bluuebunny/crossref_metadata_2025 huggingface dataset:
Two-dimensional 1H NMR study of the lambda operator site OL1: a sequential assignment strategy and its application.
Authors: Weiss M A , Patel D J , Sauer R T , Karplus M
Published: May 2006

The solution structure and dynamics of the 17-base-pair synthetic operator site OL1, which is recognized by the cI and Cro repressors of bacteriophage lambda, is studied by two-dimensional NMR methods. A sequential assignment strategy for nucleic acids is proposed and illustrated by the assignments of the base and sugar protons of OL1.
The hydrophobic moment detects periodicity in protein hydrophobicity.
Authors: Eisenberg D , Weiss R M , Terwilliger T C
Published: May 2006

Periodicities in the polar/apolar character of the amino acid sequence of a protein can be examined by assigning to each residue a numerical hydrophobicity and searching for periodicity in the resulting one-dimensional function. The strength of each periodic component is the quantity that has been termed the hydrophobic moment. When proteins of known three-dimensional structure are examined, it is found that sequences that form alpha helices tend to have, on average, a strong periodicity in the hydrophobicity of 3.6 residues, the period of the alpha helix. Similarly, many sequences that form strands of beta sheets tend to have a periodicity in their hydrophobicity of about 2.3 residues, the period typical of beta structure. Also, the few sequences known to form 3(10) helices display a periodicity of about 2.5 residues, not far from the period of 3 for an ideal 3(10) helix. This means that many protein sequences tend to form the periodic structure that maximizes their amphiphilicity. This observation suggests that the periodicity of the hydrophobicity of the protein primary structure is a factor in the formation of secondary structures. Moreover, the observation that many protein sequences tend to form segments of maximum amphiphilicity suggests that segments of secondary structure fold at a hydrophobic surface, probably formed from other parts of the folding protein.
Simple finite-element model accounts for wide range of cardiac dysrhythmias.
Authors: Smith J M , Cohen R J
Published: May 2006

A simple finite-element model of ventricular conduction processes that explicitly incorporates spatial dispersion of refractoriness was developed. This model revealed that spatial dispersion of refractoriness is a sufficient condition to produce self-sustained reentry even in the absence of unidirectional block, inhomogeneity in local conduction velocities, or the presence of ectopic pacemakers. The model displayed a wide variety of rhythm disturbances qualitatively similar to clinically familiar cardiac dysrhythmias. Electrical stability of the model was determined as a function of the model parameters including ventricular stimulation rate, conduction velocity, and mean refractory period as well as standard deviation of refractory periods. We conclude that spatial dispersion of refractoriness is a sufficient condition to initiate reentrant dysrhythmias but that other physiologic variables such as ventricular rate and conduction velocity strongly influence the dysrhythmogenic effect of spatial dispersion of refractoriness.
Secondary structure specificity of the nuclease activity of the 1,10-phenanthroline-copper complex.
Authors: Pope L E , Sigman D S
Published: May 2006

The artificial DNase activity of the 1,10-phenanthroline-cuprous ion complex [(OP)2Cu+] and H2O2 cleaves the A, B, and Z forms of DNA at different rates. The B structure, formed by most DNAs including poly(dA-dT) and poly(dA) X poly(dT), is the most susceptible to cleavage. It is completely degraded within 1 min by 40 microM 1,10-phenanthroline/4 microM Cu2+/7 mM H2O2/7 mM 3-mercaptopropionic acid. The A structure, formed by RNA X DNA hybrids such as poly(rA) X poly(dT), is cleaved in both strands roughly 10-20% as rapidly as poly(dA-dT) under comparable conditions. In contrast, the left-handed Z structure, formed by poly(dG-dC) in 3.0 M NaCl, is completely resistant to cleavage even though the same copolymer in the B structure at 15 mM NaCl is readily degraded. Poly(dA-dT) is rendered acid soluble at both salt concentrations at similar rates. The basis for the secondary structure specificity of the DNA cleavage reaction most likely resides in the requisite formation of a productive complex between (OP)2Cu+ and DNA during the course of this reaction. Previous studies have suggested that strand scission is due to oxidative destruction of the deoxyribose by hydroxyl radicals produced by the oxidation of DNA-bound Cu+ by H2O2. Apparently, the Z and A structures are unable to form a stable noncovalent complex with the same optimal geometry for cleavage as the B structure and are less susceptible to degradation. This artificial DNase activity may provide an approach to assess the formation of non-B-DNA structures in solution.
Rearrangement of a DNA sequence homologous to a cell-virus junction fragment in several Moloney murine leukemia virus-induced rat thymomas.
Authors: Lemay G , Jolicoeur P
Published: May 2006

We have studied the integrated proviruses in Moloney murine leukemia virus-induced rat thymomas. By Southern blot analysis, we found several complete integrated proviruses in each tumor. In most tumors, we could not detect defective or recombinant proviruses. Several of these integrated proviruses (with their flanking cellular sequences) from a single tumor were cloned in Charon 4A. Their flanking cellular fragments were subcloned into pBR322 and used as a probe to screen other thymoma DNAs. With one clone (pMo-lC) used as a probe, we could detect novel fragments in 11 out of 20 thymoma DNAs analyzed. In three of these thymomas we could analyze in more detail, it appears that these novel fragments are generated by the insertion of a provirus. This specific integration of Moloney provirus in the host genome may represent an important genetic event leading to tumor formation.
Nucleotide sequence of the feline retroviral oncogene v-fms shows unexpected homology with oncogenes encoding tyrosine-specific protein kinases.
Authors: Hampe A , Gobet M , Sherr C J , Galibert F
Published: May 2006

The nucleotide sequence encoding the transforming polyprotein of the McDonough strain of feline sarcoma virus was determined. This sequence includes 231 nucleotides specifying a leader peptide, 1,377 nucleotides encoding most of the feline leukemia virus-derived gag gene, and 2,969 nucleotides representing the viral transforming gene v-fms. A single open reading frame was predicted to encode a fusion polyprotein of 160,000 daltons (P160gag-fms). Fourteen potential sites for glycosylation were predicted within the v-fms-encoded portion of the protein, consistent with previous observations that the primary translation product is rapidly glycosylated. The presence of hydrophobic signal peptides within the amino-terminal leader sequence and in the middle of the v-fms-encoded moiety suggests that the transforming glycoprotein becomes oriented with its amino terminus within the lumen of the rough endoplasmic reticulum and its carboxyl terminus protruding across the membrane of the rough endoplasmic reticulum into the cytoplasm. The latter portion of the protein shows unexpected homology to tyrosine-specific protein kinases encoded by several of the known retroviral oncogenes.
DNA methylation of three 5' C-C-G-G 3' sites in the promoter and 5' region inactivate the E2a gene of adenovirus type 2.
Authors: Langner K D , Vardimon L , Renz D , Doerfler W
Published: May 2006

The E2a gene of human adenovirus type 2 (Ad2) encodes the 72-kilodalton DNA-binding protein. We previously described perfect inverse correlations between the methylation of all 5' C-C-G-G 3' sequences in the Ad2 E2a gene in virus-transformed hamster cells containing viral DNA sequences in an integrated state and the extent to which this gene is expressed. We subsequently showed that in vitro methylation of all 14 5' C-C-G-G 3' sequences in the cloned E2a gene by prokaryotic Hpa II DNA methyltransferase leads to transcriptional inactivation after microinjection into Xenopus laevis oocytes. The unmethylated cloned E2a gene is expressed in these cells. We report here the construction of partly methylated clones of the E2a gene. In the promoter (5')-methylated construct, three 5' C-C-G-G 3' sequences at the 5' end of the subclone were methylated. One of these sites is located 215 base pairs (bp) upstream (bp 26,169 of Ad2 DNA), and two sites are located 5 and 23 bp downstream from the cap site (bp 25,931 and 25,949 of Ad2 DNA) of the E2a gene. This construct was transcriptionally inactive upon microinjection into nuclei of X. laevis oocytes. In the gene (3')-methylated construct, 11 5' C-C-G-G 3' sequences in the main part of the transcribed gene region were methylated in vitro. This construct was transcribed in X. laevis oocytes, and at least some of the Ad2-specific RNA synthesized was initiated at the same sites as in Ad2-infected human KB cells. Both mock-methylated constructs were transcribed into Ad2-specific RNA in X. laevis oocytes. These results demonstrate that DNA methylations at or close to the promoter and 5' end of the E2a gene cause transcriptional inactivation. Perhaps only one methyl group would be adequate for inactivation; in vivo methylation of more than one cytosine may be a form of safeguard or redundancy.
Intervening sequence in the thymidylate synthase gene of bacteriophage T4.
Authors: Chu F K , Maley G F , Maley F , Belfort M
Published: May 2006

The continuous sequence of 2.3 kilobases in a 3-kilobase DNA fragment encoding the structural gene for coliphage T4 thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) was determined by using the M13 dideoxy chain-termination method. From the coding information within this gene and that provided by sequence analysis of selected CNBr peptides from the protein product, the primary structure of T4 thymidylate synthase was determined. The most significant finding of these studies is the presence of a 1017-base-pair interruption two-thirds of the way through the nucleotide sequence of the structural gene. The 5'- and 3'-terminal ends of this intron are demarcated by an apparent stop and start codon, respectively. The corresponding methionine preceding the second coding region of the synthase is not incorporated into the final protein product. Structural evidence confirming the presence of the intervening sequence in the phage genome was obtained by restriction and hybridization analysis. Support for the presence of the intron was also obtained at the functional level by enzyme expression studies using selected td gene fragments. This work also confirms the findings of Purohit and Mathews [ Purohit , S. & Mathews , C. K. (1983) Fed. Proc. Fed. Am. Soc. Exp. Biol. 42, 1759], which reveal that the termination codon for the dihydrofolate reductase gene and the triplet initiating thymidylate synthase overlap by a four-base stretch, A-T-G-A. The implications of this unusual gene arrangement are discussed.
Developmental control of the heat shock response in Xenopus.
Authors: Bienz M
Published: May 2006

Xenopus cells express two major proteins on heat shock, designated hsp 70 and hsp 30. Several cDNA clones for the corresponding mRNAs were identified and sequenced. Inducibility and abundance of heat shock mRNAs in various cell types and developmental stages was determined by nuclease S1-mapping. The only cells found to contain hsp 70 mRNA without heat shock are the oocytes. The level of this stored hsp 70 mRNA is not increased by heat shock. After fertilization, hsp 70 mRNA becomes undetectable; it appears as a heat-inducible mRNA for the first time at gastrulation. After this stage, all somatic cell types accumulate hsp 70 mRNA to similar levels on heat shock, presumably by transcriptional activation of the hsp 70 genes. In contrast, hsp 30 mRNA is not detectable, even after heat shock, in oocytes or embryos that induce hsp 70 mRNA to high levels. Heat inducibility appears late in development--at the tadpole stage. However, the level of induced mRNA varies considerably in different adult tissues. This indicates that the Xenopus heat shock genes are not coordinately controlled. A long-term developmental control appears to be superimposed on the temporary heat inducibility of the heat shock genes: stage- or cell-type-specific conditions can lead to constitutive or repressed heat shock genes.
Generation of antibody diversity in the immune response of BALB/c mice to influenza virus hemagglutinin.
Authors: McKean D , Huppi K , Bell M , Staudt L , Gerhard W , Weigert M
Published: May 2006

We have examined the amino-terminal sequence of the kappa light chains of a set of monoclonal antibodies specific for one of the major antigenic determinants (Sb) on the influenza virus PR8[A/PR/8/34(H1N1)] hemagglutinin molecule. This set was believed to be structurally related from earlier serological analysis that typed these kappa chains as members of the variable (V) region V kappa 21 group [ Staudt , L. M. & Gerhard , W. (1983) J. Exp. Med. 157, 678-704]. Our sequence analysis confirms and extends this conclusion; all examples of this set belong to a subgroup of the V kappa 21 group, V kappa 21C . A special feature of this set of kappa light chains is that all examples were derived from the same mouse (designated H36 ). This sequence analysis along with the characterization of gene rearrangements at the kappa light chain loci of these hybridomas is consistent with the idea that certain members of this set are the progeny of one or two lymphocytes. Because of this potential clonal relationship, we can reach several conclusions about the diversity observed among these kappa light chains: (i) the diversity is due to somatic mutation, (ii) somatic mutations occur sequentially and accumulate in the first complementarity-determining region, and (iii) the extent of somatic variation in this sample is high, suggesting a somatic mutation rate of about 10(-3) per base pair per generation.
A simple topological method for describing stereoisomers of DNA catenanes and knots.
Authors: White J H , Cozzarelli N R
Published: May 2006

Although linking number is an effective topological invariant for describing supercoiled DNA, it is inadequate for the additional interwinding in catenated or knotted DNA. We explain how the two-bridge theory of Schubert provides a powerful yet simple method for analyzing these forms by associating them with two integral invariants, alpha and beta, that measure their geometric complexity. These integers can either be determined graphically or computed with the aid of standard tables, and they allow tabulation of all possible stereoisomers of a given knot or catenane . A complete classification can then be made via a simple theorem. Stereoisomers of representative knots and catenanes are tabulated for easy reference. There are four stereoisomers of regularly interlocked catenanes that we designate right-handed parallel, right-handed antiparallel, left-handed parallel, and left-handed antiparallel according to the helical intertwining of the rings. The biological processes that form catenanes --replication, recombination, and topoisomerase action--predict distinctly different isomers.
Biologically active protease of foot and mouth disease virus is expressed from cloned viral cDNA in Escherichia coli.
Authors: Klump W , Marquardt O , Hofschneider P H
Published: May 2006

Foot and mouth disease virus O1K cDNA had been cloned in Escherichia coli. Here we report on in vitro recombination of cDNA fragments according to the cDNA restriction map and on expression of viral proteins in E. coli. Use was made of the expression vector pPLVP1 , which is known to express the virus capsid protein VP1. Recombined cDNAs of various sizes were inserted downstream from the VP1 gene. The constructed plasmids differ from each other in the number of virus genes coding for nonstructural proteins. The effects caused by their expression in E. coli are compared. It is shown that the virus protease is expressed in E. coli as an active enzyme that recognizes the simultaneously produced virus-specific polyprotein as substrate. The virus protease gene was mapped 5'-adjacent to the virus replicase gene.
Isolation of flagellated membrane vesicles from Caulobacter crescentus cells: evidence for functional differentiation of polar membrane domains.
Authors: Huguenel E , Newton A
Published: May 2006

An immunoaffinity chromatography procedure is described for the separation of membrane vesicles from Caulobacter crescentus cells into flagellated (polar) vesicles and nonflagellated (nonpolar) vesicles. Analysis by two-dimensional gel electrophoresis shows that a number of proteins are associated primarily with either the polar or the nonpolar fraction, and this result suggests that the envelope of these cells is organized into at least two relatively stable domains. Radioimmunoassay also shows that the membrane pool of flagellin, which is known to behave as a precursor in the assembly of the flagellar filament, may be localized exclusively in the polar membrane domain. Thus, the results provide biochemical evidence for the structural and functional differentiation of the C. crescentus cell envelope. These findings are consistent with a model we proposed previously to explain the targeting of surface structures to the new cell pole of C. crescentus. The immunoadsorption approach described here should be useful in the further investigation of this problem, as well as in the fractionation of membrane domains with characteristic surface antigens in other systems.
Mutations in the dopa decarboxylase gene affect learning in Drosophila.
Authors: Tempel B L , Livingstone M S , Quinn W G
Published: May 2006

Fruit flies synthesize several monoamine neurotransmitters. Dopa decarboxylase (Ddc) mutations affect synthesis of two of these, dopamine and serotonin. Both transmitters are implicated in vertebrate and invertebrate learning. Therefore, we bred flies of various Ddc genotypes and tested their learning ability in positively and negatively reinforced learning tasks. Mutations in the Ddc gene diminished learning acquisition approximately in proportion to their effect on enzymatic activity. Courtship and mating sequences of the mutants appeared normal, except for one aspect of male courtship that had previously been shown to be experience dependent. In contrast, the effect on behavior patterns that do not involve learning--phototaxis, geotaxis, olfactory acuity, responsiveness to sucrose--was relatively slight under these conditions. Moderate Ddc mutations affected the acquisition of learned responses while leaving memory retention unaltered. This is in contrast to the mutations dunce , rutabaga , and amnesiac , which primarily affect short-term memory.
Herbicide resistance in
Chlamydomonas reinhardtii
results from a mutation in the chloroplast gene for the 32-kilodalton protein of photosystem II
Authors: Erickson Jeanne M. , Rahire Michèle , Bennoun Pierre , Delepelaire Philippe , Diner Bruce , Rochaix Jean-David
Published: May 2006

We report the isolation and characterization of a uniparental mutant of
Chlamydomonas reinhardtii
that is resistant to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2-chloro-4-ethylamino-6-isopropylamino-
s
-triazine (atrazine). Such herbicides inhibit photosynthesis by preventing transfer of electrons in photosystem II from the primary stable electron acceptor Q to the secondary stable electron acceptor complex B, which is thought to contain a protein of 32 kDa and a bound quinone. It has been proposed that herbicide binding to the 32-kDa protein alters the B complex so that electron transfer from Q is prohibited. Both whole and broken-cell preparations of the mutant alga show a resistance to the effects of herbicide on electron transfer from Q to B, as measured by fluorescence-induction kinetics. In the absence of herbicide, mutant cells exhibit a slower rate of Q to B electron transfer than do wild-type cells. The 32-kDa protein from wild-type cells, but not mutant cells, binds azido[
14
C]atrazine at 0.1 μM. We have isolated
psb
A, the chloroplast gene for the 32-kDa protein, from both wild-type and herbicide-resistant algae and sequenced the coding regions of the gene that are contained in five exons. The only difference between the exon nucleotide sequences of the wild-type and mutant
psb
A is a single T-A to G-C transversion. This mutation results in a predicted amino acid change of serine in the wild-type protein to alanine in the mutant. We suggest that this alteration in the 32-kDa protein is the molecular basis for herbicide resistance in the
C. reinhardtii
mutant.
Interactions of myristic acid with bovine serum albumin: a 13C NMR study.
Authors: Hamilton J A , Cistola D P , Morrisett J D , Sparrow J T , Small D M
Published: May 2006

Interactions of myristic acid with bovine serum albumin were studied by 13C NMR spectroscopy at 50.3 MHz using 90% isotopically substituted [1-13C]-, [3-13C]-, and [14-13C]myristic acids, either individually or in a combination of all three with albumin. At pH 7.4, two or more resonances of different intensities were observed for each 13C-enriched myristic acid. Carboxyl and methylene C-3 resonances corresponding to the major myristic acid environment(s) exhibited pH-dependent chemical shift changes indicative of protonation below pH 6.7; in contrast, carboxyl groups in minor environments were resistant to protonation. 13C NMR spectra obtained as a function of the molar ratio of [3-13C]- and [14-13C]myristic acid to bovine serum albumin (from 0.7 to 5.6) revealed at least two narrow resonances for each carbon at all molar ratios. Thus, bovine serum albumin binding sites for myristic acid are heterogeneous with respect to titration behavior and with respect to the local magnetic environment at both the polar and the nonpolar ends of the fatty acid. The narrow resonances observed for the methylene and methyl carbons are inconsistent with complete immobilization of the protein-bound acid molecules. Together with spin- lattice relaxation times and nuclear Overhauser enhancements, the linewidth results indicate that bound myristic acid has internal motions that are rapid compared with overall protein tumbling and that the C-3 methylene carbon is more restricted than the terminal methyl carbon.
Cross-linking of tRNA at two different sites of the elongation factor Tu.
Authors: Van Noort J M , Kraal B , Bosch L , La Cour T F , Nyborg J , Clark B F
Published: May 2006

Recently, we reported on the induction by kirromycin of two tRNA binding sites on elongation factor Tu. To obtain independent information on the existence of these two sites and to characterize them further, 3' oxidized tRNA was cross-linked to elongation factor Tu by [3H]borohydride reduction. Specific cross-linking occurred exclusively in the presence of kirromycin. In the case of elongation factor Tu X GDP X kirromycin, cross-linking was found at lysine-208; in elongation factor Tu X GTP X kirromycin, cross-linking was at lysine-208 and lysine-237. In both elongation factor Tu complexes, kirromycin itself was found cross-linked to lysine-357. The tRNA cross-linking sites are in agreement with the idea of two different binding sites of tRNA on elongation factor Tu.
Selective isolation of cosmid clones by homologous recombination in Escherichia coli.
Authors: Poustka A , Rackwitz H R , Frischauf A M , Hohn B , Lehrach H
Published: May 2006

A procedure for selection of specific cosmid clones by homologous recombination between cosmid clones from a library and sequences cloned into a plasmid has been developed. Cosmid libraries constructed in a rec- host strain are packaged in vivo into lambda particles. Appropriate aliquots are then introduced into a rec+ host containing the sequence used for selection cloned into a plasmid vector without sequence homology to the cosmid vector. After a short time for recombination, the cosmids are packaged in vivo. Cosmids that have taken up the plasmid by homologous recombination are isolated by plating under conditions selecting for the antibiotic resistance markers carried by both vectors. The recombined cosmids can lose the inserted sequence by another homologous recombination event and, after packaging in vivo, these revertants can be identified on appropriate indicator plates.
Differences in keratin synthesis between normal epithelial cells and squamous cell carcinomas are mediated by vitamin A.
Authors: Kim K H , Schwartz F , Fuchs E
Published: May 2006

A number of recent studies have indicated that the expression of keratins is altered upon malignant transformation of human epithelial cells. We have shown that the altered expression of 67-kDa and 40-kDa keratins in established squamous cell carcinoma lines from tongue and epidermis stems largely from a difference in their sensitivity to vitamin A apparently acquired during tumorigenesis. When the vitamin A concentration in the medium is raised, the 40-kDa keratin is produced at increased levels. Conversely, when the amount of vitamin is reduced, the 67-kDa keratin is synthesized and the cells undergo stratification and terminal differentiation. However, even when vitamin A is quantitatively removed from the medium, the maximal degree of differentiation attained by each squamous cell carcinoma cell as judged by the synthesis of 67-kDa keratin was still less than that of the normal keratinocytes. These findings suggest that the altered patterns of keratins observed for some tissues upon malignant transformation arise from a complex mixture of intracellular changes in the differentiative pathway in addition to changes in the responsiveness of cells to extracellular regulators of keratin gene expression.
Cloned cDNA to cholecystokinin mRNA predicts an identical preprocholecystokinin in pig brain and gut.
Authors: Gubler U , Chua A O , Hoffman B J , Collier K J , Eng J
Published: May 2006

Molecular cloning has established the structure of preprocholecystokinin in porcine cerebral cortex and duodenal mucosa. This precursor is 114 amino acids long, is identical in brain and gut, contains all the cholecystokinin (CCK) peptides previously isolated, and has the characteristics of a prohormone. It contains a putative amino-terminal signal peptide, basic processing sites, and a carboxyl-terminal amidation signal. The CCK mRNAs from brain and gut are approximately 850 nucleotides long and differ by only a few single base changes. This analysis establishes by a strict criterion that CCK is synthesized in both brain and gut and that the different distributions of molecular forms of CCK in the two tissues are most probably a consequence of tissue-specific posttranslational events.
Monoclonal antibodies to purified muscarinic receptor display agonist-like activity.
Authors: Leiber D , Harbon S , Guillet J G , André C , Strosberg A D
Published: May 2006

Monoclonal antibody M-35, which immunoprecipitates native calf brain acetylcholine muscarinic receptor, mimics agonist stimulation of the intact guinea pig myometrium: the antibody, just like carbamoylcholine hydrochloride, causes a rise in intracellular cyclic GMP content, an inhibition of cyclic AMP accumulation due to prostacyclin, and induces uterine contractions. Another antibody, M-23, which reacts with the denatured muscarinic receptor, is devoid of agonist-like activity at the cyclic nucleotide level but is still able to induce contractions of both rat and guinea pig myometrium. The cyclic nucleotide changes caused by both carbamoylcholine and antibody M-35 are inhibited by atropine; this antagonist, which blocks carbamoylcholine-mediated contractions, fails however, to prevent contractions induced by antibodies M-35 and M-23. These results suggest that the information necessary to transmit muscarinic signals is entirely contained in the receptor and that ligands only act to trigger the biological response. The data also imply that the muscarinic receptors of the myometrium are coupled to multiple effector systems.
Crystallization of myosin subfragment 1.
Authors: Rayment I , Winkelmann D A
Published: May 2006

Crystals of myosin subfragment 1 from avian skeletal muscle have been grown reproducibly. They diffract x-rays to at least 4.5-A resolution. The subfragment 1 crystallizes in space group P2(1)2(1)2(1) where a = 107 A, b = 117 A, and c = 278 A. The cell dimensions and intensity distribution on x-ray diffraction photographs are consistent with two molecules in the crystallographic asymmetric unit. Electrophoretic analysis shows that the myosin subfragment 1 present in the crystals contains a 95-kilodalton heavy chain fragment and both the essential and regulatory light chains.
Picosecond fluorescence decay of tryptophans in myoglobin.
Authors: Hochstrasser R M , Negus D K
Published: May 2006

The fluorescence decay characteristics of Mb, MbCO, metMb (sperm whale), metMb (yellowfin tuna), and their apo derivatives were determined by using a picosecond streak camera and time-correlated single photon counting. The emission is dominated by tryptophans that transfer their energy to the heme on a subnanosecond time scale. Sperm whale Mb and derivatives have two tryptophans and their decays can be interpreted mainly as two exponentials, one of ca. 20 ps and the other of 130 ps, whereas tuna Mb has one tryptophan and its emission is nonexponential but dominated by one component of 31 ps. These results along with Förster energy transfer calculations allow us to assign the ca. 30-ps emission to Trp-14 and the 130-ps emission to Trp-7 in Mb. The streak camera was modified to determine the decay of the fluorescence anisotropy. In metMb (tuna) the fluorescence anisotropy decays in 100 ps, which is postulated to result from rapid motion of the Trp-14. Because energy transfer was used to gate the anisotropy, the fast motion of Trp-14 is proposed to correspond to only 10% of the equilibrium distribution of molecules.
Phosphoproteins are components of mitotic microtubule organizing centers.
Authors: Vandre D D , Davis F M , Rao P N , Borisy G G
Published: May 2006

Protein phosphorylation has been suggested as an important control mechanism for the events leading toward the initiation and completion of mitosis. Using a monoclonal antibody recognizing a class of phosphoproteins abundant in mitotic cells, we demonstrated the localization of a subset of these phosphoproteins to several discrete mitotic structures. Patchy immunofluorescence was present in the interphase nuclei, but a significant increase in nuclear immunofluorescence was apparent at prophase. Subsequent mitotic stages demonstrated that immunoreactive material was particularly apparent at microtubule organizing centers, namely, centrosomes, kinetochores, and midbodies. Intense centrosomal localization occurred at the prophase-prometaphase transition and persisted until the reformation of the nuclear membrane in early G1. The cytoplasm of mitotic cells also contained immunoreactive material in sharp contrast to interphase cells that exhibited no cytoplasmic fluorescent staining. Much of the diffuse immunofluorescent cytoplasmic material was removed by a brief lysis of the cells with 0.15% Triton X-100 prior to fixation. The localization of the remaining immunoreactive material after detergent lysis to mitotic microtubule organizing centers suggests that they contain phosphoprotein structural components important, perhaps, in the mitotic phase-interphase transition.
A selection for myosin heavy chain mutants in the nematode Caenorhabditis elegans.
Authors: Anderson P , Brenner S
Published: May 2006

The unc-54 gene of Caenorhabditis elegans encodes an abundant myosin heavy chain protein expressed in body-wall muscle cells. We have designed genetic techniques that select directly for unc-54 mutants. This selection is based upon properties of the unc-54 dominant allele e1152. Mutations that eliminate dominance of e1152 are null alleles of unc-54. Deletions have been identified by their genetic properties. We have defined mutationally a number of essential genes near unc-54, and we have described the genetic fine structure of this region of linkage group I. As much as 27% of the unc-54 mutations induced by the bifunctional alkylating agent 1,2,7,8-diepoxyoctane are multisite deletions. Extrachromosomal free duplications that include unc-54 are also described.
Phosphorylation at a tyrosine residue of lipomodulin in mitogen-stimulated murine thymocytes.
Authors: Hirata F , Matsuda K , Notsu Y , Hattori T , del Carmine R
Published: May 2006

When murine thymocytes were stimulated by mitogens such as concanavalin A, the Ca2+ ionophore A23187, or 4 beta-phorbol 12-myristate 13-acetate, there was a marked increase of 32P incorporation into immunoprecipitable lipomodulin, a phospholipase inhibitory protein. These compounds enhanced 45Ca2+ influx into thymocytes, which, in turn, increased protein phosphorylation, probably by Ca2+- and phospholipid-dependent protein kinase (protein kinase C). Cyclic 8-bromo-AMP, an inhibitor of lymphocyte mitogenesis, blocked the mitogen-stimulated phosphorylation of lipomodulin, although it stimulated the protein phosphorylation via cyclic AMP-dependent kinase (protein kinase A). On electrophoresis, the hydrolysates of 32P-labeled lipomodulin showed a single radioactive spot, which comigrated with authentic phosphotyrosine. The partially purified middle-sized tumor antigen was able to phosphorylate lipomodulin after being phosphorylated by protein kinase C but not by the catalytic subunit of protein kinase A. Our findings suggest that the activity of a tyrosine-specific kinase, which phosphorylates lipomodulin in vivo as well as in vitro, is stimulated by protein kinase C and inhibited by protein kinase A.
G gamma beta+ hereditary persistence of fetal hemoglobin: cosmid cloning and identification of a specific mutation 5' to the G gamma gene.
Authors: Collins F S , Stoeckert C J , Serjeant G R , Forget B G , Weissman S M
Published: May 2006

Hereditary persistence of fetal hemoglobin (HPFH) is a benign condition in which the normal shutoff of fetal hemoglobin (Hb F) production fails to occur. In the G gamma beta+ type of HPFH, erythrocytes of adult heterozygotes contain approximately equal to 20% Hb F, which is almost exclusively of the G gamma-globin variety, without increased levels of gamma-globin chains from the nearby A gamma-globin gene. Unlike some forms of HPFH, no major deletions in the globin gene cluster have been found by genomic blotting in the G gamma beta+ variety. We report here a family with this condition, from which cosmid clones of the beta-globin gene cluster from the G gamma beta+ HPFH allele have been obtained. Sequencing around the fetal genes has identified a point mutation 202 base pairs 5' to the G gamma-globin gene that is present in genomic DNA of 3/3 unrelated individuals with G gamma beta+ HPFH but in none of more than 100 non-HPFH individuals. Although the mutation could represent a tightly linked polymorphism, its location in a region suggested by recent data to be important in tissue-specific control of gene expression suggests the possibility that the -202 mutation accounts for the phenotype. The sequence created resembles elements of other eukaryotic promoters known to be important for efficient transcription.
In vitro evidence that covalent crosslinking of neurofilaments occurs in gamma-diketone neuropathy.
Authors: Graham D G , Szakál-Quin G , Priest J W , Anthony D C
Published: May 2006

We have postulated that the toxic neuropathies associated with neurofilament-filled axonal swellings have a common pathogenesis, the covalent crosslinking of neurofilaments during anterograde transport. The newly described gamma-diketone, 3,4-dimethyl-2,5-hexanedione (DMHD), is a more potent analogue of the toxic metabolite of n-hexane, 2,5-hexanedione. The axonal swellings observed in DMHD toxicity are in the proximal axon, as seen in intoxication with beta, beta'-iminodipropionitrile, rather than in the distal axon, where neurofilamentous swellings are observed in n-hexane, carbon disulfide, and acrylamide neurotoxicity. In these studies, 14C-labeled DMHD and 2-butanone were synthesized and allowed to react with peripheral nerve. Only 14C-labeled DMHD resulted in stable radiolabeled protein polymers, which were retained by nitrocellulose filters with pore sizes as large as 12 microns. More specific evidence for covalent crosslinking of neurofilaments was obtained when DMHD was allowed to react with peripheral nerve in which the neurofilaments had been pulse-labeled with L-[35S]methionine.
Human renin gene: structure and sequence analysis.
Authors: Hobart P M , Fogliano M , O'Connor B A , Schaefer I M , Chirgwin J M
Published: May 2006

The complete protein precursor of human kidney renin has been determined from the sequence of cloned genomic DNA. The gene spans 12 kilobases of DNA and is interrupted by eight intervening sequences. The nine regions (exons) encoding the protein were mapped with a mouse renin cDNA probe, synthetic oligonucleotide probes, and by hybridization of genomic restriction fragments to a 1600-nucleotide human kidney mRNA. The predicted 403-amino acid preprorenin consists of mature renin and a 66-residue amino-terminal prepropeptide. The DNA sequence 5' to the first exon indicates the location of a transcriptional promoter (T-A-T-A-A-A) for a mRNA encoding preprorenin. An additional transcriptional promoter site is located within the first intron, which, if used, would express a shortened nonsecreted prorenin. The structure of the human renin gene is similar to that of human pepsinogen, a closely related aspartyl protease enzyme. This observation suggests that renin and pepsinogen have a common evolutionary origin.
Transfer RNA mischarging mediated by a mutant Escherichia coli glutaminyl-tRNA synthetase.
Authors: Inokuchi H , Hoben P , Yamao F , Ozeki H , Söll D
Published: May 2006

We have isolated mutations in the Escherichia coli glnS gene encoding glutaminyl-tRNA synthetase [GlnS; L-glutamine:tRNAGln ligase (AMP-forming), EC 6.1.1.18] that give rise to gene products with altered specificity for tRNA and are designated "mischarging" enzymes. These were produced by nitrosoguanine mutagenesis of the glnS gene carried on a transducing phage (lambda pglnS+). We then selected for mischarging of su+3 tRNATyr with glutamine by requiring suppression of a glutamine-requiring beta-galactosidase amber mutation (lacZ1000). Three independently isolated mutants (glnS7, glnS8, and glnS9) were characterized by genetic and biochemical means. The enzymes encoded by glnS7, glnS8, and glnS9 appear to be highly selective for su+3 tRNATyr, because in vivo mischarging of other amber suppressor tRNAs was not detected. The GlnS mutants described here retain their capacity to correctly aminoacylate tRNAGln. All three independently isolated mutant genes encode proteins with isoelectric points that differ from those of the wild-type enzyme but are identical to each other. This suggests that only a single site in the enzyme structure is altered to give the observed mischarging properties. In vitro aminoacylation reactions with purified GlnS7 protein show that this enzyme can also mischarge some tRNA species lacking the amber anticodon. This is an example of mischarging phenotype conferred by a mutation in an aminoacyl-tRNA synthetase gene; the results are discussed in the context of earlier genetic studies with mutant tRNAs.
Staphylococcal enterotoxin A gene is associated with a variable genetic element.
Authors: Betley M J , Löfdahl S , Kreiswirth B N , Bergdoll M S , Novick R P
Published: May 2006

The genetic determinant of Staphylococcus aureus enterotoxin A (SEA) has been cloned in pBR322 in Escherichia coli and found to be expressed and secreted into the periplasmic space in that organism. The SEA gene (entA) is within a 2.5-kilobase-pair HindIII fragment that is part of a discrete genetic element 8-12 kilobase pairs in length. This entA element has a standard chromosomal location [between the purine (pur) and isoleucine-valine (ilv) markers] in most S. aureus strains. In some strains it is unlinked to pur-ilv. However, its internal structure is conserved at different locations. Some naturally occurring SEA-nonproducer (EntA-) strains lack the entire entA element, and one instance of its spontaneous loss is reported. Other naturally occurring strains have EntA- structural variants of the element at the same pur-ilv location at which the intact element is most commonly found. Some of these strains are EntA-, others are EntA+; the latter have a second, unlinked copy of the element containing their functional entA gene. These results suggest that entA is associated with a structurally unstable, possibly mobile, discrete genetic element.
Chemical reduction of oxidized human lymphocytes inhibits interleukin 2 production but not induction of interleukin 2 responsiveness.
Authors: Roffman E , Sredni B , Smolinsky A , Wilchek M
Published: May 2006

Treatment with neuraminidase (NA) plus galactose oxidase (GalOxase) does not cause stimulation of human thymocytes. However, stimulation can be achieved by addition of exogenous interleukin 2 (IL-2). The IL-2-induced stimulation was inhibited with anti-Tac antibody, indicating that NA/GalOxase-oxidized cells can serve as inducers of functional IL-2 receptors on IL-2-responding T cells. The induction of IL-2 receptors by the oxidized cells was not inhibited by subsequent reduction with borohydride, since the cells could still be stimulated with IL-2. The presence of IL-2 receptors was also confirmed by flow cytometry using indirect immunofluorescence. Peripheral blood lymphocytes can be stimulated by NA/GalOxase treatment, and the conditioned medium from this treatment can support the growth of an IL-2-dependent line. This stimulation can be inhibited with borohydride and restored with IL-2. The conditioned medium derived from the borohydride-reduced cells cannot support the growth of the IL-2-dependent line, indicating that borohydride inhibits the oxidation-induced IL-2 production. The results suggest that NA/GalOxase-oxidized sites can be modified chemically without losing the potential to induce IL-2 receptors.
Expression of specific mRNAs during adipose differentiation: identification of an mRNA encoding a homologue of myelin P2 protein.
Authors: Bernlohr D A , Angus C W , Lane M D , Bolanowski M A , Kelly T J
Published: May 2006

To identify and characterize specific mRNAs that increase in abundance during differentiation of mouse 3T3-L1 preadipocytes, a cDNA library was constructed from poly(A)+RNA isolated from differentiated 3T3-L1 adipocytes. Mixed probe isotope ratio selection and RNA blot analyses have identified several unique cDNA clones that represent mRNA species expressed either exclusively or at dramatically increased levels in differentiated cells. Further characterization of one such clone (pAL422) revealed that the corresponding mRNA, detectable only after differentiation, is approximately the same length (600 +/- 150 bases) as the cDNA insert (672 bases). The complete nucleotide sequence of the cDNA insert in pAL422 revealed a single long open reading frame that encodes a 132 amino acid polypeptide (the 422 protein) of 14.6 kDa. These and other results suggest that this cDNA may represent a nearly full-length copy of the mRNA. Computer-assisted analyses showed that the 422 protein shares 69% and 64% homology with myelin P2 proteins from rabbit and bovine peripheral nerves, respectively, as well as 23% and 30% homology with fatty-acid binding proteins from rat liver and intestine, respectively. Moreover, the mRNA hybrid selected by pAL422 DNA directs the in vitro translation of an approximately equal to 13 kDa polypeptide, and this protein is specifically immunoprecipitated by antiserum against bovine myelin P2. These observations strongly suggest that the 422 protein is a structural, and possibly functional, analog of myelin P2.
Biochemical and physiological correlates of deer mouse alpha-chain hemoglobin polymorphisms.
Authors: Chappell M A , Snyder L R
Published: May 2006

The alpha-hemoglobin chains in adult deer mice are usually encoded by two tightly linked loci. Because of strong linkage disequilibrium, almost all alpha-globin haplotypes fall into just two classes. The a0c0 class predominates in high-altitude populations, whereas the a1c1 class is generally fixed in low-altitude populations. Here we show that the alpha-globin genotype has effects at both the biochemical level [on blood oxygen affinity (P50)]and at the level of whole-animal physiology [on maximum rate of oxygen consumption (VO2max) during both exercise and cold exposure]. The a1c1/a1c1 genotype mice have the highest P50 values and show the highest Vo2max values at low altitude (340 m) but the lowest VO2max values at high altitude (3800 m). The a0c0/a0c0 mice have the lowest P50, and show the highest VO2max at high altitude but usually have the lowest VO2max at low altitude. The a0c0/a1c1 heterozygotes have an intermediate P50 and are generally intermediate in VO2max at both altitudes. Since high VO2max is advantageous for aerobic exercise and thermogenesis, the physiological data provide a potential explanation for the correlation of haplotype frequencies with altitude.
Isolation of cDNA clones for the chicken neural cell adhesion molecule (N-CAM).
Authors: Murray B A , Hemperly J J , Gallin W J , MacGregor J S , Edelman G M , Cunningham B A
Published: May 2006

Enriched mRNA coding for the neural cell adhesion molecule (N-CAM) was prepared from 9-day embryonic chicken brains by immunoprecipitation of polysomes with antibodies to N-CAM. This mRNA programmed the translation in vitro of N-CAM polypeptide chains in a rabbit reticulocyte lysate system. Two independent N-CAM cDNA clones (designated pEC001 and pEC020) were derived from the enriched RNA. The specificity of pEC001 for N-CAM mRNA was verified by hybrid selection experiments. Both plasmids hybridized to two discrete 6- to 7-kilobase-long RNA species in poly(A)+ RNA from embryonic chicken brain and to lesser amounts of polydisperse material of smaller sizes (probably degradation products of the large RNAs). No hybridization was detected to poly(A)+ RNA from embryonic liver. Southern blotting experiments with pEC001 detected only one hybridizing fragment in chicken genomic DNA digested with several different restriction enzymes, suggesting that sequences corresponding to those within this region of N-CAM mRNA are present at most only a few times, and possibly only once, in the chicken genome.
Binding of transferrin to the core protein of fibroblast proteoheparan sulfate.
Authors: Fransson L A , Carlstedt I , Cöster L , Malmström A
Published: May 2006

Cell-surface-associated proteoheparan sulfate from confluent human skin fibroblasts appears to consist of two disulfide-bonded polypeptides of Mr approximately equal to 90,000. The transferrin receptor, a ubiquitous cell-surface component of proliferating cells, also consists of two subunits of Mr 90,000 linked by S--S bonds. Radiolabeled proteoheparan sulfate mixed with holotransferrin or apotransferrin at pH 4-5 followed by rabbit anti-human transferrin was adsorbed onto protein A-Sepharose to approximately equal to 80-90%. At pH 7.5 apotransferrin bound approximately equal to 40% of the proteoglycan, whereas approximately equal to 80% was bound to holotransferrin. Trypsin digestion of the proteoglycan markedly lowered its ability to bind transferrin. However, binding was essentially unaffected by heparan-sulfate lyase treatment and after reduction and alkylation. Over 90% of the 3H activity of an L-[3H]leucine-labeled proteoglycan was recovered by immunoprecipitation (transferrin.antitransferrin) of a heparan-sulfate lyase digest of the proteoglycan. The immunoprecipitated core protein had an apparent Mr of 150,000 before reduction and Mr of 90,000 after reduction of disulfide bonds. The core protein of the proteoglycan was recognized by the monoclonal antibody B3/25, which is known to be receptor specific. The present findings suggest that the core polypeptides of proteoheparan sulfate and the transferrin receptor may be identical or closely similar.