter, a region coding for a 32-amino-acid polypeptide containing multiple isoleucine, valine, and leucine codons, and a transcription termination site preceding the first structural gene. The mutually exclusive secondary structures of the leader RNA have been analyzed. On the basis of these data, a model for the multivalent attenuation of the ilvGEDA operon is proposed.
Immunoglobulin chain loss in hybridoma lines.
Authors: Köhler G
Published: May 2006

Hybrid cells secreting one, two, or three different immunoglobulins were constructed. The loss of immunoglobulin heavy or light chain expression was monitored. Chain loss was random only in lines with an excess of active light chain genes over heavy chain genes. In all other combinations preferential heavy chain loss was observed. Variant cells altered in heavy or light chain synthesis exhibited an altered chain loss pattern. It is therefore proposed that free immunoglobulin heavy chain is toxic for the cells. The interdependence of the two gene products gives a possible molecular explanation of apparent directed chromosome loss in hybrid cells.
Tumorigenic transformation induced by a specific fragment of DNA from herpes simplex virus type 2.
Authors: Jariwalla R J , Aurelian L , Ts'o P O
Published: May 2006

Transfection of Syrian hamster embryo cells with limit digests of Bgl II-, Hpa I-, or Bgl II/Hpa I-cleaved DNA from herpes simplex virus type 2 (strain S-1) but not with salmon sperm DNA resulted in the appearance of refractile, morphologically altered cells at a frequency of 10(-5)/0.005 microgram of viral DNA within two to four passages. Transformed lines manifested reduced serum requirement and anchorage-independent growth and were tumorigenic in newborn hamsters. They expressed ICP10, a viral protein immunologically identical to the cervical-tumor-associated AG-4 antigen. Transforming activity was localized in the 16.5 x 10(6)-dalton Bgl II/Hpa I double-digest fragment CDs-1, which exhibited sequence homology to the Bgl II/Hpa I fragment CD of DNA from herpes simplex virus type 2 strain 333, mapping between coordinates 0.43 and 0.58 on the physical map of strain 333 DNA. This fragment, CD333, was also shown to induce neoplastic transformation.
Sex differences in cooperative silk-spinning by weaver ant larvae
Authors: Wilson Edward O. , Hölldobler Bert
Published: May 2006

Final-instar larvae of weaver ants (
Oecophylla longinoda
) use their silk for nest construction rather than for cocoon spinning; this commitment represents a shift from entirely selfish to entirely cooperative behavior that occurred after the emergence of the phylogenetically advanced subfamily Formicinae. Male larvae were found to have smaller silk glands and to contribute substantially less silk to nest construction. The hypothesis of kin selection appears to be the most plausible and parsimonious explanation of this sex difference, but other, individual-level modes of selection have not been eliminated.
Nucleotide sequence of the Salmonella typhimurium origin of DNA replication.
Authors: Zyskind J W , Smith D W
Published: May 2006

Construction of deletion derivative plasmids and cloning of restriction fragments from plasmids containing the Salmonella typhimurium origin of replication (ori) were used to locate the functional origin to within a DNA fragment of 296 base pairs between the genes uncB and asn. The nucleotide sequence of the S. typhimurium ori region was determined and compared with the Escherichia coli ori sequence. In the 296-base pair fragment, 85.8% of the bases are conserved between the two species. A nearly equal number of transition and transversion type differences, with no insertions or deletions, occurs between the two bacterial origins, such that the relatively high percentage (adenine plus thymine) of 59.5% is conserved. The 296-base pair fragment contains 14 GATC sequences, all of which are conserved. The high frequency of occurrence of GATC, which is the site of methylation under control of the dam gene, may explain in part why the bacterial ori region appears to be so highly conserved. A large number of secondary structures are possible. One such structure, with a "cloverleaf," is favored by ori nucleotide sequence comparisons and leads to potential novel macromolecular interactions.
Evidence for a "mute" catalytic subunit of cyclic AMP-dependent protein kinase from rat muscle and its mode of activation.
Authors: Gagelmann M , Reed J , Kübler D , Pyerin W , Kinzel V
Published: May 2006

An isoenzyme of the catalytic subunit of type II cyclic AMP-dependent protein kinase from rat muscle is reported which coelutes with the classical catalytic subunit but differs from it in isoelectric point (pI 8.7 vs pI 9.1) and is enzymmatically inactive. After reaction with a heat- and acid-stable component of the protein kinase modulator fraction from the same tissue, the "mute" isoenzyme displays a high activity when assayed on isoelectric focusing gels. This activation process does not occur through proteolytic degradation and is not characteristic of a turnover-type reaction. The data imply direct interaction between the isoenzyme and a modulating protein which may subsequently be separated from the enzyme without reversal of the activation. The modulator protein thus appears to act as a template, inducing a conformational change. The implications of such a mute isoenzyme and its control through small modulator proteins are discussed.
Integration of bacteriophage Mu at host chromosomal replication forks during lytic development.
Authors: Fitts R A , Taylor A L
Published: May 2006

The target site for bacteriophage Mu integration in a lytic cycle of infection was investigated. DNA synthesis in five Hfr strains of Escherichia coli K-12 was synchronized by amino acid starvation and was allowed to proceed for 0, 8, or 15 min before infection. The Hfr cells were then infected with Mu and were subsequently mated with nonimmune F- recipient cells. Mating was interrupted mechanically at 5-min intervals and samples were assayed for infective centers. Conjugal transfer of Mu was delayed in Hfr strains that have transfer origins 15 map units or more from the E. coli replication origin, and the delays increased as the distance between an Hfr point of origin and the replication origin increased. When a gene A mutant of Mu was used for the infection, no infective centers were generated. Infection with a gene B mutant resulted in infective center formation only after long periods of mating. These data are most consistent with a model in which infecting Mu DNA or its progeny integrate at host chromosomal replication forks.
Benzoate catalysis in the hydrolysis of
endo
-5-[4′(5′)imidazolyl]-bicyclo[2.2.1]hept-
endo
- 2-yl
trans
-cinnamate: Implications for the charge-transfer mechanism of catalysis by serine proteases
Authors: Roberts John D. , Kanamori Keiko
Published: May 2006

The acceleration, by a factor of 2500, of the hydrolysis of
endo
-5-[4′(5′)imidazolyl]bicyclo[2.2.1]hept-
endo
- 2-yl
trans
-cinnamate by 0.5 M sodium benzoate in 42 mol% dioxane in water can be explained without resort to operation of a “charge-relay” mechanism similar to that often postulated to account for the enzymatic activity of serine proteases. The degree of ionization of 4-methylimidazole and of sodium benzoate in 42 mol% dioxane in water at 60°C have been measured by NMR spectroscopy.
Chromosome-mediated gene transfer results in two classes of unstable transformants.
Authors: Klobutcher L A , Miller C L , Ruddle F H
Published: May 2006

The human thymidine kinase gene has been transferred from HeLa S3 cells to mouse LM(TK-) cells via isolated metaphase chromosomes. Efficient transfer of the thymidine kinase gene (1.8 X 10(-5) colonies per recipient cell) was obtained when the donor chromosomes were precipitated with calcium phosphate and the recipient cells were treated with 10% (vol/vol) dimethyl sulfoxide. Thirty-five independent cell lines were analyzed in detail. Cytologically detectable donor chromosome fragments were observed in 14% of the cell lines. Many of the transformed cell lines were also found to express the human genes for galactokinase (23% of the transformed cell lines) and procollagen type I (69% of the transformed cell lines), which are syntenic to thymidine kinase on human chromosome 17. On the basis of stability analyses, three classes of transformed cell lines were defined and characterized. One class of transformants was stable, showing no loss of the transferred phenotype in the absence of selection. A second group of transformants was unstable, losing the thymidine kinase phenotype at a rate of 1.5-2.5% per day. This group of transformants was found to possess large donor chromosome fragments (macrotransgenomes) and relatively low levels of donor gene activity. The third group of transformants lost the thymidine kinase phenotype rapidly, at a rate of 6-10% per day. These cell lines contained small, cytologically undetectable transgenomes (microtransgenomes) and overexpressed the transferred thymidine kinase gene.
Further characterization of brain cholecystokinin-converting enzymes.
Authors: Ryder S W , Straus E , Yalow R S
Published: May 2006

The brain cholecystokinin-converting enzymes that cleave intact cholecystokinin to its COOH-terminal dodecapeptide and octapeptide also cleave the synthetic dipeptides Arg-Ile (or Arg-Val or Arg-Leu) and Arg-Asp, respectively. Thus, they are not hormone-specific enzymes but are bond-specific. Ultracentrifuge studies demonstrate that there is Arg-Ile hydrolase activity associated with a protein greater in molecular weight than gamma globulin and that both Arg-Ile and Arg-Asp hydrolase activities are associated with one or more proteins between albumin and gamma globulin in molecular weight.
Cloning of DNA complementary to the measles virus mRNA encoding nucleocapsid protein.
Authors: Gorecki M , Rozenblatt S
Published: May 2006

Double-stranded cDNA synthesized from total poly(A)-containing mRNA, extracted from monkey cells infected with measles virus, has been inserted into Pst cleavage site of Escherichia coli plasmid pBR322 and cloned. A clone containing measles virus DNA sequences was identified by hybridization to a measles virus-specific 32P-labeled cDNA probe prepared from the mRNA of measles virus-infected cells. Cellular sequences in the probe were neutralized by prehybridization with an excess of unlabeled mRNA from uninfected monkey cells. The insert of cloned cDNA isolated contans 1420 base pairs, as shown by agarose gel electrophoresis and electron microscopy. The size of the mRNA complementary to this cloned cDNA is 1750 nucleotides, as determined by the reverse Southern technique. The cloned DNA fragment was further identified as the reverse transcript of the mRNA coding for the nucleocapsid protein of measles virus on the basis that the major cell-free translation product of mRNA selected by hybridization to the cloned DNA comigrated with the nucleocapsid protein and was immunoprecipitated by measles virus-specific antibodies. Subsequently, the cloned DNA was used to detect specific measles virus sequences in the poly(A)-RNA extracted from brain autopsy material from a patient with subacute sclerosing panecephalitis. The cloned DNA can thus be used as a probe to study the structure and expression of the measles genome, and in particular, to study diseases of the central nervous system in which persistent infection with measles virus has been implicated.
Characterization of Drosophila DNA-binding protein DB-2: demonstration of its sequence-specific interaction with DNA.
Authors: Weideli H , Brack C , Gehring W J
Published: May 2006

A DNA-binding protein (DB-2) was isolated from unfertilized Drosophila eggs by DNA-cellulose chromatogrphy. In competition assays with DNA from other species, DB-2 preferentially binds to Drosophila DNA. This binding protein can also be isolated from pupal nuclei and comprises only a small fraction ( < 0.01%) of the total nonhistone chromosomal proteins. In order to investigate the specificity of the interaction between DB-2 and the DNA, we attempted to isolate the DNA sequences to which DB-2 binds. DB-2 was used as a probe to screen our gene bank established by inserting randomly sheared fragments of Drosophila DNA into bacterial plasmids. Groups of plasmids were tested for binding to DB-2 by a filter binding assay. The plasmids bound to the nitrocellulose filter were eluted and used for bacterial transformation. After several cycles of transformation and cloning, two plasmids, A17 and B10, were isolated that bind DB-2 specifically, as measured by filter binding and competition assays. In plasmid A17, binding of DB-2 protects two short DNA segments of approximately 13 and 30 base pairs from digestion by DNase I. By filter hybridization according to Southern, these sequences were mapped to a defined restriction fragment. Further evidence for the binding specificity was obtained by visualizing the protein-DNA complex in the electron microscope. In salivary gland giant chromosomes, A17 DNA hybridizes to a single site (95A/B) on chromosome 3.
Sensitive assay for plasminogen activator of transformed cells.
Authors: Liu H Y , Peltz G A , Leytus S P , Livingston C , Brocklehurst J , Mangel W F
Published: May 2006

A sensitive in situ assay for the plasminogen activator of transformed cells is described; it uses the fluorogenic molecule 3',6'-bis(4-guanidinobenzoyloxy)-5-(N'-4-carboxylphenyl)thioureidospiro[isobenz ofuran-1(3H),9'-[9H]xanthen]-3-one. This fluorescein derivative is an excellent active-site titrant of the esterase activity of plasmin. When transformed cells are incubated with purified plasminogen and the resulting plasmin is titrated with the fluorogenic substrate, the amount of plasmin formed is linearly proportional to time and cell number. The assay is sensitive enough to detect quantitatively the plasminogen activator activity of as few as 250 transformed cells. This substrate should be useful in studying quantitatively the correlation between increased levels of plasminogen activator activity and cellular transformation and as a general active site titrant of serine proteases.
Regulatory region of the gene for the ompA protein, a major outer membrane protein of
Escherichia coli
Authors: Movva N. Rao , Nakamura Kenzo , Inouye Masayori
Published: May 2006

The ompA protein, an outer membrane protein required for conjugation, is one of the most abundant proteins in
Escherichia coli
. The structural gene for the ompA protein cloned in a plasmid vector, pMF21, conferred sensitivity to ompA protein-specific phages. We have determined the DNA sequence of a fragment of 533 base pairs encompassing the regulatory region of the
ompA
gene: the promoter region, the 5′-untranslated region, and the region corresponding to the signal peptide for this secretory protein. The promoter region has a sequence that is remarkably homologous with the
lac
and
gal
promoters. Particularly, both the
ompA
and
gal
promoters have the same octanucleotide sequence, T-C-A-C-A-C-T-T, in their RNA polymerase recognition site, which has been shown to be involved in the binding of cyclic AMP receptor protein to the
gal
promoter. Analogous with the observations in the
gal
operon, a specific RNA transcript was produced only when glycerol, a DNA-destabilizing agent, was added to a cell-free system directed by a DNA fragment of the
ompA
gene. These data indicate that the
ompA
mRNA has an untranslated region at the 5′ end of about 140 nucleotides. In this region there are two additional initiation codons (II and III) besides the initiation codon (I) for the pro-ompA protein. AUG-III is located 30 bases upstream from AUG-I and accompanies a ribosome-binding site. Therefore, AUG-III is likely to begin the synthesis of a pentapeptide. The termination codon for the peptide overlaps with AUG-II, so that the ribosomes could reinitiate from AUG-II without being released from the mRNA. This reinitiation leads to the synthesis of a heptapeptide. The termination codon for this peptide also overlaps with AUG-I, which initiates the production of the pro-ompA protein. Because AUG-I also has an adjacent ribosome-binding site, the tandem repeat of initiation codons and ribosome-binding sites may be an important mechanism for facilitating the rate of initiation of translation. Extensive secondary structures exist in the 5′ end as well as in the coding region of the
ompA
mRNA, which may also play a role in the function of the mRNA.
Amplification of integrated viral DNA sequences in polyoma virus-transformed cells.
Authors: Colantuoni V , Dailey L , Basilico C
Published: May 2006

Polyoma virus (Py) transformation of rat cells requires integration of viral genomes into the host DNA, which generally occurs in a partial or full head-to-tail tandem arrangement. The instability of this structure was previously demonstrated by the high rate of loss of integrated Py genomes in the presence of viral large tumor (T) antigen. We now show that integrated Py DNA sequences can also undergo amplification. We studied two rat cell lines transformed by the ts-a Py mutant, which codes for a thermolabile large T antigen. In a derivative of the ts-a H6A cell line, we have observed loss of full-length Py DNA molecules from the integrated tandem ("curing"), accompanied by the creation of new tandem repeats of two segments of viral DNA corresponding to 38% and 10% of the viral genome, each containing the origin of DNA replication. In the ts-a H3A cell line, which contains an integrated partial tandem of about 1.3 viral genomes with three distinct deletions, propagation at 33 degrees C resulted in the generation of full tandem repeats of a 94% Py DNA "unit" (including two 3% deletions), an 85% "unit" (including a 3% and the 12% deletion), or both. Amplification of integrated viral DNA was not observed in cells propagated at 39.5 degrees C, the nonpermissive temperature for large T antigen function. Amplification of integrated Py DNA sequences thus requires an active large T antigen and can generate a full tandem of integrated viral DNA molecules long after the initial integration event.
Isolation, structure, biological characterization, and synthesis of beta-[Tyr9]melanotropin-(9-18) decapeptide from pig hypothalami.
Authors: Schally A V , Chang R C , Huang W Y , Coy D H , Kastin A J , Redding T W
Published: May 2006

A decapeptide with the amino acid sequence H-Tyr-Phe-Arg-Trp-Gly-Ser-Pro-Pro-Lys-Asp-OH was isolated from acid extracts of porcine hypothalami, structurally and biologically characterized, and synthesized. Except for the NH2-terminal tyrosine, this decapeptide corresponds to the amino acid sequences 9-18 of porcine beta-melanotropin (beta-MSH) and 49-58 of porcine beta-lipotropin (beta-LPH); it also has a tetrapeptide sequence of amino acids (Phe-Arg-Trp-Gly) common to the 7-10 sequences in corticotropin (ACTH) and alpha-MSH. beta-MSH, beta-LPH, alphaMSH, and ACTH from various species all have a histidine residue in the position immediately preceding the common sequence, and the occurrence of a natural peptide with the tyrosine residue in the corresponding site has not been previously reported. This suggests that the beta-[Tyr9]MSH-(9-18) decapeptide might be a fragment of a still larger precursor (prohormone) possibly related to beta-LPH.
Clonal antibodies for core protein of chondroitin sulfate proteoglycan.
Authors: Dorfman A , Hall T , Ho P L , Fitch F
Published: May 2006

Antibodies have been prepared to the core protein of cartilage chondroitin sulfate proteoglycan from clones isolated after fusion of rat spleen cells and mouse myeloma cell lines. Antibodies produced by five clones were studied in detail by a radioimmunoassay utilizing 35SO42-labeled hyaluronidase-digested chondroitin sulfate proteoglycan. Preparations of antigen were shown to contain at least two antigenic determinants, one of which was restricted to a portion of antigen in these preparations. One clone was shown to react with proteoglycan synthesized in a cell-free system. It is proposed that such clonal antibodies can be used for structural and biosynthetic studies of core protein.
Light adaptation and temperature effects in rat PIII retinal response: analysis with a two-state model.
Authors: Gale J G , Williams T P
Published: May 2006

Aspartate-isolated PIII responses from excised perfused retinas of albino rats were studied, with emphasis on background adaptation and temperature effects. When responses are measured at their peaks, it is seen that the proportion of response elicited by the background is equal to the proportion by which a test-flash response is suppressed. This supports the notion that rat PIII, although a complex response, seems to approximate a simple two-state ("compression") system and that it is therefore subject to analysis according to our recently proposed model. This model predicts that (i) responses from two-state systems should increase with decreasing temperature within limits; (ii) delta, the semisaturation constant of voltage--log intensity functions should shift to lower intensities with decreasing temperature; and (iii) the exact magnitude of the delta shift should follow the temperature dependence of the rate of rapid "neural" adaptation of the response. All of these predictions are verified for rat PIII. This suggests that rat PIII, although produced by at least two cell types, is being controlled by only two processes: a light-driven excitation and a rapid first-order "neural" dark adaptation.
Cloning of reiterated and nonreiterated herpes simplex virus 1 sequences as BamHI fragments.
Authors: Post L E , Conley A J , Mocarski E S , Roizman B
Published: May 2006

Over 95% of the herpes simplex virus type 1 (strain F) DNA sequences have been cloned as BamHI fragments in the pBR322 plasmid. With one exception, all of the cloned fragments have the same electrophoretic mobilities and restriction enzyme cleavage sites as do the authentic fragments derived from the BamHI digests of the viral genome. The exception is the BamHI B fragment mapping at the right end of L component in the prototype arrangement of the DNA. Thus, a small deletion mapping near the left end of the fragment was present in two independently derived plasmids. Included in the collection of plasmids are several clones containing DNA sequences that span the junction between the L and S components of the virus DNA. Several plasmids containing the junction fragment were found to be sufficiently stable to permit the preparation of large amounts of the DNA fragment for fine-structure mapping of the restriction enzyme cleavage sites. Preliminary studies on one cloned fragment (BamHI G) have shown that it is biologically active in marker rescue of a temperature-sensitive mutation and in transfer of a plaque morphology marker.
Phenotypic evolution of cells resistant to bromodeoxyuridine.
Authors: Harris M , Collier K
Published: May 2006

Variants resistant to bromodeoxyuridine (BrdUrd) and deficient in thymidine kinase (ATP:thymidine 5'-phosphotransferase; EC 2.7.1.21) have been obtained from V79 Chinese hamster cells by a combination of spontaneous and drug-induced change. Initial mutations take place in wild-type populations as a facilitating step to give partially resistant clones that can be isolated by one-step selection in BrdUrd. When these tolerant populations are maintained for extended periods in BrdUrd-containing medium, a gradual phenotypic transition occurs in which BrdUrd appears to act as an inductive as well as selective agent. Thymidine kinase activity declines logarithmically over an interval of 8-10 weeks as the growth rate rises and the cells become completely resistant to BrdUrd. Relative plating efficiency in hypoxanthine/aminopterin/thymidine medium also decreases, but the decrease is not coordinate with shifts in thymidine kinase activity. The potential for colony formation in hypoxanthine/aminopterin/thymidine continues to decrease exponentially for at least 18 weeks after thymidine kinase deficiency and complete resistance to BrdUrd have been established. These phenotypic modifications are continuous or multistep in character; by clonal analysis they are found to occur in most, if not all, cells maintained in the presence of BrdUrd. Populations in transition thus come to be complex mosaics of different phenotypes that are comparatively stable if isolated in drug-free medium. The progressive evolution of cells resistant to BrdUrd will require new models for an underlying explanation.
Cloning and characterization of DNA sequences surrounding the human gamma-, delta-, and beta-globin genes.
Authors: Kaufman R E , Kretschmer P J , Adams J W , Coon H C , Anderson W F , Nienhuis A W
Published: May 2006

The human gamma-, delta-, and beta-globin genes are located within a 30-kilobase (kb) region of DNA, of which only 20% represents the globin genes. We have attempted to define the nature of flanking and intergenic sequences by isolating recombinants containing the human epsilon, both gamma-, or the 3' end of the beta-globin gene from a bacteriophage library of cloned human DNA. Comparison of these recombinants and a recombinant containing the delta- and beta-globin genes (H beta G1) has provided the following results. The epsilon-globin gene is located 14 kb 5' to the G gamma gene. DNA sequence homology between the region containing the two G gamma genes and the delta nd beta gene region is limited to only a few hundred nucleotides which include the globin coding sequences. Repetitive DNA sequences have been found in the region 3' to the beta-globin gene. Sequences located adjacent to the beta-globin gene are repeated in the globin gene region. A repetitive DNA sequence more than 3.2 kb long is repeated frequently in the human genome but is not repeated in the globin gene region in the clones examined.
Structure of the heavy chain of the H-2Kk histocompatibility antigen.
Authors: Rothbard J B , Hopp T P , Edelman G M , Cunningham B A
Published: May 2006

We have used radiochemical techniques to characterize the heavy chain (Mr 46,000) of the murine H-2Kk histocompatibility antigen in terms of six fragments (I-VI) obtained after cleavage of the polypeptide chain with CNBr. The tentative order of the fragments, which account for more than 90% of the heavy chain, was assigned by radiochemical sequence analysis of the intact heavy chain and of each purified CNBr fragment and by analysis of the CNBr fragments obtained from the large papain fragment of the heavy chain. Treatment of cells with tunicamycin yielded H-2 molecules with heavy chains of molecular weight 40,000, suggesting that the carbohydrate moieties have a combined molecular weight of approximately 6000. CNBr cleavage of H-2Kk heavy chains labeled with [3H]fucose indicated that the carbohydrate moieties are located on fragments II and IV.. Incubation of cells with 32PO4 gave H-2 molecules with radioactive phosphoserine in the carboxyl-terminal CNBr fragment (VI) of the heavy chain and in the fraction containing beta 2-microglobulin. Sequence analysis of each CNBr fragment intrinsically labeled with 3H- and 35S-labeled amino acids identified a total of 87 residues in the H-2Kk heavy chain. The sequence closely resembles that of the H-2Kb molecule, and the 11 differences are scattered throughout the polypeptide chain. Comparison with HLA sequences indicates that the two allelic H-2 sequences are more closely related to each other (88% identity) than either is to the HLA-B7 or A2 antigens (approximately 70%). Similarly, the nonallelic HLA antigens are more closely related to each other (83%) than either is to the H-2Kk or H-2Kb molecules.
Immunochemical determination of the configuration of a haptenic substituent.
Authors: Heidelberger M , Kvarnström I , Eriksen J , Nimmich W , Dudman W F
Published: May 2006

Quantitative inhibition of specific immune precipitation, a rapid microanalytical technique requiring no expensive equipment, was used to determine the stereoconfiguration of pyruvyl groups attached as acetals to two hydroxyls of nonreducing lateral end groups of the capsular polysaccharides of Klebsiella serotypes K11 and K21. The R and S isomers of 4,6-O-pyruvyl-D-alpha-methylgalactoside were used as inhibitors with appropriate polysaccharide antigens and antisera to the two serotypes. The R isomer was a potent inhibitor of precipitation in both antisera, showing that the pyruvylgalactosyl residues in the polysaccharides of both K11 and K21 are in the R form, in which the methyl group of pyruvyl is equatorial to the plane of the acetal ring.
kappa Chain joining segments and structural diversity of antibody combining sites.
Authors: Rudikoff S , Rao D N , Glaudemans C P , Potter M
Published: May 2006

Immunoglobulin kappa light chains are coded for by at least three distinct gene segments designated variable, joining, and constant. The joining gene codes for the 13 amino acid segment linking the variable and constant regions. This peptide includes the last amino acid (96) in the third complementarity-determining region and thus could introduce structural diversity. We have determined the light chain variable region sequences from three myeloma proteins with beta(1,6)galactan-binding specificity, bringing to six the number of light chains sequenced from proteins demonstrating this specificity. Five of these have isoleucine at position 96 and the sixth tryptophan. This substitution appears to be accommodated with no significant change in association constant for a beta(1,6)galactan hapten. Additionally, as many as nine substitutions are found in both light and heavy chain complementarity-determining regions between members of this group although only minimal variations in hapten binding affinity are observed. The isoleucine found at position 96 in five of the kappa chains could not be coded for by any of the joining gene nucleotide sequences previously observed and would require a novel nucleotide sequence at the recombination site between variable and joining genes to produce the observed protein structure. Alternatively, there may exist joining gene segments not yet detected.
Chronic, relapsing myelitis in hamsters associated with experimental measles virus infection.
Authors: Carrigan D R , Johnson K P
Published: May 2006

Chronic, relapsing myelitis has been induced in golden Syrian hamsters by the intracerebral inoculation of measles virus when the animals were less than 1 day old. No acute illness was seen in animals that developed myelitis, and the onset of the myelitis was at 5 to 50 weeks after infection. In most animals the illness was slowly progressive, with hindquarter myoclonus being the most common clinical sign. Occasionally the disease involved episodic limb paresis with nearly total recovery of limb function between periods of paralysis. In most animals pathologic changes were confined to the spinal cord and involved mononuclear cell infiltration, marked gliosis, widespread demyelination, and necrosis. Virus could not be isolated from nervous tissue by cocultivation techniques, and virus-specific immunofluorescence could not be detected. The strain of virus used was distinctive in that it contained high levels of a naturally occurring viral variant that differs from typical measles virus in several distinctive ways.
Tyrosine administration increases striatal dopamine release in rats with partial nigrostriatal lesions.
Authors: Melamed E , Hefti F , Wurtman R J
Published: May 2006

Partial, unilateral nigrostriatal lesions of varying severity were produced in rats by injecting graded doses of 6-hydroxydopamine into the substantia nigra. Formation of the dopamine metabolites dihydroxyphenylacetic acid and homovanillic acid in each surviving nigrostriatal neuron (estimated by the ratios of dihydroxyphenylacetic acid to dopamine and homovanillic acid to dopamine in the striatum) increased significantly when dopamine concentrations in striata containing lesions had been reduced to 25% or less of control values, but remained unchanged in rats with less severe lesions. These findings suggest that, in rats with severe damage of nigrostriatal dopaminergic neurons, surviving neurons increase their firing rates and accelerate dopamine synthesis and release. In rats that had lesions and enhanced striatal dopamine release, but not in rats with lesser lesions (i.e., which reduced ipsilateral dopamine concentrations by less than 75%), administration of tyrosine (250 mg/kg) caused further significant increases in formation of dihydroxyphenylacetic acid and homovanillic acid. These findings provide further evidence that tyrosine availability can enhance dopamine synthesis in and release from nigrostriatal neurons if the firing rates of these neurons are accelerated.
Possible role of distinct morphine and enkephalin receptors in mediating actins of benzomorphan drugs (putative kappa and sigma agonists).
Authors: Chang K J , Hazum E , Cuatrecasas P
Published: May 2006

The binding of many opiates and enkephalins to enkephalin (delta) and morphine (mu) receptors was compared by using three different binding assays: (i) 125I-labeled[D-Ala2, D-Leu5]enkephalin or 125I-labeled[D-Ala2,N-Me-Phe4,Met(O)5ol]-enkephalin to brain membranes; (ii) [3H]ethylketocyclazocine to brain membranes; and (iii) [3H]diprenorphine and [3H]naloxone to neuroblastoma cell and brain membranes, respectively. According to their relative binding potencies and the effects of Na+ and GTP on the binding to these two receptors, opiates and enkephalins can be classified into seven classes: (i) morphine-type mu agonists; (ii) enkephalin-type delta agonists; (iii) mixed agonists-antagonists; (iv) putative kappa agonists; (v) putative sigma agonists; (vi) nalorphine-type antagonists; and (vii) opiate antagonists. Studies with [3H]ethylketocyclazocine do not reveal specific kappa receptors distinct from those already described that bind morphine and enkephalins. The benzomorphan analogs ketocyclazocine and ethylketocyclazocine (putative kappa agonists) and N-allylnormetazocine (putative sigma agonist) bind to morphine (mu) and enkephalin (delta) receptors with similarly high affinities. The potency of putative kappa agonists, measured by competition with binding of the 3H-labeled antagonist, is greatly reduced by the presence of Na+ and GTP; the "Na+ and GTP ratios" are similar to those of morphine and enkephalins. However, Na+ and GTP greatly decrease the potency of binding of putative sigma agonists to enkephalin receptors but only slightly decrease the binding to morphine receptors. These data suggest that putative kappa agonists have agonistic activity toward both receptors, whereas putative sigma agonists behave as agonists for enkephalin receptors but have antagonist activity for morphine receptors. Mixed agonist-antagonists also show smaller difference in affinity to both receptors. These findings may have important implications for understanding the differences in the pharmacological effects of these drugs.
Integration in vivo into simian virus 40 DNA of a sequence that resembles a certain family of genomic interspersed repeated sequences.
Authors: Dhruva B R , Shenk T , Subramanian K N
Published: May 2006

The isolation and characterization of a viable mutant of simian virus 40 (SV40) called "in1449" are described. the mutant DNA is found to have a 157-nucleotide-long insertion at map position 0.649 within the 5' untranslated sequence of the early region of SV40. The complete nucleotide sequence of the insert is presented. Sequence comparisons show that the insert is not of SV40 origin. The insert is presumably of monkey origin since in1449 was produced within monkey kidney cells. The sequence of the in1449 insert matches remarkably well with sequences of a certain predominant family of interspersed repeated sequences in human DNA (called the Alu family) and cloned members thereof. This high degree of sequence homology suggests that the in1449 insert is derived from a member of a family of interspersed repeated sequences in monkey DNA related to the human Alu family. The in1449 insert (and the Alu family members) contain certain oligonucleotide sequences that also are found conserved in the replication origins of papovaviruses and certain other oligonucleotides found in repetitive double-stranded regions of mammalian heterogeneous nuclear RNAs. Sequences around the two recombinant joints in in1449 exhibit a definite pattern of homology. An octanucleotide present in the SV40 part of the first recombinant joint is exactly repeated 15 nucleotides away within the insert; another octanucleotide present within the insert at the second joint is exactly repeated 21 nucleotides away in the viral DNA. The viral DNA sequences flanking the insert in in1449 also exhibit some homology.
Adrenocorticotropic hormone increases specific proteins of the mitochondrial fraction that are translated inside or outside this organelle in cultured adrenal tumor cells.
Authors: Ray D B , Horst I A , Kowal J
Published: May 2006

In addition to its stimulatory effects on steroidogenesis, adrenocorticotropic hormone (ACTH) also has a trophic action on the adrenal cell. This is manifested in part by increases in the levels of key mitochondrial steroidogenic enzymes. The mechanism by which this trophic action of ACTH occurs has been studied in monolayer cultures of mouse adrenal cortical tumor cells. ACTH treatment of these cells stimulates the relative incorporation of amino acids into at least eight specific proteins in mitochondrial preparations. Two of these ACTH-responsive proteins are among the nine major adrenal polypeptides that fulfill the criteria of mitochondrial translation products: (i) their synthesis in intact cells is specifically resistant to inhibition by cycloheximide yet uniquely sensitive to chloramphenicol and (ii) they are synthesized in vitro by isolated mitochondria. The other six ACTH-responsive proteins are within the much larger category of mitochondrial proteins that are synthesized on cytoplasmic ribosomes. One of the proteins synthesized in the cytoplasm electrophoretically comigrates with purified beef adrenodoxin reductase and another with beef adrenodoxin. These findings indicate that ACTH regulates the synthesis (and turnover, or both) of specific mitochondrial proteins that are synthesized inside as well as outside the mitochondria of these adrenal cells.