bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloned DNA sequences complementary to mRNAs encoding precursors to the small subunit of ribulose-1,5-bisphosphate carboxylase and a chlorophyll a/b binding polypeptide
Authors: Broglie Richard , Bellemare Guy , Bartlett Sue G. , Chua Nam-Hai , Cashmore Anthony R.
Published: May 2006

Double-stranded cDNA was synthesized from pea poly(A)-containing mRNA and inserted into the
Pst
I site of the bacterial plasmid pBR322 by the addition of synthetic oligonucleotide linkers. Bacterial colonies containing recombinant plasmids were detected by hybridization to partially purified mRNAs and further characterized by cell-free translation of hybridization-selected mRNAs. To confirm the identity of cDNA clones encoding chloroplast polypeptides, we incubated translation products derived from complementary mRNAs with intact chloroplasts
in vitro
. After uptake, precursor polypeptides were converted to their mature size and identified by fractionation of the chloroplast stroma and thylakoid membranes. By using these procedures, we have isolated and characterized cDNA clones encoding the two major cytoplasmically synthesized chloroplast proteins: the small subunit of ribulose-1,5-bisphosphate carboxylase and a constituent polypeptide (polypeptide 15) of the light-harvesting chlorophyll a/b-protein complex. Similarly, a third cDNA clone was isolated and shown to encode a 22,000-dalton thylakoid membrane polypeptide.
A "bulged" double helix in a RNA-protein contact site.
Authors: Peattie D A , Douthwaite S , Garrett R A , Noller H F
Published: May 2006

The binding of ribosomal protein L18 affects specific nucleotides in Escherichia coli 5S RNA as detected by dimethyl sulfate alkylation and RNase A digestion of the 5S-L18 complex. Most of the affected nucleotides are clustered and localize a site of RNA-protein interaction in and around the defined central helix [Fox, G. E. & Woese, C. (1975) Nature (London) 256, 505-507] of 5S RNA. Chemical carbethoxylation of the native 5S RNA with diethyl pyrocarbonate shows that a striking feature of this region is an unstacked adenosine residue at position 66. We propose that this residue exists as a singly bulged nucleotide extending the Fox and Woese central helix by two base pairs in the E. coli sequence (to positions 16-23/60-68) as well as in each of 61 (prokaryotic and eukaryotic) aligned 5S RNA sequences. In each case, the single bulged nucleotide is at the relative position of adenosine-66 in the RNA sequences. The presence of this putative bulged nucleotide appears to have been conserved in 5S RNA sequences throughout evolution, and its identity varies with major phylogenetic divisions. This residue is likely involved in specific 5S RNA-protein recognition or interaction in prokaryotic and eukaryotic ribosomes. The uridine-65 to adenosine-66 internucleotide bond is protected from RNase A digestion in the complex, and carbethoxylation of E. coli adenosine-66 prior to L18 binding affects formation of a stable RNA-protein complex. Thus, we identify a region of E. coli 5S RNA protected by the ribosomal protein L18 and propose that it contains a bulged nucleotide residue important in stable formation of this RNA-protein complex. This bulged residue appears to be evolutionarily conserved and phylogenetically defined in 5S RNA sequences in general, and consideration of other known RNA-protein binding sites shows that such a "bulged helix" may be a common feature of RNA-protein contact sites.
Role of two of the influenza virus core P proteins in recognizing cap 1 structures (m
7
GpppNm) on RNAs and in initiating viral RNA transcription
Authors: Ulmanen Ismo , Broni Barbara A. , Krug Robert M.
Published: May 2006

Purified influenza viral cores catalyze the entire process of viral RNA transcription, which includes the endonucleolytic cleavage of heterologous RNAs containing cap 1 (m
7
GpppNm) structures to generate capped primers 10-13 nucleotides long, the initiation of transcription via the incorporation of a guanosine residue onto the primers, and elongation of the viral mRNAs [Plotch, S. J., Bouloy, M., Ulmanen, L & Krug, R. M. (1980)
Cell
23, 847-858]. To identify which viral core protein (nucleocapsid protein, P1, P2, or P3) recognizes the cap 1 structure on the RNA primer, we irradiated (UV) endonuclease reactions carried out by viral cores in the absence of ribonucleoside triphosphates, with a primer RNA labeled in its cap 1 structure with
32
P. The labeled cap was crosslinked to a protein that had a mobility similar to that of the P3 protein, the smaller of the two basic P proteins, in both one- and two-dimensional gel electrophoresis. This strongly suggests that this crosslinked protein is the viral P3 protein. Competition experiments with unlabeled RNAs containing or lacking a cap 1 structure established that this protein recognizes the cap 1 structure on RNAs. This protein remained associated with the cap throughout the transcription reaction, even after the viral mRNA molecules were elongated. To identify the viral core protein that catalyzes the initiation of transcription via the incorporation of a guanosine residue onto primer fragments, we irradiated transcription reactions carried out by viral cores in the presence of [α-
32
P]GTP as the only ribonucleoside triphosphate with an unlabeled primer RNA. A labeled guanosine residue was crosslinked to a protein that had a mobility similar to that of the P1 protein, the larger of the two basic P proteins, in both one-and two-dimensional gel electrophoresis. The transcription reaction conditions required to bring this protein in close association with a labeled guanosine residue so that crosslinking could occur indicated that this association most likely occurred coincident with the guanosine residue's being incorporated onto the primer. These results suggest that the viral P1 protein catalyzes this incorporation and hence initiates transcription.
Specific in vitro initiation of transcription on the adenovirus type 2 early and late EII transcription units.
Authors: Mathis D J , Elkaim R , Kédinger C , Sassone-Corsi P , Chambon P
Published: May 2006

Three transcription units are present in the adenovirus type 2 region EII. Transcription units EIIaE and EIIaL encode the mRNA for the 72,000-dalton DNA binding protein, early and late in the lytic cycle, respectively, and transcription unit EIIb encodes the mRNA for the protein that binds to the 5' termini of adenovirus DNA. By using a cell-free transcription system in the presence of purified RNA polymerase B (or II), we have obtained specific initiation of transcription from both the EIIaE promoter, which does not contain a T-A-T-A box, and the EIIaL promoter, which does. In addition, we have identified a new EII T-A-T-A box promoter that is located close to the early non-T-A-T-A box promoter and is used both in vivo and in vitro.
Enzymatic reduction of oxidized alpha-1-proteinase inhibitor restores biological activity.
Authors: Abrams W R , Weinbaum G , Weissbach L , Weissbach H , Brot N
Published: May 2006

The major serum inhibitor of proteolytic activity, alpha-1-proteinase inhibitor (alpha-1-PI), (or alpha-1-antitrypsin) can be readily inactivated by oxidation [Carp, H. & Janoff, A. (1978) Am. Rev. Resp. Dis. 118, 617-621]. This inactivation appears to be due to the oxidation of a critical methionine(s) in alpha-1-PI that is required for the inhibition of elastase activity. An enzyme from Escherichia coli that reduces methionine sulfoxide residues in protein [Brot, N., Weissbach, L., Werth, J. & Weissbach, H. (1981) Proc. Natl. Acad. Sci. USA 78, 2155-2158] can restore the biological inhibitory activity of canine oxidized alpha-1-PI.
traG protein of the F sex factor of
Escherichia coli
K-12 and its role in conjugation
Authors: Manning Paul A. , Morelli Giovanna , Achtman Mark
Published: May 2006

The traG protein of the F sex factor is an inner membrane protein with a molecular weight of 116,000. Mutants in
traG
(or in
traN
) are able to trigger conjugal DNA replication even though they cannot efficiently form stable mating aggregates with F
-
cells. The traG protein (and the traN protein) probably acts in the donor at the stabilization stage of conjugation.
Immunoglobulin heavy chain genes: demethylation accompanies class switching.
Authors: Rogers J , Wall R
Published: May 2006

The methylation of immunoglobulin heavy chain genes was examined before and after class switching, by using the Hpa II/Msp I restriction mapping technique. The mu, delta, and gamma 1 genes all are methylated in cells that do not express them but are demethylated when they are expressed. In particular, the delta gene remains methylated, and thus presumably untranscribed, in a cell line that probably represents an early stage of B-cell differentiation and produces only mu heavy chains. Because mu and delta RNAs are cotranscribed from a single complex transcription unit at a later stage of B-cell differentiation, this finding implies that the mu-plus-delta complex transcription unit is of variable length.
Mutations in the lacY gene of Escherichia coli define functional organization of lactose permease.
Authors: Mieschendahl M , Büchel D , Bocklage H , Müller-Hill B
Published: May 2006

Mutations in the lacY gene of Escherichia coli have been used to analyze the functional organization of lactose permease. Deletions suggest that the NH2 terminus of lactose permease is not essential and can be replaced by residues of the cytoplasmic enzyme beta-galactosidase. Negative dominant mutations in the lacY gene can be explained by the assumption that membrane-associated lactose permease is active as a dimer or oligomer. The map positions of these mutations and other point mutations that lower or alter the sugar specificity define regions of lactose permease involved in sugar or proton binding and transport.
Somatic diversification is required to generate the V kappa genes of MOPC 511 and MOPC 167 myeloma proteins.
Authors: Gershenfeld H K , Tsukamoto A , Weissman I L , Joho R
Published: May 2006

The immune response to phosphocholine in BALB/c mice involves one group of heavy chain variable region (VH) genes and at least three groups of light chain variable region (V kappa) genes, represented by the gene products of the myelomas TEPC 15, MOPC 603, and MOPC 167/MOPC 511. The amino acid sequences of BALB/c myeloma kappa chains MOPC 167 and MOPC 511 are known, and they differ by six amino acids. We have isolated several closely related V region genes of immunoglobulin light chains from a mouse sperm DNA phage library, selecting clones that cross-hybridize with a cDNA plasmid probe encoding the light chain of MOPC 167. We identified six strongly hybridizing clones, representing three separate cloning events. We determined the sequence of the coding and immediate flanking regions of three clones, representing the three separate cloning events, and they proved to be identical. This germ-line sequence encoded the amino acid sequence of neither MOPC 167 nor MOPC 511, but required four base pair changes to generate the V kappa M167 cDNA sequence and five base pair changes to generate the V kappa M511 gene. By Southern hybridization experiments, we demonstrated that neither MOPC 511 nor MOPC 167 germ-line genes exist. We conclude that the V kappa M167 and V kappa M511 genes are created somatically.
Complete amino acid sequence of light chain variable regions derived from five monoclonal anti-p-azophenylarsonate antibodies differing with respect to a crossreactive idiotype.
Authors: Siegelman M , Capra J D
Published: May 2006

The induced antibody response to the hapten p-azophenylarsonate in the A/J mouse has provided a model system for the detailed examination of a heritable crossreactive idiotype and its fine structural and serologic analysis. While earlier studies used to apparent homogeneity in the serum response for structural studies, a more complete understanding of the arsonate idiotypic system became possible with the development of monoclonal antibodies differing with respect to these determinants. Five monoclonal antibodies, four crossreactive idiotype positive and one crossreactive idiotype negative, were selected for complete amino acid sequence analysis. The sequences of the light chain variable regions of these molecules are presented here. The data indicate considerable sequence divergence of the monoclonal light chains from the serum light chains. However, there is a striking degree of homology among the monoclonal light chains regardless of the idiotype character of the parent molecule. Although minor variations are apparent throughout the variable regions, the joining regions are identical among light chains in all of these anti-arsonate antibodies. A particularly notable focus of variation is found at positions 92 and 93 in the third hypervariable region. The possible role of this region in the contribution of the light chain to the arsonate crossreactive idiotype is discussed. These data are consistent with the concept that the anti-arsonate monoclonal light chains originate from the joining of a specific J kappa gene segment to a single germ-line V kappa gene segment. These coding segments are likely further subject to a variety of somatic alterations that generate the modest sequence diversity found among the final protein products.
Establishment and characterization of human T hybrid cells secreting immunoregulatory molecules.
Authors: Okada M , Yoshimura N , Kaieda T , Yamamura Y , Kishimoto T
Published: May 2006

Hybridization of human T cells with an azaguanine-resistant human T cell line gave rise to T hybrid cell lines secreting several immunoregulatory molecules. Analyses of karyotypes, HLA phenotypes, and other surface phenotypes, such as T-cell-specific antigens or receptors for sheep erythrocytes and the patterns of mitogen responsiveness confirmed that the hypoxanthine/aminopterin/thymidine-resistant cell lines were human T-T hybridomas. One of the established hybrid clones (24-A) secreted human interleukin-2 (IL-2). The culture supernatants induced the proliferation of concanavalin A-stimulated murine T cells and supported the proliferation of an IL 2-dependent human cytotoxic T-cell line. In a clone (38-B) that did not show any IL-2 activity in culture supernatants, the addition of macrophages induced IL-2 production in the presence of phytohemagglutinin, suggesting that interleukin-1 induced IL-2 production in T hybrid cells. Hybrid cells secreting killer helper factor were also established. The culture supernatants from this clone, 55-A, helped the induction of cytotoxic T cells against UV-treated human B-blastoid cells but did not show any IL-2 activity.
Nerve growth factor stimulates phospholipid methylation in growing neurites.
Authors: Pfenninger K H , Johnson M P
Published: May 2006

Cultures of neurons from rat superior cervical ganglia were deprived of nerve growth factor, loaded with [methyl-3H]methionine, and then challenged with nerve growth factor for different periods of time. Growing neurites and perikarya were separated microsurgically and extracted with chloroform/methanol. Lipid-incorporated radioactivity in the extracts was measured and expressed on the basis of the amount of phospholipid present. The methylated species in the neurite fraction were identified by thin-layer chromatography as mono-, di-, and trimethylphosphatidylethanolamine (phosphatidylcholine). Furthermore, a small peak of lysophosphatidylcholine was detected. In the neurites, but not in the perikarya, phospholipid methylation was found to reach a peak at 10 sec after onset of stimulation. Stimulated levels were at least 4 times higher than levels of unstimulated controls. The peak was followed by rapid decline of phospholipid-incorporated radioactivity. Our result indicates that phospholipid methylation is part of a nerve-growth-factor-activated secondary messenger system in growing sympathetic neurites. The potential significance of this conclusion for directed neuritic growth and membrane expansion is discussed.
Plasminogen activator secretion by granule neurons in cultures of developing cerebellum.
Authors: Krystosek A , Seeds N W
Published: May 2006

Dispersed cell cultures were established from 7- to 9-day postnatal mouse cerebellum. The fibrin slice method was used to obtain a localization of plasminogen activator production to specific cells. Fibrinolytically active cells were small (5- to 8-micrometer diameter), round, and occurred singly or in aggregates. Fibrinolysis was both plasminogen and time dependent, inhibitable by epsilon-aminocaproic acid and soybean trypsin inhibitor and did not occur when cells were fixed in formalin prior to the fibrin overlay. Strong fibrin degradation occurred only when granule neurons were abundant in the cultures. These plasminogen activator secreting cells were identified as granule neurons by cell separation methods, nuclear morphology, and their ability to bind tetanus toxin and rabbit antiserum against mouse cerebellum (anti-Cbl-1 antiserum). Plasminogen activator also could be quantified in fractionated tissue homogenates or in cell culture medium by the 125I-labeled fibrin plate assay. Fibrinolysis in cerebellar extracts was 95% dependent on the presence of added plasminogen; furthermore, the activity was greater in cerebellar extracts as compared to cerebral cortex of the same age. At the age examined, the cerebellum contains many migratory neurons, and plasminogen activator production may be involved in the process of cell movement.
Isolated horizontal cells from carp retina demonstrate dopamine-dependent accumulation of cyclic AMP.
Authors: Van Buskirk R , Dowling J E
Published: May 2006

Horizontal cells of the carp retina were separated from other retinal cell types by using enzymatic dissociation and velocity sedimentation at unit gravity. Fractions containing horizontal cells were tested for their ability to accumulate cyclic AMP in the presence of various putative neurotransmitters. Micromolar concentrations of dopamine, when added in the presence of 3-isobutyl-1-methylxanthine, stimulated cyclic AMP accumulation in these isolated cells. The dopamine-dependent accumulation of cyclic AMP in intact isolated horizontal cells was blocked by nanomolar concentrations of dopamine antagonists such as haloperidol, (+)-butaclamol, and fluphenazine. The results indicate that there is a postsynaptic dopamine receptor on carp horizontal cells that is associated with adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1].
On the entropy function in sociotechnical systems
Authors: Montroll Elliott W.
Published: May 2006

The entropy function
H
= -Σ

p
j

log

p
j

(

p
j

being the probability of a system being in state
j
) and its continuum analogue
H
= ∫
p(x)
log
p(x) dx
are fundamental in Shannon's theory of information transfer in communication systems. It is here shown that the discrete form of
H
also appears naturally in single-lane traffic flow theory. In merchandising, goods flow from a whole-saler through a retailer to a customer. Certain features of the process may be deduced from price distribution functions derived from Sears Roebuck and Company catalogues. It is found that the dispersion in logarithm of catalogue prices of a given year has remained about constant, independently of the year, for over 75 years. From this it may be inferred that the continuum entropy function for the variable logarithm of price had inadvertently, through Sears Roebuck policies, been maximized for that firm subject to the observed dispersion.
Biosynthetic labeling of insulin receptor: studies of subunits in cultured human IM-9 lymphocytes.
Authors: Van Obberghen E , Ksauga M , Le Cam A , Hedo J A , Itin A , Harrison L C
Published: May 2006

We have identified the subunits of the insulin receptor in cultured human lymphocytes (IM-9 line) by biosynthetic labeling with [35S]methionine and specific precipitation with autoantibodies against the insulin receptor. IM-9 lymphocytes were cultured with [35S]methionine and extracted with Triton X-100. Insulin receptors were concentrated and purified 20-fold by chromatography of the cell extract on wheat germ agglutinin-agarose, and then specifically precipitated by receptor antibodies after addition of a second antibody. Analysis of the immunoprecipitates by sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing conditions followed by autoradiography revealed specific precipitation of two major bands with molecular weights of 130,000 and 90,000. Both species were precipitated by receptor antibodies from four different patients with the syndrome of extreme insulin resistance and acanthosis nigricans. In accord with previous data that insulin bound to receptor reduces the affinity of receptor for anti-receptor antibody, we found that preincubation of the wheat germ-purified cell extract with insulin (1.7 microM) prior to immunoprecipitation caused a decrease in the appearance of both species. The decrease in insulin binding seen after incubation of the lymphocytes with insulin for 12 hr ("down regulation") was associated with a decrease in the labeling of both the 130,000 and 90,000 bands. The apparent molecular weight of both subunits was decreased after pretreatment with mixed glycosidases. In conclusion, we have biosynthetically labeled the insulin receptor with [35S]methionine and showed that the receptor consists of two major glycoprotein subunits with apparent molecular weights of 130,000 and 90,000.
Isolation and characterization of mouse mutant embryonal carcinoma cells which fail to differentiate in response to retinoic acid.
Authors: Schindler J , Matthaei K I , Sherman M I
Published: May 2006

Murine embryonal carcinoma cells from line PCC4.aza1R differentiate readily in response to retinoic acid. By treating PCC4.aza1R cells with the mutagen N-methyl-N' -nitro-N-nitrosoguanidine, we derived two embryonal carcinoma lines in which the cells failed to differentiate during exposure to retinoic acid. Although these dif(RA)- cells maintained the tumorigenic potential of the parental cells, they differentiated poorly in tumor form. Similarly, the tendency of dif(RA)- cells to differentiate when aggregated in vitro was diminished relative to that of PCC4.zaz1R cells. The rate of retinoic acid uptake in cells from the two mutant lines did not appear to be reduced compared with the rate in cells from the parental line; however, specific cytoplasmic retinoic acid-binding protein activity was virtually absent in both mutants. These results strengthen the view that differentiation of embryonal carcinoma cells in response to retinoic acid requires formation of retinoic acid-cytoplasmic retinoic acid-binding protein complexes.
A mechanism of DNA transposition.
Authors: Harshey R M , Bukhari A I
Published: May 2006

Bacteriophage Mu and many other transposable elements undergo transposition by a process that involves replication of the element. We describe here a mechanism by which such integrative replication may take place. We hve examined electron microscopically the DNA structures generated in host cells after Mu induction and have deduced the following steps in the transposition process, (i) Association. A protein-mediated association is brought about between the transposable element and the target DNA. (ii) Attachment. One end of the element is nicked and attached to a site that undergoes a double-stranded cleavage. (iii) Roll-in replication. While one strand of the target DNA is linked to the nicked strand of the element, the complementary strand of the target DNA is used as a primer for replication into the element such that the replicating DNA is threaded through the replication complex. (iv) Roll-in termination. When the distal end of the element arrives at the replication complex, replication is terminated. The roll-in replication mechanism can also explain laying down of tandem repeats--i.e., amplification of circular DNA sequences.
Nonrandom patterns of codon usage and of nucleotide substitutions in human alpha- and beta-globin genes: an evolutionary strategy reducing the rate of mutations with drastic effects?
Authors: Modiano G , Battistuzzi G , Motulsky A G
Published: May 2006

Nucleotide substitutions within a structural gene can cause two principal "drastic" phenotypic effects at the protein level: translatable leads to untranslatable and nonpolar hydrophobic in equilibrium hydrophilic amino acid substitutions. The sequence of nucleotides in the structural human alpha- and beta-globin genes and their variants were examined to determine whether codon usage, patterns of nucleotide substitutions, or both, reduced the relative and absolute rates of these unfavorable mutations. Based on translation of abnormal hemoglobins, it is likely that all 61 nontermination codons are potentially translatable, though only 47 are normally used. Moreover, codons that can mutate to a termination codon are never used whenever the corresponding amino acid is specified also by triplets that cannot mutate to termination by a single-step mutation. Thus, the number of opportunities to mutate to an untranslatable codon is reduced to the minimum compatible with the amino acid composition of these chains. The relative rates of U in equilibrium non-U substitutions were much lower than those of other substitutions. Because U residues must be involved in most termination mutations and in all nonpolar hydrophobic in equilibrium hydrophilic amino acid substitutions, there is a considerable reduction of mutational events, causing drastic phenotypic effects. These findings are likely to be the end result of evolutionary selection by yet unknown mechanisms.
Influence of anti-mouse interferon serum on the growth and metastasis of tumor cells persistently infected with virus and of human prostatic tumors in athymic
nude
mice
Authors: Reid Lola M. , Minato Nagahiro , Gresser Ion , Holland John , Kadish Anna , Bloom Barry R.
Published: May 2006

Baby hamster kidney or HeLa cells form tumors in 100% of athymic
nude
mice. When such cells are persistently infected (PI) with RNA viruses, such as mumps or measles virus, the tumor cells either fail to grow or form circumscribed benign nodules. Neither the parental nor the virus PI tumor cells form invasive or metastatic lesions in
nude
mice. Previous studies have indicated a correlation between the susceptibility of virus-PI tumor cells
in vitro
and the cytolytic activity of natural killer (NK) cells and their failure to grow
in vivo
. Because interferon (IF) is the principal regulatory molecule governing the differentiation of NK cells, it was possible to test the relevance of the IF-NK cell system
in vivo
to restriction of tumor growth by treatment of
nude
mice with anti-IF globulin. This treatment was shown to reduce both IF production and NK activity in spleen cells. Both parental and virus-PI tumor cells grew and formed larger tumors in
nude
mice treated with anti-IF globulin than in control
nude
mice. The viral-PI tumor cells and the uninfected parental cells formed tumors in treated mice that were highly invasive and often metastatic. Some human tumor types have been notoriously difficult to establish as tumor lines in
nude
mice (e.g., primary human prostatic carcinomas). When transplanted into
nude
mice treated either with anti-IF globulin or anti-lymphocyte serum, two prostatic carcinomas grew and produced neoplasms with local invasiveness and some metastases. The results are consistent with the view that interferon may be important in restricting the growth, invasiveness, and metastases of tumor cells by acting indirectly through components of the immune system, such as NK cells.
Fine structural analysis of the chicken pro alpha 2 collagen gene.
Authors: Wozney J , Hanahan D , Morimoto R , Boedtker H , Doty P
Published: May 2006

Forty-two kilobase pairs of cloned chicken DNA containing 80% of the pro alpha 2 (type I) collagen gene and 8 kilobase pairs of 3' flanking sequences have been isolated. Detailed analysis of these clones indicates that this collagen gene spans approximately 40 kilobase pairs of DNA and contains on the order of 50 introns. The fine structure of 40% of the pro alpha 2 gene, including its 3' end, was determined by Southern blot restriction endonuclease mapping using a 2.6-kilobase pair procollagen cDNA clone pCg45, as a probe, and by DNA sequence determination of more than 2 kilobase pairs of this part of the genome. Exons in the triple-helical coding region are all multiples of the 9 base pairs coding for the Gly-X-Y triplet and vary in size from 45 to 108 base paris. The sequences of all six exons in a 3.8-kilobase pair EcoRI fragment were determined. One of these, a 249-base pair exon, joins the collagen domains; it codes for the last 15 amino acids of the triple-helical coding region, the telopeptide, and the first 53 amino acids of the carboxy-terminal propeptide.
Immunological identification of a common precursor to arginine vasopressin and neurophysin II synthesized by in vitro translation of bovine hypothalamic mRNA.
Authors: Schmale H , Richter D
Published: May 2006

mRNA from membrane-bound polysomes of bovine hypothalamus was translated in an mRNA-dependent cell-free system from reticulocyte lysate or wheat germ. The translation products were identified by immunoprecipitation with antibodies to either neurophysin II or arginine vasopressin followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. An immunoreactive polypeptide was obtained with an apparent Mr of 21,000. Sequential immunoprecipitation studies indicated that the Mr 21,000 product is a common precursor to neurophysin II and arginine vasopressin. The specificity of the immunoprecipitation was demonstrated by competition with excess amounts of unlabeled neurophysin II or arginine vasopressin; little or no competition was observed with unlabeled neurophysin II or arginine vasopressin; little or no competition was observed with unlabeled neurophysin I or oxytocin. Processing experiments with microsomal membranes from dog pancreas or tunicamycin-treated ascites tumor cells showed that the Mr 21,000 polypeptide is the prepro form. It was converted to a pro form with Mr 19,000 suggesting a pre sequence of approximately 15 amino acids. The Mr 19,000 polypeptide was coreglycosylated to an apparent Mr of 23,000, indicating that the neurophysin II-arginine vasopressin precursor is a glycopolypeptide.
HeLa cell DNA polymerase alpha is tightly associated with tryptophanyl-tRNA synthetase and diadenosine 5',5"'-P1,P4-tetraphosphate binding activities.
Authors: Rapaport E , Zamecnik P C , Baril E F
Published: May 2006

The purified high molecular weight form of HeLa cell DNA polymerase alpha (deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7) was shown to associate tightly with several aminoacyl-tRNA synthetase activities. Fractionation of the high molecular weight enzyme on hexylagarose followed by gel filtration, chromatography on phosphocellulose, or polyacrylamide gel electrophoresis under nondenaturing conditions demonstrated copurification of only tryptophanyl-tRNA synthetase [L-tryptophan:tRNATrp ligase (AMP-forming), EC 6.1.1.2] along with DNA polymerase alpha. The high molecular weight (660,000) and low molecular weight (145,000) forms of DNA polymerase alpha were shown to possess a highly specific, noncovalent, diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) binding activity. The dissociation constants were determined to be 16 and 22 microM, respectively, by utilization of a charcoal adsorption procedure. No high-affinity binding of ATP could be detected. These findings suggest a link between the amino acid activation process and DNA replication in mammalian cells.
Specific 5′ flanking sequences are required for faithful initiation of
in vitro
transcription of the ovalbumin gene
Authors: Tsai Sophia Y. , Tsai Ming-Jer , O'Malley Bert W.
Published: May 2006

An
in vitro
system [Weil, P. A., Luse, D. S., Segall, J. & Roeder, R. G. (1979)
Cell
18, 469-484 and Manley, J. L., Fire, A., Cano, A., Sharp, P. A. & Gefter, M. L., (1980)
Proc. Natl. Acad. Sci USA
77, 3855-3859] was adapted for studying initiation of transcription of the ovalbumin gene. The DNA template was a cloned ovalbumin gene fragment that contained 5′ flanking sequences, the first structural sequence region, and a portion of the first intervening sequence. A HeLa cell crude extract was used as the source of RNA polymerase and initiation factors. Correct initiation was judged by the sizes of the transcription products generated from ovalbumin templates truncated at various positions before the 3′ end of the gene. Transcription of the specific product was carried out by RNA polymerase II, as judged from α-amanitin sensitivity. A series of deletion mutants was constructed by trimming 5′ flanking sequences of the ovalbumin DNA template by using exonuclease III. The DNAs generated were then cloned in pBR322 and used as templates to determine which sequences were necessary for initiation of transcription. Specific initiation of the ovalbumin gene was unaffected by deletion of all but 61 nucleotides of the 5′ flanking sequence but completely abolished by deletion of all but 26 nucleotides of 5′ flanking sequence. Thus, a region between 61 and 26 nucleotides upstream from the cap site, which includes the Hogness box (T-A-T-A-T-A-T) at position 32-26, is essential for the correct initiation of the ovalbumin gene. Nevertheless, natural DNA fragments containing false Hogness boxes (T-A-T-A-A-A-A and T-A-T-A-T-A-T) not normally located in the immediate 5′ flanking region of an authentic gene did not serve as promoters for initiation of transcription. These results suggest that the Hogness box is essential, but not sufficient, for specific initiation of RNA synthesis.
Effect of phenobarbital on the level of translatable rat liver epoxide hydrolase mRNA.
Authors: Pickett C B , Lu A Y
Published: May 2006

Liver poly(A)+RNA isolated from untreated and phenobarbital-treated rats has been translated in the rabbit reticulocyte cell-fre system in order to determine the level of translationally active epoxide hydrolase (EC 3.3.2.3) mRNA. The in vitro translation systems were immunoprecipitated with rabbit IgG prepared against purified epoxide hydrolase, and the amount of epoxide hydrolase synthesized by the lysate programmed with control and phenobarbital poly(A)+RNA was quantitated. The level of translatable epoxide hydrolase mRNA is increased 3-fold after chronic phenobarbital administration. This level of induction correlates well with the 5-fold induction in catalytic activity of epoxide hydrolase (using styrene 7,8-oxide as substrate) in microsomes isolated from phenobarbital-treated rats. Therefore, we suggest that chronic phenobarbital administration increases the amount of functional epoxide hydrolase in rat liver microsomes by way of an increase in the translatable mRNA level encoding for the enzyme. We do not know whether the increase in mRNA is the result of increased transcription or messenger stability.
Characterization of a cDNA clone coding for the beta chain of bovine fibrinogen.
Authors: Chung D W , Rixon M W , MacGillivray R T , Davie E W
Published: May 2006

Recombinant plasmids containing bovine cDNA have been screened with a radiolabeled cDNA enriched for bovine fibrinogen. A number of plasmids containing cDNAs for fibrinogen were identified by this assay. One plasmid, designated pBI beta 1, was found to contain a cDNA insert of 1372 base pairs. The sequence of the cDNA insert for this plasmid was then determined. It was shown to code for 424 amino acids of the beta chain of fibrinogen, starting with residue 44. This and other data made it possible to construct the complete amino acid sequence of the beta chain of the protein. Comparison of the amino acid sequence of the beta chain of bovine fibrinogen with the corresponding chain of the human molecule indicated that the two chains are greater than 80% homologous.
Relationship between the Na+/H+ antiporter and Na+/substrate symport in Bacillus alcalophilus.
Authors: Guffanti A A , Cohn D E , Kaback H R , Krulwich T A
Published: May 2006

The Na+/H+ antiporter of the obligate alkalophile Bacillus alcalophilus facilitates growth at alkaline pH and precludes growth below pH 8.5. Thus, nonalkalophilic mutant strains do not exhibit Na+/H+ antiport activity and, interestingly, such strains concomitantly lose the ability to catalyze Na+-dependent accumulation of alpha-aminoisobutyrate [Krulwich, T. A., Mandel, D. G. Bornstein, R. F. & Guffanti, A. A. (1979) Biochem. Biophys. Res. Commun. 91, 58-62]. Several other Na+-dependent transport systems are now documented in vesicles from the wild-type strain, and it is demonstrated that these systems are defective in vesicles from the nonalkalophilic mutant KM23. Surprisingly, the defect seems to result not from the loss of Na+/H+ antiport activity per se but from a pleiotropic defect in the Na+/substrate symporters themselves. Monensin, an ionophore that catalyzes Na+/H+ exchange, does not restore respiration-driven Na+/substrate symport in KM23 vesicles. Moreover, with KM23 vesicles, efflux of alpha-aminoisobutyrate, L-malate, and L-aspartate down their respective concentration gradients is not stimulated by Na+, in contrast to the observations with wild-type vesicles. Because monensin should ameliorate a simple defect in Na+/H+ antiport activity and the antiporter should not be required for Na+/substrate symport down a concentration gradient, the results suggest that there may be a direct relationship between the antiporter and various Na+/substrate symporters. One possibility is that the systems share a Na+-translocating subunit.
Activation of plasminogen to plasmin by a protease associated with the outer membrane of Escherichia coli.
Authors: Leytus S P , Bowles L K , Konisky J , Mangel W F
Published: May 2006

Preparations of outer membrane of two strains of Escherichia coli contain a protease that can activate the serum zymogen plasminogen to the active protease plasmin. The amount of plasmin formed is proportional to the membrane concentration. The kinetics of plasminogen activation are linear and obey the Michaelis--Menten rate equation. The Km(app) for the activation of dog plasminogen by E. coli outer membrane preparations is similar to the Km(app) for the activation of dog plasminogen by human urokinase. The E. coli enzyme is active in a membrane-associated form, as opposed to a secreted or soluble form, and is most likely a serine protease because it is inhibited by diisopropyl fluorophosphate. It is also inhibited from activating plasminogen by p-nitrophenyl-p-guanidinobenzoate and aprotinin. Analysis of the activation of plasminogen by the E. coli enzyme by NaDodSO4/polyacrylamide gel electrophoresis showed that the cleavage of plasminogen to plasmin was as specific as that exhibited in the activation of plasminogen to plasmin by urokinase. Possible in vivo roles for this plasminogen activator in E. coli outer membranes are discussed.
Nucleotide sequence of the initiation site for ribosomal RNA transcription in Drosophila melanogaster: comparison of genes with and without insertions.
Authors: Long E O , Rebbert M L , Dawid I B
Published: May 2006

The sequence of 470 nucleotides surrounding the initiation site for rRNA transcription in Drosophila melanogaster has been determined. The precise initiation site was determined first by measuring the DNA fragment protected by the rRNA precursor against digestion by the single-strand specific nuclease S1 and second by direct sequence determination of the first 13 nucleotides of the rRNA precursor. Because greater than 80% od rRNA precursor molecules have been shown previously to bear pppA or ppA 5' termini, we assume that they represent the primary transcription product. Short sequence homologies exist with the initiation regions for rRNA transcription of Xenopus laevis and Saccharomyces cerevisiae. We have determined the nucleotide sequence of the initiation region in four cloned ribosomal genes from D. melanogaster which are not interrupted and in four cloned ribosomal genes in which the 28S rRNA coding region is interrupted by a 5-kilobase type 1 insertion. Three uninterrupted genes and three interrupted genes have identical sequences in the entire analyzed region. The remaining two genes have a single identical base substitution at position -17. We have shown previously that interrupted ribosomal genes in D. melanogaster are not effectively transcribed. Because the nucleotide sequences of the region where transcription initiates are identical in genes with or without insertions, we postulate that the presence of the insertion itself may be responsible for the inactivity of the interrupted genes.
Specific binding of eukaryotic initiation factor 2 to satellite tobacco necrosis virus RNA at a 5′-terminal sequence comprising the ribosome binding site
Authors: Kaempfer Raymond , van Emmelo John , Fiers Walter
Published: May 2006

The mRNA-binding property of eukaryotic initiation factor 2 (eIF-2) was examined by studying its interaction with satellite tobacco necrosis virus (STNV) RNA carrying a
32
P-labeled 5′ end. The RNA molecules bound by limiting amounts of eIF-2 were isolated and digested with pancreatic and T1 RNases. Digestion patterns showed that the labeled STNV RNA preparation offered to eIF-2 was heterogeneous, containing more than 30 different 5′ ends; by contrast, the RNA selected by eIF-2 possessed predominantly one 5′ end, pApGpUp..., the 5′-terminal sequence of intact STNV RNA. Binding analysis of individual 5′-terminal fragments generated from isolated, intact, STNV RNA by partial digestion with T1 RNase showed that eIF-2 does not bind detectably to the 32-nucleotide fragment ending with the initiation codon AUG or to shorter ones, but it does bind the 44-nucleotide fragment that contains the ribosome binding site. In addition to the structural features localized at the 5′ end of STNV RNA, eIF-2 appears to recognize a conformation found only in larger molecules, because intact RNA and large 5-′-terminal fragments are bound preferentially over smaller ones. However, binding of short 5′-terminal STNV RNA fragments to eIF-2 is specific, as judged by competition with STNV and ribosomal RNA. Finally, binding of eIF-2 to intact STNV RNA leads to a conformational change in the RNA that greatly facilitates cleavage by T1 and P1 RNases at sites in the vicinity of the initiation region. These results show that eIF-2 interacts specifically with the 5′-terminal region of STNV RNA that contains the ribosome binding site and causes local unfolding of the RNA structure.
Immunological evidence for the transmembrane nature of the rat liver receptor for asialoglycoproteins.
Authors: Harford J , Ashwell G G
Published: May 2006

Antibodies raised in goats against the rat hepatic receptor for desialylated glycoproteins were perfused through a rat liver and were specifically retained by the liver. These antireceptor antibodies also bound specifically to hepatocyte plasma membranes oriented with their cytoplasmic surface outward on polylysine-derivatized beads. These two phenomena were judged to be properties of distinct subpopulations of the antibody preparation because: (i) maximal adsorption of antibodies with membranes on polylysine beads did not affect subsequent retention by the perfused liver, and (ii) whereas perfusion resulted in a depletion of antibodies capable of blocking ligand binding, adsorption by the everted membrane preparation led to a relative enrichment of blocking antibodies. These results are interpreted as indicative of distinct antigenic determinants of the receptor being present on the two faces of the membrane and demonstrate a transbilayer disposition of the asialoglycoprotein receptor.
Site-specific cleavage by T1 RNase of U-1 RNA in u-1 ribonucleoprotein particles.
Authors: Epstein P , Reddy R , Busch H
Published: May 2006

The structures and functions of small nuclear ribonucleoprotein particles have become of interest because of their suggested role in processing heterogeneous nuclear RNA [Lerner, M. R., Boyle, J. A., Mount, S. M., Wolin S. L. & Steitz, J. A. (1980) Nature (London) 283, 220-224]. To determine the conformation of U-1 RNA in U-1 ribonucleoprotein particles and whether proteins of these particles protect segments of U-1 RNA, intact particles and isolated U-1 RNA were digested with T1 RNase. The digested particles were immunoprecipitated with anti-Sm antibodies. A 5'-end fragment containing nucleotides 1-107 and 3'-end fragments containing nucleotides 108-165 and 108-153 were recovered in nearly quantitative yield from digestion of the particles, suggesting that position 107 is the principal cleavage site in them. At the same T1 RNase concentrations, deproteinized U-1 RNA was cleaved into many fragments. At low T1 RNase concentrations, major cleavage site of deproteinized U-1 RNA was at nucleotide 69. Comparison of the cleavage sites of free U-1 RNA and of U-1 RNA in U-1 ribonucleoprotein particles suggested similar secondary structures. The resistance of the 5' end of U-1 RNA to T1 RNase was unexpected inasmuch as this region has been implicated in hydrogen bonding with heterogeneous nuclear RNA splice junctions.
The rapidly metabolized 32,000-dalton polypeptide of the chloroplast is the "proteinaceous shield" regulating photosystem II electron transport and mediating diuron herbicide sensitivity.
Authors: Mattoo A K , Pick U , Hoffman-Falk H , Edelman M
Published: May 2006

Mild trypsin treatment of Spirodela oligorrhiza thylakoid membranes leads to partial digestion of the rapidly metabolized, surface-exposed, 32,000-dalton protein. Under these conditions, photoreduction of ferricyanide becomes insensitive to diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea], an inhibitor of photosystem II electron transport. Preincubation of thylakoids with diuron leads to a conformational change in the 32,000-dalton protein, modifying its trypsin digestion and preventing expression of diuron insensitivity. Finally, light affects the susceptibility of the 32,000-dalton protein to digestion by trypsin. In other experiments, thylakoids specifically depleted in the 32,000-dalton protein were found to be deficient in electron transport at the reducing side of photosystem II but not at the oxidizing side or in photosystem I activities. Thus, the rapidly metabolized 32,000-dalton thylakoid protein in Spirodela chloroplasts fulfills the requirements of the hypothesized "proteinaceous shield" [Renger, G. (1976) Biochim. Biophys. Acta 440, 287-300] regulating electron flow through photosystem II and mediating diuron sensitivity.
Conformational activation of the yeast phenylalanyl-tRNA synthetase catalytic site induced by tRNAPhe interaction: triggering of adenosine or CpCpA trinucleoside diphosphate aminoacylation upon binding of tRNAPhe lacking these residues.
Authors: Renaud M , Bacha H , Remy P , Ebel J P
Published: May 2006

Adenosine or CpCpA trinucleoside diphosphate can be aminoacylated by phenylalanyl-tRNA synthetase [L-phenylalanine:tRNAPhe ligase (AMP forming), EC 6.1.1.20] when the reaction takes place in the presence of tRNAPhe deprived of its 3' adenosine or pCpCpA terminus. This shows that, upon interaction with tRNA, a structural alteration of the enzyme's active site is achieved. This process may be a determining step in the specificity of the aminoacylation reaction.
Effects of methylation on a synthetic polynucleotide: the B--Z transition in poly(dG-m5dC).poly(dG-m5dC).
Authors: Behe M , Felsenfeld G
Published: May 2006

We have compared the behavior in solution of the synthetic polynucleotide poly(dG-m5dC).poly(dG-m5dC) with that of the unmethylated polynucleotide poly(dG-dC).poly(dG-dC). In solutions containing high concentrations of salt, poly(dG-dC).poly(dG-dC) is known to exhibit altered circular dichroic and absorption spectra correlated with formation of a left-handed Z DNA structure. Poly(dG-m5dC).poly(dG-m5dC) behaves similarly, but the spectral transition from the B to the Z form occurs at much lower salt concentrations, close to usual physiological conditions. Divalent and polyvalent ions are particularly effective: The B--Z transition of poly(dG-m5dC).(dG-m5dC) can be induced at a Mg2+ concentration three orders of magnitude lower than that required for the unmethylated polymer. We have also studied mixed copolymers containing both dC and m5dC. Our results suggest that the sequence m5dC-dG, which occurs in eukaryotic DNA, can have a disproportionately large effect on the B--Z transition.