bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular protein topogenesis
Authors: Blobel Günter
Published: May 2006

Concurrently with or shortly after their synthesis on ribosomes, numerous specific proteins are unidirectionally translocated
across
or asymmetrically integrated
into
distinct cellular membranes. Thereafter, subpopulations of these proteins need to be sorted from each other and routed for export or targeted to other intracellular membranes or compartments. It is hypothesized here that the information for these processes, termed “protein topogenesis,” is encoded in discrete “topogenic” sequences that constitute a permanent or transient part of the polypeptide chain. The repertoire of distinct topogenic sequences is predicted to be relatively small because many different proteins would be topologically equivalent—i.e., targeted to the same intracellular address. The information content of topogenic sequences would be decoded and processed by distinct effectors. Four types of topogenic sequences could be distinguished: signal sequences, stop-transfer sequences, sorting sequences, and insertion sequences.
Signal
sequences initiate translocation of proteins across specific membranes. They would be decoded and processed by protein translocators that, by virtue of their signal sequence-specific domain and their unique location in distinct cellular membranes, effect unidirectional translocation of proteins across specific cellular membranes.
Stop-transfer
sequences interrupt the translocation process that was previously initiated by a signal sequence and, by excluding a distinct segment of the polypeptide chain from translocation, yield asymmetric integration of proteins into translocation-competent membranes.
Sorting
sequences would act as determinants for posttranslocational traffic of subpopulations of proteins, originating in translocation-competent donor membranes (and compartments) and going to translocation-incompetent receiver membranes (and compartments). Finally,
insertion
sequences initiate unilateral integration of proteins into the lipid bilayer without the mediation of a distinct protein effector. Examples are given for topogenic sequences, either alone or in combination, to provide the information for the location of proteins in any of the intracellular compartments or for the asymmetric orientation of proteins and their location in any of the cellular membranes. Proposals are made concerning the evolution of topogenic sequences and the relationship of protein topogenesis to the precellular evolution of membranes and compartments.
Cotransfer of linked eukaryotic genes and efficient transfer of hypoxanthine phosphoribosyltransferase by DNA-mediated gene transfer.
Authors: Peterson J L , McBride O W
Published: May 2006

The efficiency of DNA-mediated transfer of the gene (hprt) for hypoxanthine phosphoribosyltransferase (HPRT; IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) is dependent upon the recipient cell used. hprt has been transferred into mouse TG8 or Chinese hamster CHTG49 cells at a high frequency, similar to the frequency of the gene (tk) for thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) transfer into mouse LMTK- cells (i.e., 10(-6)). In contrast, the frequency of transfer of hprt into mouse A9 cells was about two orders of magnitude less. The identification of efficient recipient cells for hprt transfer permits the use of DNA-mediated transfer as a bioassay for the gene. Cotransfer of the linked tk gene and the gene (galk) for galactokinase (ATP: D-galactose 1-phosphotransferase, EC 2.7.1.6) to LMTK- cells has been detected once among 87 tk transferrents. This suggests that the distance between the tk and galk genes in the Chinese hamster genome may be smaller than was previously thought. Significant differences between chromosome-mediated and DNA-mediated gene transfer were observed with respect to both the size of the transferred functional genetic fragment and the recipient cell specificity.
Elementary transition state theory of the Soret and Dufour effects
Authors: Mortimer Robert G. , Eyring Henry
Published: May 2006

The elementary transition state approach has been used to obtain a simple model theory for the Soret effect (thermal diffusion) and the Dufour effect. The flow of heat in the Dufour effect is identified as the transport of the enthalpy change of activation as molecules diffuse. The theory as now formulated applies only to thermodynamically ideal mixtures of substances with molecules of nearly equal size. The results of the theory conform to the Onsager reciprocal relationship. When the results were fit to data on thermal diffusion for two liquid systems, a close fit was obtained and yielded reasonable values of between 2 and 3 kcal mol
-1
for enthalpy changes of activation and differences between the entropy changes of activation for the two components of between 0 and 1 cal K
-1
mol
-1
.
Isolation, characterization, and comparison of recombinant DNAs derived from genomes of human hepatitis B virus and woodchuck hepatitis virus.
Authors: Cummings I W , Browne J K , Salser W A , Tyler G V , Snyder R L , Smolec J M , Summers J
Published: May 2006

The human hepatitis B virus (HBV) and the woodchuck hepatitis virus (WHV) are closely related by several criteria and belong to the same class of DNA viruses. The DNA genomes from these viruses are difficult to obtain in quantities required for biochemical analysis. We have, therefore, cloned these two DNAs in the vector lambda gtWES and subcloned into the kanamycin resistance plasmid pA01. Comparison of the recombinant DNAs with authentic viral DNAs by specific hybridization, size, and restriction enzyme analysis suggests that the recombinants contain the complete genome of each virus. The nominal size of the cloned HBV genome was 3150 base pairs, compared to 3200 base pairs for the cloned WHV genome. The small amount of nucleic acid homology previously reported between the HBV and WHV DNAs could be demonstrated between the cloned viral DNAs.
Yeast mating pheromone alpha factor inhibits adenylate cyclase.
Authors: Liao H , Thorner J
Published: May 2006

The pheromone alpha factor, secreted by Saccharomyces cerevisiae cells of the alpha mating type, serves to synchronize the opposite mating type (a cells) at G1 as a prelude to fusion of the two cell types. We found that, in vitro, alpha factor inhibited the membrane-bound adenylate cyclase of these cells in a dose-dependent manner. Moreover, one class (ste5) of a cell mutants that grow normally at either 23 degrees or 34 degrees C but that are unable to respond to alpha factor or to mate at the higher temperature possessed an adenylate cyclase activity that was not inhibited by alpha factor at 34 degrees C but was fully sensitive to inhibition at 23 degrees C. Furthermore, addition of cyclic AMP to a cell culture medium shortened the period of pheromone-induced G1 arrest. We conclude that inhibition of adenylate cyclase activity by alpha factor may constitute, at least in part, the biochemical mode of action of the pheromone in vivo.
Genes for three Drosophila heat-shock-induced proteins at a single locus.
Authors: Wadsworth S C , Craig E A , McCarthy B J
Published: May 2006

Two plasmids containing cDNA inserts complementary to the messenger RNAs for the 26,000 and 23,000 molecular weight heat-shock-induced proteins from Drosophila melanogaster were identified. Both of these probes hybridized to a site of a major heat-shock-induced puff, 67B. Hybridization by the gel transfer technique with genome DNA that had been cleaved by a restriction endonuclease indicated that the two genes are closely linked. This expectation was validated through isolation of a DNA fragment generated by BamHI endonuclease containing genes for the 26,000 and 23,000 molecular weight heat-shock-induced proteins and also a gene encoding the 27,000 molecular weight heat-shock-induced protein.
Chromatin repeat length in somatic hybrids.
Authors: Sperling L , Tardieu A , Weiss M C
Published: May 2006

In order to study the mechanisms by which a characteristic repeat length is inherited in somatic cells, it was necessary to develop a method for determining repeat length with a precision of 1 to 2 base pairs. Hybrid clones between parental cell lines differing in repeat length by 6 base pairs were isolated. The four independent hybrid clones characterized had repeat lengths intermediate between those of the parental lines; however, it could be demonstrated that these repeat lengths are unique values and do not arise from a double distribution of the parental repeat lengths. It therefore is concluded that repeat length in somatic cells is determined by a common pool of diffusible substances.
Synthesis and circularization of N- and B-tropic retroviral DNA Fv-1 permissive and restrictive mouse cells.
Authors: Yang W K , Kiggans J O , Yang D M , Ou C Y , Tennant R W , Brown A , Bassin R H
Published: May 2006

Production of various forms of nonintegrated viral DNA was measured in cultured mouse cells carrying different Fv-1 alleles early after infection with N-tropic or B-tropic retroviruses. Quantitative analyses were performed by agarose gel electrophoresis, transfer to diazobenzyloxymethyl-paper, and molecular hybridization. In permissive infection of Fv-1n cells (NIH Swiss and DBA mouse strains) with N-tropic virus and of Fv-1b cells (BALB/c and C57BL/6 strains) with B-tropic virus, form III (double-stranded linear) DNA first appeared at 3-4 hr and reached a maximum at 8-10 hr; two form I (closed circle) DNAs appeared at 7-8 hr and reached a maximum at or beyond 12 hr. In the two Fv-1b cells infected with N-tropic virus and in DBA (Fv-1n) cells infected with B-tropic virus, formation of the two form I DNAs was quantitatively restricted but formation of form III DNA was unaltered. In Fv-1n NIH Swiss mouse embryo cells infected with B-tropic virus, the level of form III DNA was markedly depressed and hence the two form I DNAs were not detectable. In C57BL/6 cells as well as in DBA/2 cells 12 hr after infection, the quantity of form III DNA varied directly with the amount of restricted virus, whereas the quantity of form I DNA varied according to the square of the amount of restricted virus. The significance of these results for understanding the molecular basis of retrovirus replication and its restriction by the Fv-1 gene is discussed.
Relative binding affinities of monovalent cations for double-stranded DNA
Authors: Bleam M. Louise , Anderson Charles F. , Record M. Thomas
Published: May 2006

The competition between sodium and various other monovalent cations that bind to helical DNA in aqueous solution has been studied by
23
Na NMR. Variations in the sodium linewidth with the concentration of the other ion have been analyzed with an equation that describes the competitive binding in terms of two parameters:
r
, the total extent of counterion binding, and
D
, a measure of the binding affinity of a cation relative to sodium. The concentration dependence of these parameters was found to be minimal. In the absence of a competing cation the constancy of
r
has been demonstrated over a range of DNA phosphate concentrations (0.0025-0.015 M) and NaCl concentrations (0.003-1.3 M). For the cations investigated the range in
D
values is small (0.5-0.9), and the relative binding affinities follow the order: NH
4
+
> Cs
+
> K
+
> Li
+
> Na
+
.
Role of the chlorophyll dimer in bacterial photosynthesis
Authors: Warshel Arieh
Published: May 2006

The role of a special dimer (D) of bacteriochlorophyll molecules in bacterial photosynthesis was examined by calculations of the rates of electron transfer reactions in a system of the dimer and a bacteriopheophytin (BPh) molecule. It was found that the dependence of the potential surfaces of D on the distance between the monomers allows a fast light-induced electron transfer from D to BPh but only a slow back reaction (reduction of D
+
by BPh
-
). The same potential surfaces allow efficient reduction of D
+
by cytochrome
c
. Possible advantages of greatly different values of the electronic matrix elements for the forward and back reactions are pointed out. It is suggested that the electrostatic interaction between D
+
and an ionized group of the protein might play an important role in the photosynthetic reaction.
Transforming growth factors: isolation of polypeptides from virally and chemically transformed cells by acid/ethanol extraction.
Authors: Roberts A B , Lamb L C , Newton D L , Sporn M B , De Larco J E , Todaro G J
Published: May 2006

Polypeptides characterized by their ability to confer a transformed phenotype on an untransformed indicator cell have been isolated directly from tumor cells growing both in culture and in the animal, by using an acid/ethanol extraction procedure. Assay of these polypeptides is based on their ability to induce normal rat kidney fibroblasts to form colonies in soft agar. Peptides from murine sarcoma virus-transformed mouse 3T3 cells grown in culture had the highest specific activity in this assay; peptides from sarcomas produced from these cells or from chemically induced transplantable bladder carcinomas of mice were one-third as active; and peptides from a chemically induced rat tracheal carcinoma had only one-tenth the activity. Treatment with either trypsin or dithiothreitol destroyed the activity of all of these materials. The properties of these intracellular polypeptides from both virally and chemically transformed cells are similar to those described for the sarcoma growth factors (SGFs) previously isolated from the conditioned medium of sarcoma virus-transformed mouse 3T3 cells, suggesting the definition of a class of transforming growth factors common to tumor cells of different origins. The transforming peptides from the cultured sarcoma virus-infected cells were separately by gel filtration into two fractions of apparent molecular weight 7000 and 10,000. The major fraction at molecular weight 7000 represented approximately 0.1% of the original cell protein and had a specific activity 50 times that of the original acid/ethanol extract.
Microtubule assembly and conanavalin A capping in lymphocytes: reappraisal using normal and abnormal human peripheral blood cells.
Authors: Oliver J M , Gelfand E W , Pearson C B , Pfeiffer J R , Dosch H M
Published: May 2006

We have analyzed the assembly of microtubules and the distribution of concanavalin A(Con A)-receptor complexes in the same populations of human peripheral blood T and B lymphocytes. We hoped to resolve the prolonged controversy over the relationship of microtubules to Con A cap formation in lymphocytes and to explain the abnormally high spontaneous and colchicine-induced Con A capping that was observed recently in lymphocytes from a patient with an inherited form of severe combined immunodeficiency disease (SCID) characterized by total immunologic dysfunction despite normal numbers and distribution of T and B cells. The data establish that (i) microtubule disassembly is correlated with enhanced Con A cap formation on normal human lymphocytes; (ii) T and B cells differ significantly from each other and from circulating polymorphonuclear leukocytes with respect to their capping responses after exposure to colchicine; and (iii) there is an abnormal relationship of microtubule assembly to surface topography in the functionally defective SCID cells.
Evolution of a new enzymatic function by recombination within a gene.
Authors: Hall B G , Zuzel T
Published: May 2006

Mutations that alter the ebgA gene so that the evolved beta-galactosidase (ebg) enzyme of Escherichia coli can hydrolyze lactose fall into two classes: class I mutants use only lactose, whereas class II mutants use lactulose as well as lactose. Neither class uses galactosylarabinose effectively. In this paper we show that when both a class I and a class II mutation are present in the same ebgA gene, ebg enzyme acquires a specificity for galactosylarabinose. Although galactosylarbinose utilization can evolve as the consequence of sequential spontaneous mutations, it can also evolve via intragenic recombination in crosses between class I and class II ebgA+ mutant strains. We show that the sites for class I and class II mutations lie about 1 kilobase, or about a third of the gene, apart in ebgA. Implications of these findings with respect to the evolution of new metabolic functions discussed.
Genetics of cell surface receptors for bioactive polypeptides: Binding of epidermal growth factor is associated with the presence of human chromosome 7 in human-mouse cell hybrids
Authors: Shimizu Nobuyoshi , Behzadian M. Ali , Shimizu Yoshiko
Published: May 2006

Mouse A9 cells, L-cell-derived mutants deficient in hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) were found to be incapable of binding
125
I-labeled epidermal growth factor (EGF) to the cell surface. The A9 cells were fused with human diploid fibroblasts (WI-38) possessing EGF-binding ability, and human-mouse cell hybrids (TA series) were isolated after hypoxanthine/aminopterin/thymidine/ouabain selection. Analyses of isozyme markers and chromosomes of four representative clones of TA hybrids indicated that the expression of EGF-binding ability is correlated with the presence of human chromosome 7 or 19. Four subclones were isolated from an EGF-binding-positive line, TA-4, and segregation of EGF-binding was found to be concordant with the expression of human mitochondrial malate dehydrogenase (MDHM; L-malate:NAD
+
oxidoreductase, EC 1.1.1.37), a marker for chromosome 7, but not with glucosephosphate isomerase (GPI; D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9), a marker for chromosome 19. Furthermore, evidence from 27 clones of AUG hybrids that were produced between A9 and another human fibroblast line, GM1696, carrying an X/7 chromosome translocation indicated that EGF-binding ability segregates together with human MDHM and two X-linked markers, HPRT and glucose-6-phosphate dehydrogenase (G6PD; D-glucose-6-phosphate:NADP
+
1-oxidoreductase, EC 1.1.1.49), that are located on the translocation chromosome 7p
+
. These results permit assignment of the gene, designated
EGFS
, which is associated with the expression of EGF-binding ability, to human chromosome 7 and its localization to the p22-qter region. Because the EGF receptor is reported to be a glycoprotein the
EGFS
could be either a structural gene(s) for receptor protein or a gene(s) for modifying the receptor protein through glycosylation.
Apolipoprotein B variant derived from rat intestine.
Authors: Krishnaiah K V , Walker L F , Borensztajn J , Schonfeld G , Getz G S
Published: May 2006

A variant of apolipoprotein B has been observed in the lymph lipoproteins [chylomicrons, very low density lipoproteins (VLDL), and low density lipoproteins (LDL)] of rats, in the plasma VLDL of fed rats, and in the plasma VLDL and LDL of rats fed a high-fat, high-cholesterol diet. It is the sole apolipoprotein B in the chylomicrons and VLDL of lymph. It differs from the apolipoprotein B of normal plasma LDL in its immunological properties and in its apparent molecular weight from electrophoresis on 3.5% NaDodSO4/polyacrylamide gel.
Copy-number mutants of the plasmid carrying the replication origin of the Escherichia coli chromosome: evidence for a control region of replication.
Authors: Ogura T , Miki T , Hiraga S
Published: May 2006

A composite plasmid (pXX11) was constructed by joining of an oriC plasmid (pMCR115) carrying the replication origin (oriC) of the Escherichia coli chromosome and a mini-F plasmid (pSC138) carrying the ampicillin-resistance gene (bla). Plasmid pXX11 can replicate, by using oriC, in Hfr cells and mafA mutant cells that cannot support replication of an F plasmid. This plasmid is stably maintained in these host cells during cell growth even under nonselective conditions by use of the partition mechanism of the mini-F genome. In contrast to other oriC plasmids reported previously, pXX11 has no detectable effect on host cell growth. Higher copy-number (Cop-) mutants of pXX11 were isolated, and some of them were found to carry an insertion or deletion within a region derived from the E. coli chromosome. This region, designated cop (copy number), covers about 0.7 kilobase pair and is located approximately 3 kilobase pairs away from the oriC region at the side opposite the asn gene. Evidence suggests that the normal cop region locted on the oriC plasmid acts to reduce the copy number of the plasmid. Plasmid pXX11 complements the uncB402 mutation located on the host chromosome, but some of the Cop- plasmids do not, suggesting that the cop region is vey closely linked to uncB.
T-DNA from
Agrobacterium
Ti plasmid is in the nuclear DNA fraction of crown gall tumor cells
Authors: Chilton Mary-Dell , Saiki Randall K. , Yadav Narendra , Gordon Milton P. , Quetier Francis
Published: May 2006

The crown gall teratoma tumor line BT37, incited by
Agrobacterium tumefaciens
strain T37, has been found to contain part of the tumor-inducing plasmid, pTi T37, of the inciting strain. This foreign DNA segment, called T-DNA, is maintained at several copies per diploid tumor cell. We have examined subcellular DNA fractions from this tumor line in an effort to determine whether T-DNA is in chloroplasts, mitochondria, or nuclei. Tumor cell chloroplast DNA exhibited
Eco
RI and
Bst
I endonuclease cleavage patterns identical to those of normal tobacco chloroplast DNA. Tumor cell mitochondrial DNA exhibited a complex
Bst
I cleavage pattern that did not differ detectably from that of normal tobacco mitochondrial DNA. Southern blots of tumor chloroplast and mitochondrial cleavage products did not hybridize with labeled pTi T37 DNA, whereas blots of tumor cell nuclear DNA cleavage products hybridized strongly. We conclude that T-DNA is located not in chloroplasts or mitochondria but rather in the nuclear fraction of this tumor line.
Vinculin, an intracellular protein localized at specialized sites where microfilament bundles terminate at cell membranes.
Authors: Geiger B , Tokuyasu K T , Dutton A H , Singer S J
Published: May 2006

As intracellular protein of 130,000 molecular weight was recently isolated in this laboratory from chicken gizzard smooth muscle. By immunofluorescence observations of cultured chicken fibroblasts, it was shown to be concentrated on the ventral surfaces of the cells where they formed focal adhesions to the substratum [Geiger, B. (1979) Cell 18, 193-205]. Focal adhesions are sites where, inside the fibroblast, microfilament bundles are known to terminate at the cell membrane. The suggestion was made that this new protein (herein named "vinculin") might be involved in the linkage of the termini of microfilament bundles to membranes in various cell types. To explore this possibility, in the present study we examined several chicken tissues, including intestinal epithelium, gizzard smooth muscle, and cardiac striated muscle, by immunoelectron microscopic labeling for vinculin on ultrathin frozen sections of the specimens. In each case, the immunolabeling for vinculin was concentrated close to membrane sites where microfilament bundles terminate: at the zonula adherens in the junctional complex of the brush border of epithelial cells; at the membrane-associated sense plaques of smooth cells; and at the fascia adherens of the intercalated disk membranes of cardiac muscle cells. These results suggest therefore that vinculin may participate in the anchoring of microfilament bundles to specific membrane sites in various cells.
Inhibition of human polymorphonuclear leukocyte chemotaxis by oxygenated sterol compounds.
Authors: Gordon L I , Bass J , Yachnin S
Published: May 2006

When preincubated with certain oxygenated sterol compounds in lipoprotein-depleted serum [20% (vol/vol)], human polymorphonuclear leukocytes show inhibition of chemotaxis toward the synthetic dipeptide N-formylmethionylphenylalanine without alteration of random movement or loss of cell viability. These effects can occur at sterol concentrations as low as 6.25 microM and after as little as 5 min of preincubation, but they are increased at higher concentrations and longer preincubation times. The inhibition can be almost completely reversed by preincubation in lipoprotein-replete serum [human AB serum, 20% (vol/vol)] and may be partially corrected by addition of free cholesterol (0.125 mM) to the medium. These effects are unlikely to be due to inhibition of cellular sterol synthesis, competition for chemotaxin membrane binding sites, or deactivation of the leukocytes but they may be a consequence of insertion of the sterol molecule into the leukocyte plasma membranes.
Brain size and ecology in small mammals and primates.
Authors: Harvey P H , Clutton-Brock T H , Mace G M
Published: May 2006

Comparisons of brain-body size relationships within small mammal and primate families reveal intergeneric differences related to diet and foraging strategy. These same associations between relative brain size and ecology are also evident among interfamily comparisons.
Isolation and structure of pro-somatostatin: a putative somatostatin precursor from pig hypothalamus.
Authors: Schally A V , Huang W Y , Chang R C , Arimura A , Redding T W , Millar R P , Hunkapiller M W , Hood L E
Published: May 2006

An octacosapeptide that we named pro-somatostatin has been isolated from acid extracts of porcine hypothalami and found to have the amino acid sequence Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys. This octacosapeptide possesses high somatotropin (growth hormone) and prolactin release-inhibiting activity in vitro. It also crossreacts strongly with antisera generated against the somatostatin tetradecapeptide. This octacosapeptide is most likely a precursor (pro-hormone) of somatostatin in the hypothalamus. The existence of still larger molecular size precursors of somatostatin was also observed.
High molecular weight polypeptides (270,000-340,000) from cultured cells are related to hog brain microtubule-associated proteins but copurify with intermediate filaments.
Authors: Pytela R , Wiche G
Published: May 2006

High molecular weight polypeptides (HMWPs) of 270,000 to 340,000 were found to be major components of intermediate filaments prepared by Triton X-100 extraction after spreading of rat glioma C6, HeLa, Chinese hamster ovary, and simian virus 40-transformed Chinese hamster lung cells. C6 HMWPs were shown to resemble high molecular weight microtubule-associated proteins from hog brain by four criteria: (i) comigration in electrophoresis on high-resolution sodium dodecyl sulfate/polyacrylamide gels, (ii) one-dimensional peptide mapping, (iii) phosphorylation in vitro with [gamma-32P]ATP, and (iv) ability to promote microtubule assembly in vitro. HMWPs were also found to be major components of one-time polymerized C6 microtubule preparations, which contained a sizable amount of intermediate filaments. The predominant part of HMWPs present in these microtubule preparations was found not to copurify with microtubules in cycles of temperature-dependent assembly/disassembly but to remain with the cold-insoluble intermediate filaments. These results provide an explanation for the low yields that have hampered attempts to purify microtubule-associated porteins, in particular HMWPs, from cultured cells in the past. Moreover, they suggest that HMWPs might have a dual role in the cell, serving not only as regulators of microtubule assembly but also as linker components between microtubules and intermediate filaments.
5'-Terminal nucleotide sequence of Semliki forest virus 18S defective interfering RNA is heterogeneous and different from the genomic 42S RNA.
Authors: Pettersson R F
Published: May 2006

An 18S defective interfering (DI) RNA population was isolated from the cytoplasm of baby hamster kidney (BHK-21) cells infected with Semliki Forest virus from the 10th undiluted passage. The RNA was approximately 2000 nucleotides long and contained a 5'-terminal cap with the structure 7mGpppAp and a poly(A) tract. The DI RNA contained large TI oligonucleotides derived from both the 42S RNA-specific region and the 3' one-third of the genome common to 42S and 26S RNA. Several of the large oligonucleotides were present in nonequimolar ratios, suggesting that the RNA population is heterogeneous. As this population is approximately uniform in size, this suggests that the DI RNAs may be generated by internal deletions involving different regions of the genome. The 5'-terminal cap-containing RNase T1 oligonucleotide was isolated by two-dimensional gel electrophoresis from uniformly 32P-labeled RNA and shown to be heterogeneous. Five T1 caps with the structure 7mGpppA-U(A-U)nC-A-U-G(n = 4-8) were identified. The two major T1 caps (n = 4 and 6) comprised about 75% of the total yield of T1 caps. The T1 caps were different from the genomic 42S RNA T1 cap (7mGpppA-U-G), suggesting that the extreme 5' end of the genome is not conserved in this defective interfering RNA.
Time-resolved protein fluorescence studies of intermediates in the photochemical cycle of bacteriorhodopsin.
Authors: Fukumoto J M , Hopewell W D , Karvaly B , El-Sayed M A
Published: May 2006

The photolysis-induced changes in the protein fluorescence intensity (at 320 nm) during the proton-pumping cycle of bacteriorhodopsin were examined by a delayed two-pulse technique in the time range 1 microsecond-20 msec at room temperature. No detectable change in the protein fluorescence intensity was observed on the earliest time scale within the lifetime of the intermediate K590, when retinal apparently undergoes the largest structural changes. The time dependence of the relative changes in fluorescence intensity did, however, display a close correlation with the population of the L550 and M412 intermediates. From a computer numerical fit of the data, with available published kinetic parameters, the protein fluorescence quantum yields of the K590, L550, and M412 intermediates are found to be 1.0, 0.92, and 0.80 of that for native bR570, respectively. The probable mechanisms of the observed fluorescence quenching during the photochemical cycle are qualitatively discussed.
Intermediates of a polynuclear manganese center involved in photosynthetic oxidation of water
Authors: Dismukes G. Charles , Siderer Yona
Published: May 2006

Electron paramagnetic resonance of spinach chloroplasts given a series of laser flashes,
n
= 0, 1,..., 6, at room temperature and rapidly cooled to -140°C reveals a signal possessing at least 16 and possibly 21 or more hyperfine lines when observed below 35 K. The spectrum is consistent with a pair of antiferromagnetically coupled Mn ions, or possibly a tetramer of Mn ions, in which Mn(III) and Mn(IV) oxidation states are present. The intensity of this signal peaks on the first and fifth flashes, suggesting a cyclic change in oxidation state of period 4. The multiline signal produced on the first flash is not affected by the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea but is abolished by agents that influence the state of bound manganese, such as incubation with alkaline Tris, or dithionite, and by extraction with cholate detergent in the presence of ammonium sulfate. These results indicate that the paramagnetic signal is monitoring oxidation state changes in the enzyme involved in oxidation of water.
One-trial associative learning modifies food odor preferences of a terrestrial mollusc
Authors: Sahley Christie , Gelperin Alan , Rudy Jerry W.
Published: May 2006

We present evidence of rapid and reliable associative learning by the terrestrial mollusc,
Limax maximus
. Slugs were exposed once to a pairing of a highly attractive food odor (potato or carrot) and a saturated solution of quinidine sulfate, a bitter-tasting plant substance. In comparison with control slugs, the exposed slugs subsequently displayed a markedly reduced preference for the odor paired with quinidine. This reduced odor preference was limited to the specific odor paired with quinidine and did not generalize to other food odors.
RNAs involved in copy-number control and incompatibility of plasmid R1.
Authors: Stougaard P , Molin S , Nordström K
Published: May 2006

Replication of plasmid R1 is controlled by the products of two genes, copA and copB, that act as inhibitors of replication. Here it is shown that one small RNA synthesized from the copA gene acts as replication inhibitor. This RNA molecule was identified from analyses of RNAs synthesized in EScherichia coli minicells carrying R1 miniplasmids or chimeric plasmids containing the copA gene. In minicells, This RNA was found to be unstable with a half-life of less than a few minutes. Two mutant hybrid plasmids lacking the inhibitor function did not express the RNA normally made from plasmids carrying the wild-type copA allele. Nucleotide sequence analysis of one of the copA mutants showed that a base substitution had occurred within the promoter sequence in front of the copA gene. DNA sequence analysis of the other mutant showed that a putative transcription-termination sequence was affected. The DNA sequence analysis also showed that the RNA molecule synthesized from the copA gene is untranslatable but has the potential for a high degree of secondary structure.
Low molecular weight RNAs transcribed in vitro by RNA polymerase III from Alu-type dispersed repeats in Chinese hamster DNA are also found in vivo.
Authors: Haynes S R , Jelinek W R
Published: May 2006

An Alu-type dispersed repeat previously identified in a cloned fragment of Chinese hamster DNA [Haynes, S. R., Toomey, T. P., Leinwand, L. & Jelinek, W. R. (1981) Mol. Cell. Biol. 1, 573-583] serves as a template for cell-free transcription of discrete low molecular weight RNAs by RNA polymerase III [RNA nucleotidyltransferase (DNA-directed), EC 2.7.7.6]. A class of analogous RNAs has been isolated from growing Chinese hamster cells by hybridization of total low molecular weight nuclear RNAs to the cloned DNA fragment from which cell-free transcription occurs. Two-dimensional analysis of RNase digestion products of these RNAs suggests that they are transcribed from multiple members of the Alu-type dispersed repeat family.
Pathway of protein glycosylation in the trypanosomatid Crithidia fasciculata.
Authors: Parodi A J , Quesada Allue L A , Cazzulo J J
Published: May 2006

Cells of the insect parasite Crithidia fasciculata incubated with [14C]glucose were found to possess only one lipid-bound oligosaccharide with solubility in chloroform/methanol/water mixtures and net charge similar to the charges of dolichol pyrophosphate derivatives. The saccharide moiety could be released from lipid by mild acid hydrolysis. Several enzymatic and chemical treatments of the oligosaccharide indicated that the latter had the structure Man alpha leads to Man alpha leads to Man alpha leads to [Man alpha leads to Man alpha leads to Man (alpha 1 leads to 6)]Man leads to GlcNAc(beta 1 leads to 4)GlcNAc. Two labeled oligosaccharides were liberated from proteins by a sequential treatment with a protease and endo-beta-N-acetylglucosamindase H. One of the protein-bound oligosaccharides had the same structure as the lipid-linked compound, whereas in the second oligosaccharide some mannose residues had been replaced by galactose units, but both compounds migrated as did a Man7GlcNAc standard. These were the largest oligosaccharides obtained even after short labeling periods. It is suggested that glycosylation of proteins in the protozoan Crithidia fasciculata does not involved glucosylated lipid-bound oligosaccharides as intermediates.
Yeast transformation: a model system for the study of recombination.
Authors: Orr-Weaver T L , Szostak J W , Rothstein R J
Published: May 2006

DNA molecules that integrate into yeast chromosomes during yeast transformation do so by homologous recombination. We have studied the way in which circular and linear molecules recombine with homologous chromosomal sequences. We show that DNA ends are highly recombinogenic and interact directly with homologous sequences. Circular hybrid plasmids can integrate by a single reciprocal crossover, but only at a low frequency. Restriction enzyme digestion within a region homologous to yeast chromosomal DNA greatly enhances the efficiency of integration. Furthermore, if two restriction cuts are made within the same homologous sequence, thereby removing an internal segment of DNA, the resulting deleted-linear molecules are still able to transform at a high frequency. Surprisingly, the integration of these gapped-linear molecules results in replacement of the missing segment using chromosomal information. The final structure is identical to that obtained from integration of a circular molecule. The integration of linear and gapped-linear molecules, but not of circular molecules, is blocked by the rad52-1 mutation. Consideration of models for plasmid integration and gene conversion suggests that RAD52 may be involved in the DNA repair synthesis required for these processes. Implications of this work for the isolation of integrative transformants, fine-structure mapping, and the cloning of mutations are discussed.
Molecular basis for familial isolated growth hormone deficiency.
Authors: Phillips J A , Hjelle B L , Seeburg P H , Zachmann M
Published: May 2006

Nuclear DNA from four individuals with familial isolated growth hormone (somatotropin) deficiency (IGHD) type A was studied by restriction endonuclease analysis. By using 32P-labeled human growth hormone (hGH) cDNA sequences as a probe, patterns seen after various digestions indicated that these individuals were homozygous for a deletion of at least 7.5 kilobases (kb) of DNA. This deletion includes the gene that encodes the normal growth hormone but does not include the variant growth hormone gene. Restriction patterns of DNAs from all family members agreed with an autosomal recessive mode of inheritance of the deletion that correlates with the clinical phenotype. Furthermore, independent assortment of the two types of hGH genes suggests that these genes are nonallelic. These findings indicate that, in these families, IGHD type A is caused by deletion of the normal hGH genes and that this disorder can occur in the presence of variant hGH genes.
Primary structure of corticotropin-releasing factor from ovine hypothalamus.
Authors: Spiess J , Rivier J , Rivier C , Vale W
Published: May 2006

Sequence analysis was performed of an ovine hypothalamic 41-residue polypeptide that had been postulated to be a putative corticotropin-releasing factor (CRF) because of its high intrinsic corticotropin releasing activity. The NH2-terminal 39 residues of CRF were determined by Edman degradation of 0.6-3.5 nmol of peptide in a Wittmann-Liebold modified Beckman 890C spinning cup sequencer with reverse-phase high-pressure liquid chromatography for the identification of amino acid phenylthiohydantoins (direct micro-sequence analysis). Evidence for residue 40 (isoleucine) was provided by direct micro-sequence analysis of 2.0 nmol of acetylated CRF selectively cleaved at its arginine residues by trypsin prior to analysis. The thermolytic COOH-terminal fragment isoleucyl-alanineamide was characterized as its dansyl derivative. Based on the analytical data, the following primary structure is proposed for ovine hypothalamic CRF: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp -Ile-Ala-NH2. In agreement with this proposal, the synthetic replicate of CRF is highly potent in stimulating secretion of both corticotropin and beta-endorphin-like immunoactivities.
Base insertion and deletion mutations induced in an Escherichia coli plasmid by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide.
Authors: Mizusawa H , Lee C H , Kakefuda T , McKenney K , Shimatake H , Rosenberg M
Published: May 2006

Mutations induced by (+/-)trans-benzo-[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BaP-diol epoxide-1) were selected by using a recombinant plasmid vector system designed for the study of transcription termination signals. The plasmid contains a transcription terminator positioned between a promotor signal and the Escherichia coli galactokinase structural gene (galK). By selections for the expression of galK (i.e., galK- to galK+), mutations are obtained in the terminator region that allow transcription from the promotor to read the galK gene. These mutations were characterized by direct DNA sequence of the terminator region. The DNA sequence changes caused by BaP-diol epoxide-1 were demonstrated for three different mutants. Two were found to be single-base-pair insertions of T.A into a cluster of consecutive T.A base pairs and the other change was a single-base-pair deletion of G.C from a cluster of consecutive G.C base pairs.
Cloning and sequence of cDNA coding for alpha 1-antitrypsin.
Authors: Kurachi K , Chandra T , Degen S J , White T T , Marchioro T L , Woo S L , Davie E W
Published: May 2006

Recombinant plasmids containing human and baboon cDNA have been screened for alpha 1-antitrypsin, a major serine protease inhibitor present in blood. One plasmid, designated pBa alpha 1a2, was found to contain a cDNA insert of 1352 base pairs coding for the baboon inhibitor. It included 45 nucleotides that code for 15 amino acids present in the amino-terminal signal sequence of the protein, 1182 nucleotides that code for 394 amino acids in the mature protein, a stop codon, and a noncoding region of 76 nucleotides. Comparison of the amino acid sequences of baboon alpha 1-antitrypsin, human antithrombin III, and chicken ovalbumin indicated that these three proteins are about 230% homologous. A second plasmid, designated pH alpha 1a1, was found to contain a human cDNA insert of 306 base pairs. This plasmid coded for 69 amino acids present in the carboxyl-terminal region of human alpha 1-antitrypsin. The human and baboon cDNAs and their amino acid sequences are greater than 96% homologous.
Movement of antibody-coated latex beads attached to the spirochete Leptospira interrogans.
Authors: Charon N W , Lawrence C W , O'Brien S
Published: May 2006

Antibody-coated latex beads (Ab-beads) were attached to Leptospira interrogans serovars illini 3055 and icterohaemorrhagiae SC1157. The movement of the Ab-beads relative to the motion of the cells was observed by direct darkfield microscopy or was recorded on videotape. When the Ab-beads were attached to the front end of motile cells, the Ab-beads were displaced towards the back end of the cells. When the cells reversed direction, the Ab-beads also reversed direction. A number of hypotheses were proposed and tested to account for this Ab-bead displacement. The one best supported by the evidence states that the Ab-beads are attached to antigens of the outer membrane sheath. These antigens are dragged laterally through the sheath due to the forward motion of the cells and the retarding forces of the medium acting on the beads. The results obtained provide information on the nature of the outer membrane sheath of L. interrogans, the basis for certain movements of spirochetes, and insight on how spirochetes attach to eukaryotic cells and tissues. In addition, the results indicate that antigens can move laterally through membranes as rapidly as 11 micrometers/sec.
Growth of purified astrocytes in a chemically defined medium.
Authors: Morrison R S , de Vellis J
Published: May 2006

Astrocytes purified from primary cultures of neonatal rat cerebrum can now be grown in a synthetic medium supplemented with putrescine, prostaglandin F2 alpha, insulin, fibroblast growth factor, and hydrocortisone. These five supplements have a marked synergistic effect on growth when used in combination but have little effect when used individually. Astrocytes grown in the defined medium exhibit dramatic changes in morphological characteristics in comparison to cells grown in serum-free or serum-supplemented medium. In addition, these cells express the astrocyte-specific marker glial fibrillary acidic protein and are estimated by several criteria to be greater than 95% astrocytes.
Metabolism of arachidonate through NADPH-dependent oxygenase of renal cortex.
Authors: Morrison A R , Pascoe N
Published: May 2006

In normal kidneys the renal medulla very efficiently converts arachidonic acid to prostaglandins. Although the renal cortex has only trace amounts of cyclooxygenase activity, we report here the existence of an active cortical NADPH-dependent monooxygenase that converts arachidonate primarily into 19-hydroxy- and 20-hydroxyarachidonate as well as 19-ketoarachidonate and a dicarboxylic acid. The enzyme is presumably a cytochrome P-450 monooxygenase and demonstrated marked resistance to inhibition by 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF-525A), metyrapone, and carbon monoxide. In the rabbit kidney these products are produced only by the cortex in the presence of NADPH and represent the major metabolic products of arachidonate metabolism.
Interactions among iron(II) bleomycin, Lewis bases, and DNA.
Authors: Antholine W E , Petering D H , Saryan L A , Brown C E
Published: May 2006

The interaction of sulfur-containing ligands with complexes of Fe(II) bleomycin [Fe(II)Blm] was investigated by comparing DNA strand-scission activity with the structural characteristics of the complex. The bithiazole ring of excess Blm can form intermolecular complexes with Fe(II)Blm and with NO-Fe(II)Blm, and the bithiazole groups of CO-Fe(II)Blm interact with the metal center. This binding can be reversed by glutathione, CO-Fe(II)Blm binds to poly(dA-dT) . poly(dA-dT) via its bithiazole group to change the environment of the Fe2+ ion. Glutathione binds to the CO-Fe(II)Blm-poly(dA-dT) . poly(dA-dT) complex under conditions in which it stimulates DNA strand scission by Fe(II)Blm.
Molecular basis of valine resistance in Escherichia coli K-12.
Authors: Lawther R P , Calhoun D H , Adams C W , Hauser C A , Gray J , Hatfield G W
Published: May 2006

The relationship of valine resistance to the expression of the ilvGEDA operon of Escherichia coli K-12 has been determined. DNA sequence and in vivo protein analyses indicate that in wild-type E. coli K-12 there is a frameshift site within the gene (ilvG) for valine resistance. The ilvG+2096 (formerly designated ilv02096) mutation displaces this frameshift site, resulting in the expression of ilvG and the relief of transcriptional polarity on the distal genes of this operon. Thus, the "ilv0" mutation, which concomitantly confers valine resistance and increased expression of the ilvEDA genes, is, in fact, the "reversion" of a polar site within the first structural gene of the ilvGEDA operon.
Specific reaction rate of acetylcholine receptor-controlled ion translocation: a comparison of measurements with membrane vesicles and with muscle cells.
Authors: Hess G P , Aoshima H , Cash D J , Lenchitz B
Published: May 2006

The specific reaction rate (J) of the acetylcholine receptor-controlled ion translocation has been determined. In eel Ringer's solution (pH 7.0) at 1 degrees C, J = 3 X 10(7) M-1 sec-1. J is an intrinsic constant that is characteristic of the receptor and independent of other properties of a receptor-containing cell that also determine the rates of ion translocation. Membrane vesicles (prepared from the electric organ of Electrophorus electricus) and a flow-quench technique that has a millisecond time resolution were used to measure the receptor-controlled ion translocation. Using the value of J and the molar concentrations of receptor sites and inorganic ions, we calculated that 6 X 10(3) ions are translocated per msec per receptor. Analysis of electrical noise in frog muscle cells at temperatures above 8 degrees C [Nether, E. & Stevens, C. F. (1977) Annu. Rev. Biophys. Bioeng. 6, 345-381] gave a value of about 1 X 10(4) ions msec-1 per channel. Thus, each technique gives essentially the same result. It is now possible, therefore, to correlate the results obtained when receptor function is measured in two different ways in membrane vesicles and in muscle cells: (i) chemical kinetic measurements, using membrane vesicles, which relate the ligand binding and ion translocation processes and (ii) analysis of acetylcholine noise in muscle cells [Katz, B. & Miledi, R. (1972) J. Physiol. (London) 224, 665-699], which allows one to measure elementary steps in the formation of ion channels through the cell membrane.