bluuebunny/crossref_metadata_2025 huggingface dataset:
The distribution of interspersed repeats is nonuniform and conserved in the mouse and human genomes.
Authors: Soriano P , Meunier-Rotival M , Bernardi G
Published: May 2006

We investigated the genomic distribution of mouse and human repeated sequences by assessing their relative amounts in the four major components into which these genomes can be resolved by density gradient centrifugation techniques. These components are families of fragments that account for most or all of main-band DNAs, range in dG + dC content from 37% to 49%, and are derived by preparative breakage from long DNA segments (greater than 300 kb) of fairly homogeneous composition, the isochores. The results indicate that the short repeats of the B1 family of mouse and of the Alu I family of man are most frequent in the heavy components, whereas the long repeats of the BamHI family of mouse and of the Kpn I family of man are mainly present in the two light components. These results show that the genomic distribution of repeated sequences is nonuniform and conserved in two mammalian species. In addition, we observed that the base composition of two classes of repeats (60% dG + dC for short repeats; 39% dG + dC for long repeats) is correlated with the composition of the major components in which they are embedded. Finally, we obtained evidence that not only the short repeats but also the long repeats are transcribed, these transcripts having been found in mouse poly(A)+ mRNA.
Identification of the hypusine-containing protein hy+ as translation initiation factor eIF-4D.
Authors: Cooper H L , Park M H , Folk J E , Safer B , Braverman R
Published: May 2006

A single protein of Mr 17,000-19,000 and pI approximately equal to 5.1, found in all animal cells we have studied to date, undergoes post-translational modification in growing cells to form the unusual amino acid hypusine. Because of the association of this modification with the increasing rate of protein synthesis during lymphocyte growth stimulation, its subcellular distribution, and its widespread occurrence and structural conservation among animal cells, we considered the possibility that this protein might be a translation initiation factor. Purified rabbit reticulocyte factors (eukaryote initiation factors) eIF-4C and eIF-4D were chosen for study because of their Mr (17,000-19,000) and acidic pI. The hypusine-containing protein and purified eIF-4D showed identity of electrophoretic mobility in both isoelectric focusing and NaDodSO4/polyacrylamide gel electrophoresis dimensions, while eIF-4C was clearly nonidentical. Purified eIF-4D contained approximately 1 mol of hypusine per mol of protein. Since only one protein has thus far been observed to contain hypusine, we conclude that eIF-4D is the hypusine-containing protein. On the basis of relative synthesis among lymphocyte proteins and detection by Coomassie blue staining, we also conclude that eIF-4D is a major cell protein. It is possible that the activity of this factor is modulated by It is possible that the activity of this factor is modulated by post-translational hypusine formation, which may play a role in regulation of protein synthesis during lymphocyte growth stimulation.
Bathorhodopsin intermediates from 11-cis-rhodopsin and 9-cis-rhodopsin.
Authors: Spalink J D , Reynolds A H , Rentzepis P M , Sperling W , Applebury M L
Published: May 2006

Bathorhodopsin-rhodopsin difference spectra of native 11-cis-rhodopsin and regenerated 9-cis-rhodopsin were measured at room temperature with a double-beam laser spectrophotometer after excitation at 532 nm. A detailed analysis of data obtained at 85 psec after excitation suggests that the bathorhodopsins generated from 11-cis- and 9-cis-rhodopsin differ in their extinction coefficients and that their absorption maxima are shifted in wavelength by about 10 nm from one another. The ratio of quantum yields for photochemical production of the 11-cis-bathorhodopsin and the 9-cis-bathorhodopsin approximates 1. Implications that the early photochemical processes in vision are more complex than previously considered are explored.
Evolution: questions for the modern theory.
Authors: Darlington P J
Published: May 2006

The blind spot of the present generation of evolutionists is failure to see the consequences and limits of natural selection. Darwinian natural selection is a costly process of differential elimination of individuals. The widely accepted misdefinition of natural selection as differential reproduction mistakenly hides the Darwinian process and its cost. And current theories of selfish genes, inclusive fitness, and kin selection are incompatible with Darwinian selection. Implicitly, if not explicitly, they postulate genes that favor themselves but reduce the Darwinian fitness of the individuals carrying them. Such genes would not survive; they would eliminate themselves by causing the selective elimination of their carriers. Critical questions that evolutionists should be asked are suggested. My own "unhappy conclusion" is that, because most biologists have forgotten what natural selection is, much current evolutionary and sociobiological theory presented by the most influential evolutionists is mistaken and dangerous. Anthropologists and sociologists are wise to distrust it.
Adenosine receptor binding: structure-activity analysis generates extremely potent xanthine antagonists.
Authors: Bruns R F , Daly J W , Snyder S H
Published: May 2006

Structure-activity analysis of alkylxanthine derivatives at adenosine receptor binding sites has been employed to design more potent adenosine receptor antagonists. Receptor affinities of xanthines were determined by measuring inhibition of the binding of N6-[3H]cyclohexyladenosine to bovine brain membranes. 1,3-Dipropyl substitutions enhance potency compared to the 1,3-dimethyl substitution in theophylline. An 8-phenyl substituent produces a considerable increase in potency, which is augmented by certain para substitutions on the 8-phenyl ring. Combining an ortho amino with a para-chloro substituent on the 8-phenyl ring affords further increases in potency. Combining all of these substituents results in 1,3-dipropyl-8-(2-amino-4-chlorophenyl) xanthine, a compound of extraordinary receptor affinity, with a Ki for adenosine A1 receptors of 22 pM. It is 4,000,000 times more potent than xanthine itself and 70,000 times more potent than theophylline.
Chemical evidence that catecholamines are transported across the chromaffin granule membrane as noncationic species.
Authors: Ramu A , Levine M , Pollard H
Published: May 2006

Catecholamines are transported into chromaffin granules by a Mg2+/ATP-driven process under conditions in which the substrate exists primarily as a positively charged or neutral species. In order to distinguish between these two states, we studied the transport properties of a permanently charged quaternary analogue of epinephrine, (R,S)-dimethylepinephrine. We found that this compound was a classical competitive inhibitor of (R)-[3H]norepinephrine uptake, with a Ki of 3.8 mM for the racemic form, or 1.9 mM for the R form. However, the [3H]dimethylepinephrine was not transported at all into granules. Our control for steric hindrance as an explanation for deficient translocation was analysis of the transport properties of isoproterenol, a secondary catecholamine with an isopropyl group around the amine residue. (R)-Isoproterenol was an effective competitive inhibitor of (R)-[3H]norepinephrine transport, with a Ki of 91 microM. In contrast to dimethylepinephrine, (R,S)-[3H]isoproterenol was clearly translocated across the granule membrane, with a Km of 123 microM, or 61.5 microM for the R isomer. Thus, the positive charge on dimethylepinephrine and not the size of the amine moiety appeared to be responsible for the lack of translocation. We interpret these data to indicate that, although the positively charged species can interact with the transport site, an uncharged species is the one actually transported.
Three human transforming genes are related to the viral ras oncogenes.
Authors: Shimizu K , Goldfarb M , Suard Y , Perucho M , Li Y , Kamata T , Feramisco J , Stavnezer E , Fogh J , Wigler M H
Published: May 2006

Three distinct transforming genes present in human tumor cell lines are all related to the viral oncogenes of Harvey and Kirsten murine sarcoma viruses, designated v-H-ras and v-K-ras, respectively. The transforming gene of a bladder carcinoma cell line has been shown to be a human homolog to v-H-ras [Parada, L. F., Tabin, C. J., Shih, C. & Weinberg, R. A. (1982) Nature (London) 297, 474-478; Santos, E., Tronick, S. R., Aaronson, S. A., Pulciani, S. & Barbacid, M. (1982) Nature (London) 298, 343-347]. The transforming gene common to one colon (SK-CO-1) and two lung carcinoma (SK-LU-1 and Calu-1) cell lines is the same human homolog of v-K-ras as is the transforming gene previously identified in a lung carcinoma cell line Lx-1 [Der, C. J., Krontiris, T. G. & Cooper, G. M. (1982) Proc. Natl. Acad. Sci. USA 79, 3637-3640]. The transforming gene of SK-N-SH neuroblastoma cells is weakly homologous to both v-H-ras and v-K-ras. NIH 3T3 cells transformed with the SK-N-SH transforming gene contain increased levels of a protein serologically and structurally related to the protein products of the v-H-ras and v-K-ras genes. Therefore, it represents a third member of the ras gene family, which we have called N-ras. Based on the homology with the v-ras genes, we have established the orientation of transcription and approximate coding regions of the cloned human K-ras and N-ras genes.
UV-induction of chalcone synthase mRNA in cell suspension cultures of
Petroselinum hortense
Authors: Kreuzaler Fritz , Ragg Hermann , Fautz Erich , Kuhn David N. , Hahlbrock Klaus
Published: May 2006

DNAs complementary to poly(A)
+
mRNAs from UV-irradiated cell suspension cultures of parsley (
Petroselinum hortense
) were inserted into pBR322 and used to transform
Escherichia coli
strain RR1. A clone containing a DNA complementary to chalcone synthase mRNA was identified by hybrid-selected and hybrid-arrested translation. Large and rapid changes in the amount of chalcone synthase mRNA in response to irradiation of the cells was detected by RNA blot hybridization experiments. The pattern of changes coincided with that previously determined for the rate of chalcone synthase synthesis as measured either
in vivo
or with polyribosomal mRNA
in vitro
. The results are consistent with the hypothesis that induction of chalcone synthase by UV light is due to a transient increase in the rate of synthesis of chalcone synthase mRNA.
Synthesis of spectrin in avian erythroid cells: association of nascent polypeptide chains with the cytoskeleton.
Authors: Blikstad I , Lazarides E
Published: May 2006

The site of synthesis of spectrin was investigated in erythroid cells from 10-day chicken embryos. After various periods of [35S]methionine incorporation the cells were lysed in a Triton X-100 (TX-100)-containing buffer and were separated into a TX-100-soluble and -insoluble (cytoskeletal) fraction. Analysis of these two fractions by two-dimensional gel electrophoresis after a short pulse-labeling period reveals that alpha-spectrin nascent polypeptides are present predominantly in the TX-100-insoluble fraction. These polypeptides can be immunoprecipitated with alpha-spectrin antisera and the [35S]methionine incorporated into them during a short pulse can be chased into mature alpha-spectrin molecules. The alpha-spectrin nascent polypeptide chains are released quantitatively from the TX-100 cytoskeleton by treatment of lysed cells with puromycin, suggesting that they themselves are not associated with the cytoskeleton. A small fraction of the newly synthesized mature alpha-spectrin molecules is rapidly incorporated into the cytoskeleton, as shown by the fact that they are not released by the puromycin treatment; the rest are recovered in the soluble fraction. These results suggest that alpha-spectrin is synthesized in association with the cytoskeleton during chicken erythropoiesis and assembles onto the membrane-cytoskeleton posttranslationally.
Evidence for clonal population structure in Escherichia coli.
Authors: Ochman H , Selander R K
Published: May 2006

Genotypes of 142 K1 isolates of four O serogroups of Escherichia coli from human hosts in Europe and the United States were characterized by an electrophoretic analysis of allozymic variation in 12 chromosomally encoded enzymes. The genetic structure of natural populations revealed by this analysis is closely similar to that indicated in earlier studies by Achtman and colleagues of the electrophoretic migration pattern for four outer membrane proteins and the chemical structure of the cell-wall lipopolysaccharides. The combined evidence demonstrates that most of the K1 isolates belong to a small number of geographically widespread clones. The distribution of O serogroups among the isolates does not consistently correspond to the clonal structure; O1:K1 isolates represent at least two distantly related, geographically widespread clones, one of which is genetically similar to a clone of the O18:K1 serotype. These findings for K1 isolates add to a growing body of evidence supporting the hypothesis that the genetic structure of natural populations of E. coli is basically clonal, with very limited recombination of chromosomal genes. Clonal structure has important implications for the study of the determinants of pathogenicity and disease specificity in E. coli.
Expression and regulation of chicken actin genes introduced into mouse myogenic and nonmyogenic cells.
Authors: Seiler-Tuyns A , Eldridge J D , Paterson B M
Published: May 2006

We have introduced the chicken genes for cytoplasmic beta-actin, cardiac alpha-actin, and skeletal alpha-actin into C2 cells, a murine myogenic cell line, and into L cells by using the simian virus 40-derived vector PSV2 -gpt. In each selection, the entire population of transformed cells was analyzed for the expression and regulation of the actin genes by nuclease S1 assay and primer extension. This was compared to the expression of the vector marker Eco-gpt. The beta-actin gene is transcribed accurately and efficiently both in L-cells and in undifferentiated C2 cells. In fused C2 cells, beta-actin transcripts decrease significantly in parallel with the endogenous level of mouse beta-actin mRNA. Eco-gpt RNA levels remain essentially constant during myogenesis. The alpha-actin genes are correctly expressed at low levels in L cells but at significantly higher levels in the C2 cell background. Unlike the endogenous mouse alpha-actin gene, this level of expression does not change measurably with myogenesis. The skeletal alpha-actin gene is expressed poorly in pre- and post-fusion C2 cells, displaying no induction with differentiation. These results suggest that the tissue specificity of expression is maintained but the pattern of gene regulation for the sarcomeric actins is not. Factors in addition to the sequences flanking these genes are important for modulating gene expression during development. The decrease in the levels of beta-actin RNA during C2 cell differentiation provides a model system in which to study gene repression during development.
Neurons with graded response have collective computational properties like those of two-state neurons.
Authors: Hopfield J J
Published: May 2006

A model for a large network of "neurons" with a graded response (or sigmoid input-output relation) is studied. This deterministic system has collective properties in very close correspondence with the earlier stochastic model based on McCulloch - Pitts neurons. The content- addressable memory and other emergent collective properties of the original model also are present in the graded response model. The idea that such collective properties are used in biological systems is given added credence by the continued presence of such properties for more nearly biological "neurons." Collective analog electrical circuits of the kind described will certainly function. The collective states of the two models have a simple correspondence. The original model will continue to be useful for simulations, because its connection to graded response systems is established. Equations that include the effect of action potentials in the graded response system are also developed.
Radial Fourier multipliers of

L
p

(R
2
)
Authors: Carbery Anthony , Gasper George , Trebels Walter
Published: May 2006

We give sufficient conditions (in terms of differentiability and growth properties) for a radial function to be an

L
p

(R
2
) Fourier multiplier. These conditions are in the nature of best possible.
The persistence of maternal inheritance in Chlamydomonas despite hypomethylation of chloroplast DNA induced by inhibitors.
Authors: Feng T Y , Chiang K S
Published: May 2006

We have used inhibitors of methylation to evaluate the proposal that the extent of methylation of chloroplast DNA ( cpDNA ) of the mating type-plus (mt+) parent occurring during gametogenesis in wild-type Chlamydomonas renhardtii is directly correlated with the uniparental transmission of chloroplast genes by this parent [ Sager , R., Grabowy , C. & Sano , H. (1981) Cell 24, 41-47]. As detected by high-pressure liquid chromatography, the methylation of cpDNA was at its lowest level in the vegetative stage; the mt+ cells had a deoxycytidine methylation index (the percentage of deoxycytidine methylated) of 0.5, while the mating type-minus (mt-) index was lower by at least a factor of 3. This basal level of cpDNA methylation increased more than 20-fold after gametogenesis to give a methylation index of 12.1 and 4.3 for mt+ and mt- gametes, respectively. Another striking increase was detected at the 7-hr-zygote stage, resulting in the methylation of nearly half of the total deoxycytidine residues. The extent of zygotic cpDNA methylation was shown to be dependent on the preexisting methylation level of both parental gametic cpDNAs . L-Ethionine and 5-azacytidine effectively inhibited cpDNA methylation during gametogenesis and ensuing zygotic development as shown by both Hpa II/Msp I digestion patterns and HPLC. The transmission of chloroplast genes was analyzed concomitantly with the inhibitor studies. The two inhibitors produced different patterns of inhibition of methylation in mt- and mt+ cells at a given developmental stage. Our overall results demonstrate that the extent of mating type-specific and gamete-specific methylation during gametogenesis is not correlated with the frequency of maternal transmission of chloroplast genes.
Linkage disequilibrium and evolutionary relationships of DNA variants (restriction enzyme fragment length polymorphisms) at the serum albumin locus.
Authors: Murray J C , Mills K A , Demopulos C M , Hornung S , Motulsky A G
Published: May 2006

Four additional DNA variants (restriction enzyme fragment length polymorphisms) making a total of eight polymorphic sites at the human albumin locus have been identified. These eight sites were found after screening 689 of 20,000 nucleotides by using cDNA probes for albumin with 27 different restriction enzymes. One in 85 nucleotides was therefore potentially polymorphic. The average nucleotide diversity between any two randomly chosen chromosomes was calculated to be 1/500. We observed marked linkage disequilibrium between the eight variants. Only 7 haplotypes among 256 possible combinations were observed in 160 chromosomes from Caucasoids, Blacks, and Asians. Two haplotypes were found in all three human races, indicating that their origin predated human racial divergence. The three rarest haplotypes appear to represent recombinational events between the more common haplotypes. All crossovers occurred in the same general region. Studies of several nonhuman primates indicated that the origin of one haplotype predated the human-African ape divergence. Although it is not possible to rule out maintenance of this tight linkage by selection or fixation, it is suggested that the limited number of haplotypes at the chromosomal site of the albumin gene near the centromere of chromosome 4 may be the result of decreased recombination.
Twisted states of Bacillus subtilis macrofibers reflect structural states of the cell wall.
Authors: Mendelson N H , Favre D , Thwaites J J
Published: May 2006

Static and dynamic studies of helical Bacillus subtilis macrofibers reveal that a spectrum of twisted states exists ranging from tight left-handed structures with twist equal to approximately equal to 40 left turns per mm to tight right-handed structures with twist equal to 57 right turns per mm. In the lytic-deficient strain FJ7 , twist varies as a function of growth temperature above or below 39 degrees C, where there is zero twist. The relationship between the temperature (below 39 degrees C) at which right-hand structures are produced to the time it takes for them to begin the inversion process in which they become left-handed following transfer to 48 degrees C reveals that structures with less twist are more rapidly converted to left-handedness than are those with higher values of twist. The initial response of live macrofibers to digestion by lysozyme consists of "relaxation" motions in which the twist of both left- and right-handed structures changes towards the right-hand end of the spectrum. The rate of relaxation is approximately equal to 5-fold higher at the left-hand end than at the right-hand end. These findings suggest that cell wall polymers can assume a range of structural states during helical growth and that these determine the quantitative aspects of macrofiber shape as well as the sensitivity of walls to attack by lysozyme.
Binding of the differentiation-inducer, granulocyte-colony-stimulating factor, to responsive but not unresponsive leukemic cell lines.
Authors: Nicola N A , Metcalf D
Published: May 2006

Granulocyte-colony-stimulating factor (G-CSF) is a tissue-derived 25,000 Mr glycoprotein that stimulates neutrophilic granulocyte colony formation from murine bone marrow progenitor cells in vitro. It is also a potent inducer of terminal differentiation and suppressor of stem cell renewal in the murine myelomonocytic leukemic cell line WEHI-3B. Purified G-CSF was radioiodinated to high specific radioactivity with retention of full biological activity. Iodinated G-CSF bound specifically to WEHI-3B cells, J774 macrophage tumor cells, and normal murine bone marrow cells but not to a variety of other tumor cell lines or murine thymocytes. WEHI-3B cells showed a high affinity for 125I-labeled G-CSF (Kd = 90 pM) but displayed only a small number of specific receptors (300-700 per cell) at 37 degrees C. Other purified colony-stimulating factors showed no competition for binding to these receptors. WEHI-3B (D-), a subline of WEHI-3B that cannot be induced to differentiate by G-CSF, showed no specific binding of this factor, indicating that it is deficient in receptor presentation.
Clustered genes for human U2 RNA.
Authors: Westin G , Zabielski J , Hammarström K , Monstein H J , Bark C , Pettersson U
Published: May 2006

Genes for the human small nuclear RNA U2 are present within 6.2-kilobase-pair-long tandem repeats. The haploid human genome contains approximately 20 such repeats, organized in one or a few very large clusters.
Separation of sequences defining basal expression from those conferring alpha gene recognition within the regulatory domains of herpes simplex virus 1 alpha genes.
Authors: Kristie T M , Roizman B
Published: May 2006

The genes of herpes simplex virus 1 form three major groups--alpha, beta, and gamma--whose expression is coordinately regulated and sequentially ordered in a cascade fashion. To determine how the infected cell differentiates between these gene groups, alpha-regulated chimeric genes were constructed in earlier studies by fusing the structural sequences of the thymidine kinase (TK) gene, a beta gene, to the 5' noncoding sequences of alpha genes. These studies showed that (i) one or more structural components of the virion act in trans to increase alpha gene expression and (ii) the 5' noncoding sequences of alpha genes contain cis-acting domains that promote gene expression and confer alpha-gene regulation. These two domains could be moved independently, but the regulatory domain required a promoter for its function. We report here the properties of three sequences containing features common to the regulatory regions of all alpha genes. Sequence 1, containing (G + C)-rich inverted repeats, increased the basal level of TK expression when fused 5' to either the alpha gene 4 promoter or the truncated beta TK promoter. The effect was to some extent orientation dependent. Moreover, sequence 1 restored beta regulation to the truncated beta TK promoter but did not confer alpha-specific regulation on any of the chimeric genes tested. Sequences 2 (49 base pairs) and 3 (29 base pairs), containing an (A + T)-rich homolog from alpha gene 27 and alpha gene 0, respectively, restored alpha-specific regulation to the alpha promoter gene but only sequence 2 conferred alpha regulation on the truncated beta promoter gene. Our results indicate that (i) in natural beta TK the promoter and regulatory domains overlap, (ii) sequence 1 determines basal level of expression and substitutes for a promoter component that is essential for beta but not alpha regulation, and (iii) conversion of a gene with a promoter into an alpha gene requires two elements. Sequence 2 may contain both whereas sequence 3 contains only one.
Optimal conditions for cell-free synthesis of citrus exocortis viroid and the question of specificity of RNA polymerase activity
Authors: Semancik J. S. , Harper K. L.
Published: May 2006

Cell-free synthesis of citrus exocortis viroid (CEV) in nuclei-rich preparations from infected
Gynura aurantiaca
was optimum at 18-24°C. Incubation of reaction mixtures at higher temperatures (30-36°C) resulted in an increase of CEV linear molecules and the recovery of incomplete or nicked newly synthesized RNA species. Although the Mg
2+
optimum (2.5-5 mM) for CEV synthesis was lower than that for total [
32
P]CMP incorporation (10 mM), the response to Mn
2+
ion was distinctly different. Whereas maximum total activity was observed in 1 mM Mn
2+
with a pronounced reduction (80%) in 5 mM Mn
2+
, CEV synthesis was maintained in 1-15 mM Mn
2+
. Inhibition of α-amanitin-sensitive CEV synthesis in 200 mM (NH
4
)
2
SO
4
resembles the reaction of RNA polymerase II on a free nucleic acid template. However, detection of trace levels of α-amanitin-resistant CEV synthesis activity inhibited by low (NH
4
)
2
SO
4
concentrations (25 mM) suggests the possible involvement of RNA polymerase I- and/or III-like activity.
Interallelic complementation in an inborn error of metabolism: genetic heterogeneity in argininosuccinate lyase deficiency.
Authors: McInnes R R , Shih V , Chilton S
Published: May 2006

We used complementation analysis as a probe for the detection of genetic heterogeneity within a single locus affected in a human disease, argininosuccinate lyase (L-argininosuccinate arginine-lyase, EC 4.3.2.1) deficiency. Fibroblasts cultured from 28 unrelated patients were fused in all possible pairwise combinations, and the argininosuccinate lyase activity in heterokaryons was assayed by measuring the incorporation of 14C from L-[ureido-14C]citrulline into acid-precipitable material. Partial complementation was observed in fusions involving 20 of the 28 strains, with the lyase activity increasing from 2- to 10-fold. Thirteen of the mutants were identified by the complementation analysis as being phenotypically unique. Of the 20 complementing strains, 3 were remarkable because they participated in all but 2 of the 32 positive complementation tests; 2 others constituted a unique subgroup that produced the highest increases in argininosuccinate lyase activity of all fusions. The 8 strains that did not complement any others consisted of two types: 3 mutants with the highest residual argininosuccinate lyase activity of all strains and 5 mutants with low residual activity. All of the mutants mapped to a single major complementation group. The data could be summarized as a circular complementation map with an attached linear tail, the mutants being distributed among 12 subgroups in a complex pattern. We conclude that all of these mutants are affected at a single locus, that extensive genetic heterogeneity is present in the mutant population, and that the affected locus in argininosuccinate lyase deficiency is likely to be the structural gene coding for that enzyme.
Alpha-factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae.
Authors: Brake A J , Merryweather J P , Coit D G , Heberlein U A , Masiarz F R , Mullenbach G T , Urdea M S , Valenzuela P , Barr P J
Published: May 2006

Saccharomyces cerevisiae cells were transformed with plasmids containing hybrid genes in which the sequence encoding mature human epidermal growth factor was joined to sequences encoding the leader region (preprosegment) of the precursor of the yeast mating pheromone alpha-factor. These cells accurately process the hybrid protein and efficiently secrete authentic biologically active human epidermal growth factor into the medium.
Isolation and nucleotide sequence of the gene encoding human rhodopsin.
Authors: Nathans J , Hogness D S
Published: May 2006

We have isolated and completely sequenced the gene encoding human rhodopsin. The coding region of the human rhodopsin gene is interrupted by four introns, which are located at positions analogous to those found in the previously characterized bovine rhodopsin gene. The amino acid sequence of human rhodopsin, deduced from the nucleotide sequence of its gene, is 348 residues long and is 93.4% homologous to that of bovine rhodopsin. Interestingly, those portions of the polypeptide chain predicted to form loops on the cytoplasmic face of rhodopsin are perfectly conserved between the human and bovine proteins.
Linear organization of the liver cell adhesion molecule L-CAM.
Authors: Cunningham B A , Leutzinger Y , Gallin W J , Sorkin B C , Edelman G M
Published: May 2006

A linear model of the liver cell adhesion molecule L-CAM from embryonic chickens is proposed in terms of its orientation on the cell surface, the number, type, and distribution of carbohydrate moieties, and sites of phosphorylation. L-CAM is isolated from cell membranes as a glycoprotein of Mr = 124,000. A soluble fragment (Ft1) of Mr = 81,000 can be released from cells by digestion with trypsin in the presence of calcium. Radiochemical amino acid sequence analyses indicated that both polypeptides have the same sequence for the first 10 amino acids, suggesting that fragment Ft1 contains the amino terminus of the L-CAM molecule and that the carboxyl-terminal portion of the peptide chain is associated with the cell. Digestions with endoglycosidase H and endoglycosidase F indicated that Ft1 has all of the N-linked carbohydrate groups associated with the larger species, including one high mannose oligosaccharide and three complex oligosaccharides. When hepatocytes were grown in the presence of 32PO4, 32P was detected in phosphoserine and phosphothreonine residues of intact L-CAM, but little or no 32P was detected in Ft1, suggesting that L-CAM is phosphorylated in the carboxyl-terminal region. On CNBr cleavage, the bulk of the 32P was detected in a single fragment of Mr = 20,000. The overall features of the L-CAM molecule incorporated in the model provide a basis for correlating its structure with its cell-cell binding activity and for detailed comparisons with similar molecules described in mammalian species.
Selective destruction of a host blood cell type by a parasitoid wasp.
Authors: Rizki R M , Rizki T M
Published: May 2006

Foreign objects that enter the hemocoel of Drosophila melanogaster larvae are encapsulated by one type of blood cell, the lamellocyte, yet eggs of the parasitoid wasp Leptopilina heterotoma remain unencapsulated in D. melanogaster larval hosts that have many lamellocytes. Here we demonstrate that shortly after a female wasp oviposits in the hemocoel the lamellocytes undergo morphological changes and lose their adhesiveness. These affected blood cells are eventually destroyed as the parasitoid egg continues its development. The factor responsible for lamellocyte destruction, lamellolysin, is contained in an accessory gland of the female reproductive system and is injected along with the egg into the host hemocoel. Lamellolysin does not alter the morphology or the defense functions of the other types of blood cells in the host.
Mechanism of translational control by partial phosphorylation of the alpha subunit of eukaryotic initiation factor 2.
Authors: Siekierka J , Manne V , Ochoa S
Published: May 2006

Catalysis of ternary complex formation by the GDP exchange factor (GEF), in the presence of Mg2+, is blocked by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF-2). We proposed earlier that this phosphorylation interferes with the interaction between eIF-2 and GEF (then termed ESP). If so, inhibition should be related to the extent of phosphorylation. However, work in other laboratories indicated that in fully inhibited, heme-deficient lysates only 20-40% of the eIF-2 is phosphorylated. To understand the nature of the molecular lesion in eIF-2-alpha phosphorylation we used a system of pure components in which the rate of exchange of eIF-2-bound [3H]GDP with unlabeled GDP (via the reaction eIF-2-GDP + GEF in equilibrium eIF-2-GEF + GDP) was measured by using mixtures of eIF-2(alpha P) X [eH]GDP and eIF-2 X [3H]GDP in different proportions at constant concentration of eIF-2 X GEF. If, for example, the ratio of eIF-2 X GEF to total (phosphorylated and unphosphorylated) eIF-2 X [3H]GDP was 0.25, the exchange was found to be maximally inhibited when the proportion of eIF-2(alpha P) X [3H]GDP in hte mixture reached 25%. This suggests that the reaction stops because the available GEF is trapped in an inactive complex with eIF-2(alpha P). In the absence of free GEF, eIF-2 would not be able to recycle and initiation would come to a standstill when the available eIF-2 is tied up as eIF-2 X GDP. The trapping of GEF by eIF-s(alpha P) is strongly supported by the following observation. Incubation of eIF-2 X GEF with excess [3H]GDP leads to the formation of eIF-2 X [3H] GDP and free GEF and, if eIF-2(alpha 32P) X GDP is also present, all of the GEF is converted to eIF-2(alpha 32P) X GEF. This suggests that, whereas the equilibrium of the reaction eIF-2 X GEF + GDP in equilibrium eIF-2 X GDP + GEF favors the formation of free GEF, the equilibrium of the reaction eIF-2(alpha P) X GDP + GEF in equilibrium eIF-2(alpha P) X GEF + GDP is in favor of the association of GEF to eIF-2(alpha P).
Protein HU in the enzymatic replication of the chromosomal origin of Escherichia coli.
Authors: Dixon N E , Kornberg A
Published: May 2006

A protein that stimulates the enzymatic replication of duplex DNAs of recombinant phages and plasmids bearing the Escherichia coli origin of replication (oriC) has been isolated from an extract of E. coli. The isolated protein and the well-known protein HU, a histone-like DNA-binding protein, have identical polypeptide molecular weights and saturate the oriC replication assay at less than 40 dimers per template DNA circle. This level is one-tenth that needed to coat the template. Protein HU from the blue-green alga Anabaena is similarly active. Antibody specific for protein HU from E. coli inhibits replication promoted both by the reconstituted system and by a crude enzyme extract; in both assays, activity is restored by excess of the isolated protein. Cells lysed in 1 M KCl yield 32,000 dimers of the protein per cell, a number consistent with the reported abundance of HU. These data establish the identity of the isolated factor and protein HU and provide an indication of a function for HU in replication.
Escherichia coli mutants suppressing replication-defective mutations of the ColE1 plasmid.
Authors: Naito S , Kitani T , Ogawa T , Okazaki T , Uchida H
Published: May 2006

Mutants of Escherichia coli K-12 have been isolated that suppress cer mutants, ColE1 mutants that are unable to replicate as the plasmid. These host suppressors were designated her, for host factor affecting ColE1 replication. Each her suppressor showed a characteristic pattern of suppression depending on the cer mutation used for selecting the mutant bacteria. One of the suppressors, named herA, that suppressed cer6, a single-base-pair alteration 160 base pairs upstream of the ColE1 replication origin, was genetically identified as an alteration of the rnh gene (RNase H). HerA was recessive to its wild-type allele. RNase H activity of herA cell extracts was defective. Conversely, rnh mutants that were isolated independently of ColE1 replication supported replication of cer6 DNA. Some rnh mutants manifested the HerA phenotype only above a certain transition temperature, and their RNase H activity was found to be temperature sensitive. Therefore, replication of cer6 DNA in vivo is sensitive to RNase H activity. Under the conditions that suppressed cer6, the wild-type colE1 replicon replicated normally. Then, ColE1 replication in vivo proceeds in the absence of RNase H activity, which has been shown to be required for in vitro replication of the DNA.
Charge state of His-57-Asp-102 couple in a transition state analogue-trypsin complex: a molecular orbital study.
Authors: Umeyama H , Hirono S , Nakagawa S
Published: May 2006

Ab initio molecular orbital studies have been made as a model for the deacylation step of trypsin. Ser-195 is modeled by H2O in which one H is replaced either by--PO2(OH)- (monoisopropyl phosphoryl, MIP) or by--CHO(OH)- (a transition state analogue, TSD). The quantum mechanical region includes imidazole+ and acetate- as models for His-57+ and Asp-102-, two hydrogen bonds from two formamide molecules to the oxyanion MIP or TSD, and three hydrogen bonds to Asp-102. The remainder of the enzyme is treated classically as a fractional charge model. The effect of proton transfer from His-57+ to Asp-102- is very similar for the MIP and TSD models, and the proton transfer is energetically unfavorable for all models that include at least the hydrogen bond from an H2O that models Ser-214. Thus, the several hydrogen bonds to the models of the catalytic unit (substrate, Ser-195, His-57, and Asp-102) stabilize the His-57+/Asp-102- salt link, and this indicates that proton transfer does not occur from His-57+ to Asp-102-. (Also, the similarities of energy of transfer of this proton transfer for the various models show that the model substrate analogue behaves very similarly to the MIP inhibitor.)
Alkane biosynthesis by decarbonylation of aldehydes catalyzed by a particulate preparation from Pisum sativum.
Authors: Cheesbrough T M , Kolattukudy P E
Published: May 2006

Mechanism of enzymatic conversion of a fatty acid to the corresponding alkane by the loss of the carboxyl carbon was investigated with particulate preparations from Pisum sativum. A heavy particulate preparation (sp. gr., 1.30 g/cm3) isolated by two density-gradient centrifugation steps catalyzed conversion of octadecanal to heptadecane and CO. Experiments with [1-3H,1-14C]octadecanal showed the stoichiometry of the reaction and retention of the aldehydic hydrogen in the alkane during this enzymatic decarbonylation. This decarbonylase showed an optimal pH of 7.0 and a Km of 35 microM for the aldehyde. This enzyme was severely inhibited by metal ion chelators and showed no requirement for any cofactors. Microsomal preparations and the particulate fractions from the first density-gradient step catalyzed acyl-CoA reduction to the corresponding aldehyde. Electron microscopic examination showed the presence of fragments of cell wall/cuticle but no vesicles in the decarbonylase preparation. It is concluded that the aldehydes produced by the acyl-CoA reductase located in the endomembranes of the epidermal cells are converted to alkanes by the decarbonylase located in the cell wall/cuticle region.
A gene regulating the heat shock response in Escherichia coli also affects proteolysis.
Authors: Baker T A , Grossman A D , Gross C A
Published: May 2006

The htpR locus in Escherichia coli encodes a regulator of the heat shock response. Cells containing the htpR165 mutation are defective in the induction of synthesis of heat-shock proteins at high temperature. We show that these cells are also defective in degrading two proteins that are normally unstable in htpR+ cells. The proteolytic defect is manifest at both 30 degrees C and 42 degrees C. We used a marker rescue technique to map this defect to the htpR locus. Although both proteolytic substrates are partially stabilized in lon- strains, we argue that the defect in proteolysis exhibited by the htpR165 strain does not mimic the lon- state. The htpR165 strain synthesizes Lon at the normal rate at 30 degrees C and does not show the phenotypes of mucoidy and radiation sensitivity associated with lon- strains.
Kinetics of thymidine incorporation into detergent-soluble DNA of mouse lymphocytes.
Authors: Strauss P R , Banerjee P T , LaGree K , Mui S C
Published: May 2006

We reported recently that splenocytes from concanavalin A-stimulated mice rapidly incorporated [3H]thymidine into non-mitochondrial DNA that was detergent soluble and distributed in size classes between 200 and 5000 base pairs. In this report we show that small [3H]thymidine-labeled oligonucleotides (less than 100 base pairs long) appear by 15 min. Subsequently, [3H]thymidine in small oligonucleotides diminishes as incorporation into larger size classes of detergent-soluble DNA occurs in a pattern that is stable for at least 3 hr. Although incorporation of [3H]thymidine into the oligonucleotides is not sensitive to aphidicolin or hydroxyurea, the appearance of [3H]thymidine in larger species is blocked by both drugs. These results indicate that the enzymatic process involved in synthesis of oligonucleotides is somehow distinct from the process involved in synthesis of larger detergent-soluble size classes. Synthesis of the latter may be replication related.
Synthesis of a major integral membrane polypeptide of rat liver peroxisomes on free polysomes.
Authors: Fujiki Y , Rachubinski R A , Lazarow P B
Published: May 2006

The manner of synthesis and assembly of the peroxisomal membrane proteins is unknown. Understanding these processes is essential to an understanding of the formation of the organelle. We have investigated the biogenesis of the previously identified major 21.7-kDa integral peroxisomal membrane polypeptide [Fujiki, Y., Fowler, S., Shio, H., Hubbard, A. L. & Lazarow, P. B. (1982) J. Cell Biol. 93, 103-110]. This protein was purified to apparent homogeneity and used to elicit a rabbit antiserum. In immunoblotting analysis, antibody bound only to the 22-kDa membrane polypeptide present exclusively in peroxisomal membranes. Total rat liver RNA was translated in a nuclease-treated rabbit reticulocyte cell-free protein-synthesizing system. The in vitro translation product, isolated by means of the antibody and Staphylococcus aureus cells, comigrated with the mature 22-kDa polypeptide in NaDodSO4/PAGE. Analysis of the translation products of RNAs from free and membrane-bound polysomes indicated that the mRNA for the 22-kDa membrane polypeptide is found predominantly in free polysomes. The results imply post-translational insertion of the membrane polypeptide into the peroxisomal membrane without proteolytic processing and suggest that peroxisomes, like mitochondria and chloroplasts, form by fission from preexisting organelles.
Molecular basis of DNA sequence recognition by the catabolite gene activator protein: detailed inferences from three mutations that alter DNA sequence specificity.
Authors: Ebright R H , Cossart P , Gicquel-Sanzey B , Beckwith J
Published: May 2006

Previously, we reported that substitution of Glu-181 of the catabolite gene activator protein (CAP) by lysine, leucine, or valine results in a protein that has specificity for A X T base pairs at positions 7 and 16 of the DNA recognition site, rather than G X C base pairs as is the case with the wild-type CAP. In this paper, we deduce from these genetic data both (i) the specific chemical interactions by which amino acid side chains at position 181 interact with base pairs 7 and 16 and (ii) the precise alignment between the structures of the CAP and DNA in the intermolecular CAP-DNA complex. Our analysis supports the idea that the two symmetry-related F alpha-helices of the CAP dimer interact with successive major grooves of right-handed B-type DNA [Pabo, C. & Lewis, M. (1982) Nature (London) 298, 443-447; and Steitz, T., Weber, I. & Matthew, J. (1983) Cold Spring Harbor Symp. Quant. Biol. 47, 419-426].
Related cell-surface antigens expressed with positional specificity in Drosophila imaginal discs.
Authors: Brower D L , Wilcox M , Piovant M , Smith R J , Reger L A
Published: May 2006

We have isolated three classes of monoclonal antibodies against Drosophila cell-surface antigens that are expressed with positional specificity in imaginal discs. Comparison of immunofluorescence patterns with the wing-disc fate map reveals that expression of the antigens is not directly related to the specific type of cuticular structure that a cell will make upon differentiation but depends on the position of the cell in the undifferentiated disc epithelium. On mature wing discs, each class of position-specific (PS) antibody binds nonuniformly with respect to the dorso-ventral compartment boundary, with PS1 antibodies binding primarily to dorsal cells and PS2 antibodies, to ventral cells. Antibodies of the different PS classes extract similar but nonidentical sets of large glycoproteins from cell lysates, and antibodies of the most general class, PS3, recognize the PS1 and PS2 antigens in addition to PS3-specific components. Thus, the distributions and molecular characteristics of the PS antigens suggest that the molecules are structurally and functionally related to one another.
Interspecific DNA transformation in Drosophila.
Authors: Scavarda N J , Hartl D L
Published: May 2006

A DNA fragment that includes the wild-type rosy (ry+) gene of Drosophila melanogaster has been introduced by microinjection into the germ line of the reproductively isolated species Drosophila simulans and incorporated into the D. simulans genome. Transformation was mediated by the transposable element P, which occurs in the genome of most natural populations of D. melanogaster but not in D. simulans. Rubin and Spradling [Rubin, G.M. & Spradling, A.C. (1982) Science 218, 348-353] have previously shown that the ry+ DNA fragment, which is flanked by recognition sequences of P element, can transform the germ line of D. melanogaster. Successful transformation in D. simulans indicates that the P element continues to function as a transposable element in the D. simulans genome. Moreover, the ry+ gene of D. melanogaster functions in the genome of D. simulans to produce normal eye color, despite the estimated 1 to 5 million yr of reproductive isolation since the evolutionary divergence of these species. Interspecific DNA transformation provides a useful method for the study of genetic differences affecting gene expression among related but reproductively isolated species.
Partial purification and functional expression of brain mRNAs coding for neurotransmitter receptors and voltage-operated channels.
Authors: Sumikawa K , Parker I , Miledi R
Published: May 2006

Poly(A)+ mRNAs extracted from embryonic chicken brain and from adult rat brain were fractionated on sucrose density gradients. The fractions were subsequently injected into Xenopus oocytes where the mRNA was translated. The products were processed and incorporated into the oocyte membrane where they formed functional neurotransmitter receptors and voltage-operated channels. Different mRNA fractions induced the incorporation of different transmitter receptors and voltage-operated channels into the oocyte membrane. These experiments provide a useful step towards the understanding of the structure and function of neurotransmitter receptors and channels.