bluuebunny/crossref_metadata_2025 huggingface dataset:
Promoter mapping and cell cycle regulation of flagellin gene transcription in Caulobacter crescentus.
Authors: Minnich S A , Newton A
Published: May 2006

Caulobacter crescentus contains a 25- and a 27-kDa flagellin, which are assembled into the flagellar filament, and a 29-kDa flagellin, which is related in sequence but is of unknown function. We have used DNA sequence analysis and nuclease S1 assays to map the in vivo transcription start sites of the three flagellin genes and to study their regulation. These experiments lead to several conclusions. First, copies of the 29-, 25-, and 27-kDa flagellin genes are organized in a tandem array in the flaEY gene cluster of C. crescentus. Second, flagellin genes are under transcriptional control and each gene is expressed with a characteristic periodicity in the cell cycle. Third, flagellin gene promoters contain conserved nucleotide sequence elements at -13, -24, and -100 that are homologous to the fla genes in the hook gene cluster. The -13 and -24 sequences conform to a fla gene promoter consensus sequence (C/TTGGCC/GC-N5-TTGC) that is similar in sequence to the -12, -24 consensus sequence of the Klebsiella pneumonia nif gene promoters. Fourth, the sequence element at approximately -100 in the 25- and the 27-kDa flagellin genes is homologous to a 19-base-pair sequence [designated previously as II-1; see Chen, L.-S., Mullin, D. M. & Newton, A. (1986) Proc. Natl. Acad. Sci. USA 83, 2860-2864]at -101 in the promoter of transcription unit II of the hook gene cluster; the two flagellin genes, like the fla genes examined in the hook gene cluster that contain the -100 element, are under positive control by transcription unit III of the hook gene cluster. This result supports a model in which the timing of fla gene transcription in the C. crescentus cell cycle is determined in part by a cascade of trans-acting regulatory gene products.
Ribonucleotide-induced helical alteration in DNA prevents nucleosome formation.
Authors: Hovatter K R , Martinson H G
Published: May 2006

Several polynucleotides that assume an A-form helical structure in solution are unable to form nucleosomes. We attempted to establish a relationship between the ease of the A-form----B-form helix transition and ease of nucleosome formation by reconstituting nucleosomes using ribosubstituted DNA containing various levels of ribonucleotides. Instead we discovered that, when riboadenosine is substituted for deoxyriboadenosine, even one ribonucleotide per 125 base pairs of DNA reduces nucleosome formation and that DNA containing greater than 5% ribonucleotide is completely unable to form nucleosomes. Ribosubstituted DNA restriction fragments exhibited altered mobility on native 6% polyacrylamide gels, indicating an altered helical structure (probably bending). The effects on both nucleosome formation and gel mobility are nucleotide specific and are correlated, being greatest for riboadenosine and decreasing in the order riboadenosine greater than riboguanosine greater than ribocytosine. The results are consistent with the hypothesis that the rate of nucleosome formation can be drastically reduced by isolated local perturbations, such as kinking or bending, in the helical structure of DNA.
Hydrocarbon formation in the reductive cleavage of hydroperoxides by cytochrome P-450.
Authors: Vaz A D , Coon M J
Published: May 2006

Evidence is presented that cytochrome P-450 catalyzes the reductive cleavage of hydroperoxides. For example, in a reconstituted system containing rabbit liver microsomal P-450 form 2, NADPH-cytochrome P-450 reductase, and NADPH, cumyl hydroperoxide yields acetophenone and methane, but no cumyl alcohol is formed. The stoichiometry of the reaction and similar results with alpha-methylbenzyl, benzyl, and t-butyl hydroperoxides are in accord with the following general equation, in which X represents an alkyl group and R and R' are either alkyl groups or hydrogen atoms in the starting peroxide: XRR'C-OOH + NADPH + H+----XRCO + R'H + H2O + NADP+. Because 13-hydroperoxy-9,11-octadecadienoic acid yields pentane under these conditions, we propose that the known formation of alkanes and aldehydes in membrane lipid peroxidation involves reductive cleavage by P-450 to give the products predicted by the above equation. The cleavage reaction is thought to involve stepwise one-electron transfer, resulting in homolysis of the peroxide oxygen-oxygen bond and generation of an alkoxy radical, with beta-scission of the latter followed by reduction of the secondary radical to the hydrocarbon. In accordance with this scheme, when the cleavage reaction with cumyl hydroperoxide was done in 2H2O, deuteromethane was formed.
Expression of a set of growth-related immediate early genes in BALB/c 3T3 cells: coordinate regulation with c-fos or c-myc.
Authors: Lau L F , Nathans D
Published: May 2006

We have previously identified by cDNA cloning 5 mRNAs that appear in resting BALB/c 3T3 cells soon after growth stimulation by serum or platelet-derived growth factor. Five additional mRNAs of this class are described in this report. The mRNAs reached peak levels between 40 and 120 min after serum addition and rapidly decayed thereafter. All 10 RNAs were superinduced in the presence of cycloheximide. Nuclear run-on experiments indicated that the increase in the mRNAs is the result of rapid transcriptional activation of their genes on stimulation by serum or platelet-derived growth factor. Superinducibility by cycloheximide is due to two effects: prolonged transcription and stabilization of mRNAs. This overall pattern of regulation is similar to that of the c-fos or c-myc protooncogenes reported previously. We hypothesize that these newly identified "immediate early" genes play a role in the proliferative response induced by growth factors.
Prokaryotic and eukaryotic RNA polymerases have homologous core subunits.
Authors: Sweetser D , Nonet M , Young R A
Published: May 2006

Eukaryotic RNA polymerases are complex aggregates whose component subunits are functionally ill-defined. The gene that encodes the 140,000-dalton subunit of Saccharomyces cerevisiae RNA polymerase II was isolated and studied in detail to obtain clues to the protein's function. This gene, RPB2, exists in a single copy in the haploid genome. Disruption of the gene is lethal to the yeast cell. RPB2 encodes a protein of 138,750 daltons, which contains sequences implicated in binding purine nucleotides and zinc ions and exhibits striking sequence homology with the beta subunit of Escherichia coli RNA polymerase. These observations suggest that the yeast and the E. coli subunit have similar roles in RNA synthesis, as the beta subunit contains binding sites for nucleotide substrates and a portion of the catalytic site for RNA synthesis. The subunit homologies reported here, and those observed previously with the largest RNA polymerase subunit, indicate that components of the prokaryotic RNA polymerase "core" enzyme have counterparts in eukaryotic RNA polymerases.
Depolarization-induced Ca2+ increase in isolated neurosecretory nerve terminals measured with fura-2.
Authors: Brethes D , Dayanithi G , Letellier L , Nordmann J J
Published: May 2006

The free Ca2+ concentration in isolated rat neurohypophysial nerve endings was measured using the Ca2+ indicator fura-2. Depolarization with high K, veratridine, or electrical stimulation induced an increase in intracellular Ca2+ concentration that was abolished by agents known to block voltage-sensitive Ca channels. Electrical stimulation of the isolated nerve endings with a pulse pattern similar to that recorded in vivo from the hypothalamic magnocellular neurons showed that the increase in intracellular Ca2+ concentration was not only a function of the applied frequency but also of the duration of the silent interburst intervals. The relationship between the cytoplasmic free Ca concentration and the release of neuropeptides is discussed.
Nucleolin, the major nucleolar protein of growing eukaryotic cells: an unusual protein structure revealed by the nucleotide sequence.
Authors: Lapeyre B , Bourbon H , Amalric F
Published: May 2006

Nucleolin (also called C23) is the major nucleolar protein of exponentially growing eukaryotic cells. It is found associated with intranucleolar chromatin and preribosomal particles. Through use of a polyclonal antiserum, nucleolin cDNA clones were isolated from a Chinese hamster ovary cell library constructed in the expression vector lambda gt11. The isolated cDNAs encoded a polypeptide containing 679 residues of the 713 amino acids of nucleolin. The amino acid sequence presents several unusual features: in particular, repetitive sequences are found at both ends of the molecule. A repeat, Hy-Thr-Pro-Hy-Lys-Lys-Hy-Hy, in which Hy is a nonpolar residue, is found six times in the NH2-end proximal portion, followed by three acidic stretches containing 25, 25, and 33 glutamic acid or aspartic acid residues. Four potential phosphorylation sites (serines) are also observed in this region. The COOH-terminal proximal portion of the protein carries a glycine-rich region with fairly regularly interspersed phenylalanine and dimethylarginine residues. The two terminal portions of the molecule exhibit unique potential secondary structures: alpha-helix (NH2 terminus) and extended (COOH terminus). The central region exhibits alternating hydrophobic and hydrophilic stretches. Five potential N glycosylation sites are detected. The structure of this protein may reflect two functions in preribosome biogenesis: interaction with chromatin (NH2 terminus) and with preribosomes (COOH terminus).
Distribution of calcium and potassium in presynaptic nerve terminals from cerebellar cortex.
Authors: Andrews S B , Leapman R D , Landis D M , Reese T S
Published: May 2006

The elemental composition of the presynaptic nerve terminals in rapidly frozen synapses of the cerebellar molecular layer was determined by electron probe x-ray microanalysis and elemental imaging of characteristic x-rays. Elemental imaging of thin freeze-dried cryosections from fresh cerebellar slices frozen within 20 sec of removal from the brain showed normal concentrations of potassium (95 +/- 6 mmol/liter wet tissue +/- SEM) and calcium (0.8 +/- 0.4 mmol/liter) in whole presynaptic terminals, even though mitochondrial and nonmitochondrial sites containing up to 30 mmol of calcium per liter were present elsewhere in the neuropil. Quantitative electron probe analysis of synaptic vesicle clusters and intraterminal mitochondria indicated that their calcium concentrations were 0.4 +/- 0.1 and 1.2 +/- 0.2 mmol/liter, respectively. The low calcium content of presynaptic organelles was confirmed by the absence of detectable deposits in preparations freeze-substituted so as to stabilize calcium content. Similar experiments were carried out on cerebellar slices rapidly frozen after incubation in vitro. The distribution of potassium and calcium in presynaptic terminals of resting and depolarized (55 mM potassium) slices was qualitatively and quantitatively similar to that in freshly excised cortex, although resting slices lacked the few calcium-rich sites that appeared in other areas of the neuropil after stimulation. The calcium concentrations in whole terminals, synaptic vesicles, and mitochondria of resting slices were 1.4 +/- 0.7, 0.7 +/- 0.2, and 0.9 +/- 0.2 mmol/liter, respectively. Thus, amounts of calcium typical of storage organelles in other tissues are not present within cerebellar synaptic vesicles, suggesting that they have a limited role in calcium storage and release.
Herpes simplex virus-infected cells contain a function(s) that destabilizes both host and viral mRNAs.
Authors: Kwong A D , Frenkel N
Published: May 2006

The herpes simplex virus virion contains a function that mediates the shutoff of host-protein synthesis and the degradation of host mRNA. Viral mutants affected in this function (vhs mutants) have previously been derived. Cells infected with these mutants exhibit a more stable synthesis of host as well as the immediate early (alpha)-viral proteins. We now show that a function associated with purified virions of the wild-type virus reduces the half-life of host and alpha mRNAs, whereas purified vhs-1 mutant virions lack this activity. The functional half-life of many early (beta)- and late (gamma)-viral mRNAs is also prolonged in mutant virus infections. These studies suggest that the wild-type virion brings into cells a function that indiscriminately reduces the half-life of both host and viral transcripts and that the early translational shutoff of the host is a consequence of this function. This function may facilitate rapid transitions in the expression of groups of genes that are transcriptionally turned on at different times after infection.
Graft-vs.-host disease elicits expression of class I and class II histocompatibility antigens and the presence of scattered T lymphocytes in rat central nervous system.
Authors: Hickey W F , Kimura H
Published: May 2006

In the central nervous system (CNS) of healthy animals, T lymphocytes and cellular expression of major histocompatibility complex (MHC) gene products are virtually undetectable. Yet, in CNS immunological diseases, such as multiple sclerosis in humans, these constituents of the immune response must appear by some mechanism. Immunohistochemical examination of the CNS of F1 hybrid rats following induction of graft-vs.-host disease by parental lymphocytes revealed extensive parenchymal and vascular expression of host class I and II (Ia) MHC-encoded cell surface molecules. In addition, occasional scattered T lymphocytes were detected in the CNS of these animals. F1 hybrid rats reconstituted during the neonatal period with bone marrow cells from one parental strain also expressed increased levels of MHC antigens in the CNS. Thus, evidence is presented that the "immunological privilege" of the CNS seems to decrease or disappear during a strong systemic immune response such as graft-vs.-host disease. These findings may have important implications concerning the mechanism of induction of human CNS immunological diseases.
Detection of three protein binding sites in the serum-regulated promoter of the human gene encoding the 70-kDa heat shock protein.
Authors: Wu B J , Williams G T , Morimoto R I
Published: May 2006

The basal promoter of the human gene encoding 70-kDa heat shock protein (HSP70) controls maximal transcriptional activity and serum-regulated expression. We demonstrate that the three promoter elements defined by in vivo studies--CCAAT, serum-regulated element (SRE), and "TATA"--correspond to protein-binding sites in vitro. The promoter interactions with protein factors in HeLa cell crude nuclear extracts were detected by an exonuclease III digestion assay. The sequence specificity was demonstrated with promoter probes containing wild-type sequences or unique linker-scanner mutations that alter each of the elements. We suggest that the protein factor binding to the SRE is involved in the serum-regulated expression of the human gene for HSP70.
Retroviral-mediated transfer of genomic globin genes leads to regulated production of RNA and protein.
Authors: Karlsson S , Papayannopoulou T , Schweiger S G , Stamatoyannopoulos G , Nienhuis A W
Published: May 2006

A high-titer amphotropic retroviral vector containing the neomycin resistance gene and a hybrid gamma-beta genomic human globin gene has been constructed. Mouse erythroleukemia cells infected with this virus were found to contain the full transcriptional unit of the transferred human globin gene by Southern blot analysis. These cells contain normally initiated, spliced, and terminated human globin mRNA. The human globin mRNA level increased 5- to 10-fold upon induction of the mouse erythroleukemia cells. Human globin chains were produced but only in a fraction of the cells as detected by immunofluorescent staining. A similar retrovirus containing a human beta-globin gene was used to transduce mouse erythroleukemia cells resulting in much higher levels of human globin synthesis than detected in mouse erythroleukemia cells transduced with the gamma-beta globin virus.
Adult B-cell repertoire is biased toward two heavy-chain variable-region genes that rearrange frequently in fetal pre-B cells.
Authors: Lawler A M , Lin P S , Gearhart P J
Published: May 2006

Fetal pre-B cells rearrange a very restricted set of immunoglobulin variable genes for the heavy chain (VH). To determine whether the adult B-cell repertoire is similarly skewed, we first identified the genes that rearrange in pre-B cells from BALB/c mice and then determined their frequency of rearrangement in adult B cells. In fetal pre-B cell lines, two genes, VH81X from the 7183 subfamily and VHOx2 from the Q52 subfamily, comprise 75% of the rearranged alleles of an estimated 1000 genes (P less than 0.001). Sequencing analyses revealed that rearrangements involving the two genes were both productive and nonproductive. The biased rearrangement of these two VH genes persists in B-cell hybridomas from adult mice at a frequency of 22%, as determined by Southern gel analysis and RNA sequencing. The sequence of one VHOx2 rearrangement from a hybridoma shows that the rearrangement is productive, suggesting that the gene encodes an antibody that could participate in the immune response. The data indicate that the adult B-cell repertoire is not random concerning usage of individual VH genes, and it may be shaped by the unknown mechanisms that cause preferential rearrangement of certain genes early in ontogeny.
Bacteriopheophytin
g
: Properties and some speculations on a possible primary role for bacteriochlorophylls
b
and
g
in the biosynthesis of chlorophylls
Authors: Michalski T. J. , Hunt J. E. , Bowman M. K. , Smith U. , Bardeen K. , Gest H. , Norris J. R. , Katz J. J.
Published: May 2006

Bacteriopheophytin
g
and small amounts of bacteriochlorophyll
g
have been obtained in high purity from the recently discovered photosynthetic bacterium
Heliobacterium chlorum
. Preparative methods and precautions in handling these sensitive compounds are described. The compounds have been characterized by californium-252 plasma desorption mass spectrometry, HPLC, visible absorption, and electron spin resonance spectroscopy. Our results agree with the structure of bacteriochlorophyll
g
advanced by H. Brockmann and A. Lipinski [(1983)
Arch. Microbiol
. 136, 17-19], with the exception that we find the esterifying alcohol to be farnesol and not geranylgeraniol as originally suggested. Zero field splitting parameters of triplet state bacteriopheophytin
g
and the ESR properties of the cation free radical of bacteriochlorophyll
g
are reported. The photoisomerization of the subject compounds has been studied. Bacteriopheophytin
g
undergoes photo-isomerization in white light to pheophytin
a
with a half-time of ≈42 min. We suggest that all of the chlorophylls are biosynthesized from a common intermediate containing an ethylidine group, [unk]CH—CH
3
, such as is present in bacteriochlorophylls
b
and
g
.
Hepatitis B virus (HBV) particles are produced in a cell culture system by transient expression of transfected HBV DNA.
Authors: Yaginuma K , Shirakata Y , Kobayashi M , Koike K
Published: May 2006

An in vitro system for the production of hepatitis B virus (HBV) particles was established by the transient expression of transfected HBV DNA using a human hepatocellular carcinoma cell, HuH-7, as a recipient. The 3.6- and 2.2-kilobase transcripts observed were similar to those in virus-infected liver cells. Both transcripts revealed the microheterogeneity of their 5' ends. The formation of virus-related particles subsequent to the RNA transcription was demonstrated. The core particles observed in the cytoplasm and the virus particles secreted in the culture medium contained the replicative intermediates of HBV DNA and banded at densities of 1.35-1.36 g/cm3 and 1.22-1.24 g/cm3, respectively. Furthermore, the in vitro mutagenesis of the template HBV DNA demonstrated that the P gene as well as the C gene products were essential for the production of HBV particles.
cDNA sequence of human transforming gene hst and identification of the coding sequence required for transforming activity.
Authors: Taira M , Yoshida T , Miyagawa K , Sakamoto H , Terada M , Sugimura T
Published: May 2006

The hst gene was originally identified as a transforming gene in DNAs from human stomach cancers and from a noncancerous portion of stomach mucosa by DNA-mediated transfection assay using NIH3T3 cells. cDNA clones of hst were isolated from the cDNA library constructed from poly(A)+ RNA of a secondary transformant induced by the DNA from a stomach cancer. The sequence analysis of the hst cDNA revealed the presence of two open reading frames. When this cDNA was inserted into an expression vector containing the simian virus 40 promoter, it efficiently induced the transformation of NIH3T3 cells upon transfection. It was found that one of the reading frames, which coded for 206 amino acids, was responsible for the transforming activity.
Expression of a full-length cDNA for the human "MDR1" gene confers resistance to colchicine, doxorubicin, and vinblastine.
Authors: Ueda K , Cardarelli C , Gottesman M M , Pastan I
Published: May 2006

Intrinsic and acquired multidrug resistance (MDR) is an important problem in cancer therapy. MDR in human KB carcinoma cells selected for resistance to colchicine, vinblastine, or doxorubicin (former generic name adriamycin) is associated with overexpression of the "MDR1" gene, which encodes P-glycoprotein. We previously have isolated an overlapping set of cDNA clones for the human MDR1 gene from multidrug-resistant KB cells. Here we report the construction of a full-length cDNA for the human MDR1 gene and show that this reconstructed cDNA, when inserted into a retroviral expression vector containing the long terminal repeats of Moloney leukemia virus or Harvey sarcoma virus, functions in mouse NIH 3T3 and human KB cells to confer the complete multidrug-resistance phenotype. These results suggest that the human MDR1 gene may be used as a positive selectable marker to introduce genes into human cells and to transform human cells to multidrug resistance without introducing nonhuman antigens.
Nuclear matrix proteins reflect cell type of origin in cultured human cells.
Authors: Fey E G , Penman S
Published: May 2006

The low abundance proteins of the nuclear matrix (NM) were separated from the intermediate filament (IF) proteins and analyzed by two-dimensional gel electrophoresis. Three human breast carcinoma lines had virtually identical patterns of 37 NM proteins. In contrast, cell lines derived from diverse tissues had qualitatively different NM protein patterns. Together, the five cell types examined here had a total of 205 distinguishable NM proteins with 125 of these proteins unique to a single cell type. The remaining NM proteins were shared among cell types to different degrees. Polyclonal antisera, obtained by immunization with total NM proteins as antigens, preferentially stained the nuclear interior and not the exterior IF. These observations suggest that the NM proteins, localized to the interior of the nucleus, vary in a cell-type-specific manner.
Identification and distribution of three serologically undetected alleles of HLA-DR by oligonucleotide.DNA typing analysis.
Authors: Tiercy J M , Gorski J , Jeannet M , Mach B
Published: May 2006

Recent progress in the molecular biology of human major histocompatibility complex class II genes (HLA-DP, -DQ, -DR) have shown that the genetic complexity and allelic polymorphism are greater than expected. In the case of HLA-DR, three DR beta-chain loci have been identified and linked, two of which (DR beta I and DR beta III, now assigned names HLA-DRIB and HLA-DR3B) are functional. We have shown that the HLA micropolymorphism detected at the DNA sequence level can easily be analyzed by hybridization with allele-specific oligonucleotides (HLA "oligotyping"). In the case of the HLA DRw52 supertypic specificity, which includes the DR3, DR5, DRw6, and DRw8 haplotypes, three alleles, referred to as DRw52a, DRw52b, and DRw52c, have recently been identified at the HLA-DR3B locus by DNA sequencing. Hybridization with locus- and allele-specific oligonucleotide probes (designated 52a, 52b, and 52c) has been performed on DNA from normal individuals forming a panel of 82 haplotypes to establish the distribution of these three alleles. Individuals of the DR3 haplotype had either the DRw52a or DRw52b allele, and individuals of extended haplotype HLA-A1,B8,DR3 had only the DRw52a allele. DR5 individuals all had the DRw52b allele, while individuals of DRw6 haplotype had the DRw52a, -52b, or -52c allele. None of these three alelles are found in DRw8 individuals. Analysis of this micropolymorphism, undetectable by common typing procedures, is therefore now operational for more accurate HLA matching for transplantation and for improving correlations between HLA and disease susceptibility.
Molecular cloning and amino acid sequence of human 5-lipoxygenase.
Authors: Matsumoto T , Funk C D , Rådmark O , Höög J O , Jörnvall H , Samuelsson B
Published: May 2006

5-Lipoxygenase (EC 1.13.11.34), a Ca2+-and ATP-requiring enzyme, catalyzes the first two steps in the biosynthesis of the peptidoleukotrienes and the chemotactic factor leukotriene B4. A cDNA clone corresponding to 5-lipoxygenase was isolated from a human lung lambda gt11 expression library by immunoscreening with a polyclonal antibody. Additional clones from a human placenta lambda gt11 cDNA library were obtained by plaque hybridization with the 32P-labeled lung cDNA clone. Sequence data obtained from several overlapping clones indicate that the composite cDNAs contain the complete coding region for the enzyme. From the deduced primary structure, 5-lipoxygenase encodes a 673 amino acid protein with a calculated molecular weight of 77,839. Direct analysis of the native protein and its proteolytic fragments confirmed the deduced composition, the amino-terminal amino acid sequence, and the structure of many internal segments. 5-Lipoxygenase has no apparent sequence homology with leukotriene A4 hydrolase or Ca2+ -binding proteins. RNA blot analysis indicated substantial amounts of an mRNA species of approximately equal to 2700 nucleotides in leukocytes, lung, and placenta.
Alternative transcription and splicing of the human porphobilinogen deaminase gene result either in tissue-specific or in housekeeping expression.
Authors: Chretien S , Dubart A , Beaupain D , Raich N , Grandchamp B , Rosa J , Goossens M , Romeo P H
Published: May 2006

Porphobilinogen deaminase [PBGD; porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8] is a cytosolic enzyme involved in the heme biosynthetic pathway. Two isoforms of PBGD, encoded by two mRNAs differing solely in their 5' end, are known: one is found in all cells and the other is present only in erythroid cells. We have previously shown that the human PBGD is encoded by a single gene and have now cloned and characterized this gene, which is split into 15 exons spread over 10 kilobases of DNA. We demonstrate that the two mRNAs arise from two overlapping transcription units. The first one (upstream) is active in all tissues and its promoter has some of the structural features of a housekeeping promoter; the second, located 3 kilobases downstream, is active only in erythroid cells and its promoter displays structural homologies with the beta-globin gene promoters.
Separation of positional isomers of oligosaccharides and glycopeptides by high-performance anion-exchange chromatography with pulsed amperometric detection.
Authors: Hardy M R , Townsend R R
Published: May 2006

High-performance anion-exchange (HPAE) chromatography under alkaline conditions (pH congruent to 13) has been found to efficiently separate neutral oligosaccharides (triose to undecaose) according to molecular size, sugar composition, and linkage of monosaccharide units. The method was able to resolve 1----3, 1----4, and 1----6 positional isomers of neutral oligosaccharides, which are defined as having the same number, type, sequence, and anomeric configurations of monosaccharides but differing in the linkage position of a single sugar. From correlating structural features of different oligosaccharides and retention times, we deduced that at least two factors are operative to determine the superior resolution of oligosaccharides by this type of chromatography: (i) the relative acidities of the hydroxyl groups and (ii) the accessibility of oxyanions of the oligosaccharides to the functional groups of the stationary phase. Splitting of peaks attributable to mutarotation was not observed. Reducing oligosaccharides were much more retained than their reduced counterparts. Linkage of Fuc(alpha 1-3) to GlcNAc of oligosaccharides markedly decreased retention times. Positional isomers of two branched monosaccharides, which differed by 1----6 and 1----4 linkages, were widely separated. The separation of 1----3 and 1----4 positional isomers of both tetrasaccharides and glycopeptides containing undecasaccharides demonstrated the significant improvement in resolution of HPAE compared to previous chromatographic methods by either reverse-phase or amine-bonded stationary phases. Picomole quantities of underivatized oligosaccharides have been detected by triple-pulse amperometric detection, which produced similar responses for a wide range of structures. Quantification of two triantennary glycopeptides from bovine fetuin by using either detector response or 1H NMR was comparable. The N-glycanase-catalyzed release of two 1----4 and 1----3 positional isomers of an undecasaccharide from a tryptic glycopeptide of bovine fetuin could be observed and quantified by direct injection of the enzyme mixture into the chromatograph.
The alpha and beta chains of avian retrovirus reverse transcriptase independently expressed in Escherichia coli: characterization of enzymatic activities.
Authors: Soltis D A , Skalka A M
Published: May 2006

Reverse transcriptase of the avian sarcoma and leukosis retroviruses is a heterodimer composed of a 63-kDa alpha and a 95-kDa beta polypeptide chain, both of which are encoded in the pol gene and are produced by proteolytic processing of a larger precursor. We previously constructed a bacterial expression clone of the entire pol coding region that produces a protein 4 kDa larger than the mature viral beta subunit. By use of this clone and synthetic oligonucleotides to introduce stop codons, two derivatives have been constructed: one that directs synthesis of a protein equivalent to the mature beta subunit and the other that directs synthesis of a protein equivalent to alpha subunit. Predicted amino acid sequences of these proteins differ from their viral counterparts only by an initiator methionine that was added to the N termini for expression in Escherichia coli. Both bacterially expressed proteins exhibit reverse transcriptase activity and appear to function as homodimers. The properties of these proteins resemble those of the viral reverse transcriptase heterodimer; however, the bacterially produced alpha dimer protein could be distinguished from the other proteins by its increased sensitivity to heat inactivation, which also has been reported for the corresponding viral product. These results show that correct folding and expression of enzymatic function does not require formation of a precursor. The alpha and beta clones provide a convenient source of individual pol gene products for further evaluation of their roles in the synthesis and integration of retroviral DNA.
Mechanochemical coupling in synthetic polypeptides by modulation of an inverse temperature transition.
Authors: Urry D W , Haynes B , Zhang H , Harris R D , Prasad K U
Published: May 2006

For the polypentapeptide of elastin, (L-Val-L-Pro-Gly-L-Val-Gly)n, and appropriate analogs when suitably cross-linked, it has been previously demonstrated that development of elastomeric force at fixed length and length changes at fixed load occur as the result of an inverse temperature transition, with the temperature of the transition being inversely dependent on the hydrophobicity of the polypeptide. This suggests that at fixed temperature a chemical means of reversibly changing the hydrophobicity could be used for mechanochemical coupling. Evidence for this mechanism of mechanochemical coupling is given here with a 4%-Glu-polypentapeptide, in which the valine in position 4 is replaced in 1 out of 5 pentamers by a glutamic acid residue. Before cross-linking, the temperature for aggregation of 4%-Glu-polypentapeptide is remarkably sensitive to pH, shifting from 25 degrees C at pH 2 to 70 degrees C at pH 7.4 in phosphate-buffered saline (PBS). At 37 degrees C, the cross-linked 4%-Glu-polypentapeptide matrix in PBS undergoes a pH-modulated contraction and relaxation with a change from pH 4.3 to 3.3 and back. The mean distance between carboxylates at pH 4.3 in the elastomeric matrix is greater than 40 A, twice the mean distance between negatively charged species in PBS. Accordingly, charge-charge repulsion is expected to make little or no contribution to the coupling. Mechanochemical coupling is demonstrated at fixed load by monitoring pH dependence of length and at constant length by monitoring pH dependence of force. To our knowledge, this is the first demonstration of mechanochemical coupling in a synthetic polypeptide and the first system to provide a test of the recent proposal that chemical modulation of an inverse temperature transition can be a mechanism for mechanochemical coupling. It is suggested that phosphorylation and dephosphorylation may modulate structure and forces in proteins by locally shifting the temperatures of inverse temperature transitions.
Granulocyte colony-stimulating factor enhances interleukin 3-dependent proliferation of multipotential hemopoietic progenitors.
Authors: Ikebuchi K , Clark S C , Ihle J N , Souza L M , Ogawa M
Published: May 2006

In cultures of spleen cells from normal mice, recombinant human granulocyte colony-stimulating factor (G-CSF) supported the formation of multipotential blast cell colonies. Serial replating of the blast cell colonies in the presence of G-CSF, however, failed to demonstrate any direct effect of G-CSF on murine multipotential progenitors. We therefore examined the effects of G-CSF in combination with murine interleukin 3 on proliferation of murine blast cell colony-forming cells. The time course of total colony formation and multilineage colony formation by spleen cells harvested from mice 4 days after injection of 5-fluorouracil at 150 mg/kg was significantly shortened in cultures containing both factors in contrast with cultures supported by either factor alone. Serial observations of individual multipotential blast cell colonies (mapping) revealed that blast cell colonies emerged at random time intervals in the presence of interleukin 3 or G-CSF. The appearance of blast cell colonies, however, was significantly hastened in cultures containing both factors relative to cultures grown with either factor. In cultures of day-2 post-5-fluorouracil bone marrow cells, G-CSF in concentrations as low as 1 unit/ml revealed synergism with interleukin 3 in supporting the proliferation of multipotential progenitors. This synergistic activity may explain the previous in vivo studies suggesting the effects of G-CSF on apparent multipotential stem cells.
Time for acquiring a new gene by duplication.
Authors: Ohta T
Published: May 2006

In view of the widespread occurrence of gene families in eukaryotic genomes that suggests the importance of gene duplication in evolution, a population genetic model incorporating unequal crossing-over was formulated. By using this model, the time needed for acquiring a new gene is investigated by an approximate analytical method and by computer simulations. The model assumes that natural selection favors those chromosomes with more beneficial genes than other chromosomes in the population, as well as random genetic drift, mutation, and unequal crossing-over. Starting from a single gene copy, it is found that the time for acquiring another gene with a new function is dependent on the rates of occurrence of unequal crossing-over and mutation. Within a realistic range of parameter values, the required time was at least several times 4N generations, where N is the effective population size. Interchromosomal unequal crossing-over at meiosis is more effective than intrachromosomal (between sister chromatids) unequal crossing-over for obtaining a new gene, provided that other parameters are the same. However, the genetic load for acquiring a gene is larger under the model of interchromosomal crossing-over. The relevance of this finding to the advantage of sexual reproduction is discussed.
Magnesium ion-dependent triple-helix structure formed by homopurine-homopyrimidine sequences in supercoiled plasmid DNA.
Authors: Kohwi Y , Kohwi-Shigematsu T
Published: May 2006

DNA can be chemically cleaved at the site of chloroacetaldehyde-modified residues by the chemicals used for Maxam-Gilbert sequencing reactions. Use of this technique facilitates fine structural analysis of unpaired DNA bases in DNA with non-B-DNA structure. This method was used to study the non-B-DNA structure adopted by the poly-(dG).poly(dC) sequence under torsional stress at various ionic conditions. In the presence of 2 mM Mg2+, the 5' half of the deoxycytosine tract is very reactive to chloroacetaldehyde, while the 3' half is virtually unreactive. In the poly(dG) tract, chloroacetaldehyde reaction is restricted to the center guanine residues. In the absence of Mg2+, however, it is the 5' half of the deoxyguanine tract that is reactive to chloroacetaldehyde, while the 3' half is unreactive. And chloroacetaldehyde reaction is restricted to the center cytosine residues in the poly(dC) stretch. These results strongly suggest that the poly(dG).poly(dC) sequence is folded into halves from the center of the sequence to form a tetra-stranded-like structure. Such a structure contains either a triplex consisting of poly(dG).poly(dG).poly(dC) strands in the presence of Mg2+ or a triplex consisting of poly(dC).poly(dG).poly(dC) strands in the absence of Mg2+. The fourth strand, not involved in triplex formation, is closely associated with the triplex and is positioned in such a way that DNA bases are exposed and freely accessible to the chloroacetaldehyde reaction.
Mechanisms for light-dependent regulation of ribulose-1,5-bisphosphate carboxylase activity and photosynthesis in intact leaves
Authors: Kobza John , Seemann Jeffrey R.
Published: May 2006

The mechanisms involved in the
in vivo
light-dependent regulation of ribulose-1,5-bisphosphate (Rbu
P
2
) carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] activity in intact leaves were studied. In the three species examined,
Phaseolus vulgaris, Beta vulgaris
, and
Spinacea oleracea
, the regulated level of Rbu
P
2
carboxylase activity (assayed
in vitro
with saturating substrate) was highly correlated (
r
= 0.96) with the rate of net CO
2
uptake of the corresponding leaves measured over a wide range of photosynthetic photon flux density (PPFD). However, the mechanisms by which the enzyme was regulated differed between these species. In
Phaseolus
, the inhibitor 2-carboxyarabinitol 1-phosphate (CA
P
) accounted for all of the PPFD-dependent regulation of Rbu
P
2
carboxylase activity. A similar compound was detected in
Beta
, and changes in its concentration accounted for about half of the PPFD-dependent regulation of enzyme activity in this species. No CA
P
was detected in
Spinacea
, but evidence we obtained suggests that a different inhibitor (possibly Rbu
P
2
) accounts for a significant portion of the PPFD-dependent regulation of enzyme activity in this species. Changes in the activation state of the enzyme were observed with
Beta
and
Spinacea
, while in
Phaseolus
the enzyme was apparently fully activated at all PPFD levels. These results indicate that plant species may differ markedly in the mechanisms they use to regulate Rbu
P
2
carboxylase activity as PPFD changes. The results also suggest that tight binding inhibitors are a more widespread mechanism for regulation of this enzyme than previously thought. Furthermore, the results establish the importance of such inhibitors in regulating both the activity of Rbu
P
2
carboxylase and whole leaf photosynthesis over a range of PPFD.
Regulated expression of genes inserted at the human chromosomal beta-globin locus by homologous recombination.
Authors: Nandi A K , Roginski R S , Gregg R G , Smithies O , Skoultchi A I
Published: May 2006

We have examined the effect of the site of integration on the expression of cloned genes introduced into cultured erythroid cells. Smithies et al. [Smithies, O., Gregg, R.G., Boggs, S.S., Koralewski, M.A. & Kucherlapati, R.S. (1985) Nature (London) 317, 230-234] reported the targeted integration of DNA into the human beta-globin locus on chromosome 11 in a mouse erythroleukemia-human cell hybrid. These hybrid cells can undergo erythroid differentiation leading to greatly increased mouse and human beta-globin synthesis. By transfection of these hybrid cells with a plasmid carrying a modified human beta-globin gene and a foreign gene composed of the coding sequence of the bacterial neomycin-resistance gene linked to simian virus 40 transcription signals (SVneo), cells were obtained in which the two genes are integrated at the beta-globin locus on human chromosome 11 or at random sites. When we examined the response of the integrated genes to cell differentiation, we found that the genes inserted at the beta-globin locus were induced during differentiation, whereas randomly positioned copies were not induced. Even the foreign SVneo gene was inducible when it had been integrated at the beta-globin locus. The results show that genes introduced at the beta-globin locus acquire some of the regulatory properties of globin genes during erythroid differentiation.
Primary structure of the human follistatin precursor and its genomic organization.
Authors: Shimasaki S , Koga M , Esch F , Cooksey K , Mercado M , Koba A , Ueno N , Ying S Y , Ling N , Guillemin R
Published: May 2006

Follistatin is a single-chain gonadal protein that specifically inhibits follicle-stimulating hormone release. By use of the recently characterized porcine follistatin cDNA as a probe to screen a human testis cDNA library and a genomic library, the structure of the complete human follistatin precursor as well as its genomic organization have been determined. Three of eight cDNA clones that were sequenced predicted a precursor with 344 amino acids, whereas the remaining five cDNA clones encoded a 317 amino acid precursor, resulting from alternative splicing of the precursor mRNA. Mature follistatins contain four contiguous domains that are encoded by precisely separated exons; three of the domains are highly similar to each other, as well as to human epidermal growth factor and human pancreatic secretory trypsin inhibitor. The genomic organization of the human follistatin is similar to that of the human epidermal growth factor gene and thus supports the notion of exon shuffling during evolution.
The gene encoding multidrug resistance is induced and expressed at high levels during pregnancy in the secretory epithelium of the uterus.
Authors: Arceci R J , Croop J M , Horwitz S B , Housman D
Published: May 2006

A survey of the expression of the multidrug-resistance gene (mdr) in mouse tissues revealed that a mdr mRNA species is expressed at extremely high levels in the gravid uterus. mdr mRNA expression levels increase dramatically during pregnancy compared to the relatively low levels of expression observed in the nongravid uterus. In situ hybridization experiments revealed that the increased expression of the mdr mRNA is specifically localized to the secretory epithelial cells of the endometrium. Immunocytochemistry studies with a mdr glycoprotein-specific antiserum demonstrate that the mdr glycoprotein is predominantly localized to the luminal surface of the secretory epithelial cells. These results indicate that the mdr gene expression in the uterus is controlled by the physiologic changes associated with pregnancy. Our data are consistent with a potential role for the mdr glycoprotein in the transport of substrate across the secretory epithelium of the gravid uterus.
A retrovirus carrying an MDR1 cDNA confers multidrug resistance and polarized expression of P-glycoprotein in MDCK cells.
Authors: Pastan I , Gottesman M M , Ueda K , Lovelace E , Rutherford A V , Willingham M C
Published: May 2006

A full-length cDNA for the human multidrug resistance gene 1 (MDR1) has been inserted into a retroviral vector containing a murine Harvey sarcoma virus from which the viral oncogene was deleted. Ecotropic and amphotropic virus was produced after transfection of this vector into psi-2 and PA-12 packaging cell lines. This virus conferred the full phenotype of multidrug resistance on mouse and human cell lines. Viral titers of up to 2 X 10(5) drug-resistant colonies per ml were observed. Infected cells became resistant to colchicine, vinblastine, doxorubicin, VP16 (etoposide), and puromycin, but not cisplatin, indicating that the presence of the human MDR1 gene is sufficient to cause multidrug resistance. When the dog kidney cell line MDCK was infected with the MDR1 virus, P-glycoprotein was expressed in a polarized manner on the upper surface of the cells, showing that the cloned cDNA also encodes information for polarized expression of P-glycoprotein. The MDR1 virus should be useful for introducing this drug resistance gene into a variety of cell types for biological experiments in vitro and in vivo.
Immunological identification of a high molecular weight protein as a candidate for the product of the Duchenne muscular dystrophy gene.
Authors: Kao L , Krstenansky J , Mendell J , Rammohan K W , Gruenstein E
Published: May 2006

An oligopeptide was synthesized based on translation of the nucleotide sequence of the putative exon region of clone pERT87-25 from the gene for Duchenne muscular dystrophy. Immunization of rabbits with this oligopeptide induced the formation of antibodies directed against a protein present in human, rat, and rabbit skeletal muscle. This protein, which is missing in the skeletal muscle of two patients with Duchenne muscular dystrophy, has a molecular mass of approximately equal to 320-420 kDa and is clearly different from the putative Duchenne muscular dystrophy-related protein nebulin. The data suggest that this 320- to 420-kDa protein is produced by the Duchenne muscular dystrophy gene.
High levels of heterodisperse RNAs accumulate in T cells infected with human immunodeficiency virus and in normal thymocytes.
Authors: Koga Y , Lindstrom E , Fenyo E M , Wigzell H , Mak T W
Published: May 2006

Infection by human immunodeficiency virus (HIV-1) of a human T leukemia cell line (HUT 78) leads to a rapid accumulation at an elevated level (10- to 20-fold) of heterodisperse RNAs, revealed by their containing repetitive sequences. Sequence data indicate that the repetitive elements (e.g., Alu) are associated with transcripts with no apparent long open reading frame. In contrast, a large increase of such heterodisperse RNAs is not seen in T lymphocytes activated by phytohemagglutinin or in a variety of leukemic cell lines. Examination of several cellular messages showed that the levels of some of their "fully processed" transcripts are reduced late after infection of HUT 78 cells, indicating that the levels of some mRNAs may decrease. Surprisingly, similar heterodisperse transcripts are also seen in great abundance in normal fresh thymocytes. It is possible that in HIV-infected T lymphocytes, the accumulation of high levels of such aberrant RNAs may directly or indirectly contribute to the death of HIV-infected T cells.