bluuebunny/crossref_metadata_2025 huggingface dataset:
A proposed mechanism for the self-splicing of proteins.
Authors: Clarke N D
Published: May 2006

Intervening protein sequences, called inteins, are intronlike elements that are removed posttranslationally, apparently by self-splicing. The conserved and essential residues of precursor proteins consist of an asparagine as the last residue of the intein and a hydroxyl- or thiol-containing residue immediately following both splice junctions. Evidence for a branched intermediate has been reported [Xu, M.-Q., Southworth, M., Mersha, F., Hornstra, L. & Perler, F. (1993) Cell 75, 1371-1377]; however, the chemical nature of the branched structure is unclear. I propose a mechanism that includes the formation of a branched structure, provides an explanation for the reversal of branch formation observed at high pH, and accounts for each of the essential amino acids. The branched structure is formed by nucleophilic attack of the asparagine side chain on the N-terminal splice junction. The nature of this branched structure is a distinguishing feature of the model and can be experimentally tested.
A fast random cost algorithm for physical mapping.
Authors: Wang Y , Prade R A , Griffith J , Timberlake W E , Arnold J
Published: May 2006

Ordering clones from a genomic library into physical maps of whole chromosomes presents a central computational/statistical problem in genetics. Here we present a physical mapping algorithm for creating ordered genomic libraries or contig maps by using a random cost approach [Berg, A. (1993) Nature (London) 361, 708-710]. This random cost algorithm is 5-10 times faster than existing physical mapping algorithms and has optimization performance comparable to existing procedures. The speedup in the algorithm makes practical the widespread use of bootstrap resampling to assess the statistical reliability of links in the physical map as well as the use of more elaborate physical mapping criteria to improve map quality. The random cost algorithm is illustrated by its application in assembling a physical map of chromosome IV from the filamentous fungus Aspergillus nidulans.
A Drosophila gene promoter is subject to glucose repression in yeast cells.
Authors: Hickey D A , Benkel K I , Fong Y , Benkel B F
Published: May 2006

Previous work has shown that the alpha-amylase gene of Drosophila melanogaster is subject to repression by dietary glucose. Moreover, glucose repression of this gene is mediated by promoter elements that lie upstream of the transcriptional start site. In this study, we examined the activity of the glucose-repressible Drosophila promoter in transformed yeast cells. We show that the amylase promoter region can mediate glucose repression of a heterologous reporter gene in yeast. The implication of this result is that the yeast regulatory machinery can recognize the Drosophila promoter signals. This, in turn, implies an unexpectedly high degree of evolutionary conservation in the mechanism of glucose repression among eukaryotes. It also shows that genes that have acquired complex patterns of developmental regulation-e.g., the Drosophila amylase gene, can still retain, intact, more primitive forms of regulation, such as glucose repression.
Centrally active modulators of glutamate receptors facilitate the induction of long-term potentiation in vivo.
Authors: Stäubli U , Perez Y , Xu F B , Rogers G , Ingvar M , Stone-Elander S , Lynch G
Published: May 2006

An experimental drug, 1-(1,3-benzodioxol-5-ylcarbonyl)piperidine, that facilitates glutamatergic transmission in brain after systemic administration was tested for its effects on the induction of long-term potentiation in the hippocampus of rats. Intraperitoneal injections of the drug markedly increased the degree and duration of long-term potentiation; similar results were obtained with an analogue of 1-(1,3-benzodioxol-5-ylcarbonyl)piperidine that was also found to improve retention of memory in a radial maze task and in an odor-matching problem. These results define tools for enhancing long-term potentiation in vivo and confirm an important prediction from the hypothesis that long-term potentiation is a substrate of memory.
Three discrete classes of Arabidopsis cyclins are expressed during different intervals of the cell cycle.
Authors: Ferreira P , Hemerly A , de Almeida Engler J , Bergounioux C , Burssens S , Van Montagu M , Engler G , Inzé D
Published: May 2006

We have isolated cDNAs encoding four additional mitotic-like cyclins from Arabidopsis: cyc2aAt, cyc2bAt, cyc3aAt, and cyc3bAt. Examination of amino acid sequences deduced from plant cyclin cDNAs isolated so far showed that they can be grouped into three distinct classes. The members of each plant cyclin family are more related to each other than to any animal or yeast cyclin. Reverse transcription-PCR analysis demonstrated that cyc2aAt was expressed in all plant organs, whereas cyc2bAt mRNAs were found only in roots; cyc3aAt was not expressed in leaves and was barely expressed in flowers. On the other hand, cyc3bAt transcripts were observed in all organs. Whole-mount in situ hybridizations on roots showed that the cyclin mRNAs were confined to parts of the roots with mitotic activity. Furthermore, results of whole-mount in situ hybridizations on roots treated with either oryzalin or hydroxyurea suggest that the different cyclin classes have distinct functions in the cell cycle.
Domains of Escherichia coli primase: functional activity of a 47-kDa N-terminal proteolytic fragment.
Authors: Sun W , Tormo J , Steitz T A , Godson G N
Published: May 2006

Endoproteinase Asp-N cleaves the 581-amino acid Escherichia coli primase (65,564 Da) into several major fragments. One of these, a 47-kDa fragment containing the complete N terminus and the first 422 amino acids of primase, is capable of primer RNA (pRNA) synthesis in the G4oric/single-stranded DNA binding protein/primase pRNA synthesis system. A cloned 398-amino acid N-terminal fragment of primase can also synthesize pRNA. The sizes of the pRNA synthesized by these N-terminal fragments, however, are smaller than those synthesized by intact primase, suggesting that the C-terminal region of primase plays a role in processivity or regulation of pRNA synthesis. Primase mutants with the last 10 and 40 C-terminal amino acids deleted synthesize pRNA as wild-type primase, indicating that any regulatory sequences must be internal to the C terminus of primase.
Folding of apominimyoglobin.
Authors: De Sanctis G , Ascoli F , Brunori M
Published: May 2006

The acid unfolding pathway of apominimyoglobin (apo-mini-Mb), a 108-aa fragment (aa 32-139) of horse heart apomyoglobin has been studied by means of circular dichroism, in comparison with the native apoprotein. Similar to sperm whale apomyoglobin [Hughson, F. M., Wright, P. E. & Baldwin, R. L. (1990) Science 249, 1544-1548], a partly folded intermediate (alpha-helical content approximately 35%) is populated at pH 4.2 for horse heart apomyoglobin. For this intermediate, Hughson et al. proposed a structural model with a compact subdomain involving tertiary interactions between the folded A, G, and H helices, with the remainder of the protein essentially unfolded. As described in this paper, a folding intermediate with an alpha-helical content of approximately 33% is populated at pH 4.3-5.0 also in apo-mini-Mb. The acid unfolding pathway is similarly affected in both the native and the mini apoprotein by 15% trifluoroethanol, a helix-stabilizing compound. Thus, the folding of the apo-mini-Mb intermediate is similar to that observed for the native apoprotein, in spite of the absence in the miniprotein of the A helix and of a large part of the H helix, which are crucial for the stability of apo-Mb intermediate. Our results suggest that acquisition of a folded state in apo-mini-Mb occurs through an alternative pathway, which may or may not be shared also by apo-Mb.
The genes COL4A5 and COL4A6, coding for basement membrane collagen chains alpha 5(IV) and alpha 6(IV), are located head-to-head in close proximity on human chromosome Xq22 and COL4A6 is transcribed from two alternative promoters.
Authors: Sugimoto M , Oohashi T , Ninomiya Y
Published: May 2006

The genes for the alpha 5(IV) and alpha 6(IV) chains of human basement membrane collagen type IV have been found together on chromosome X at segment q22 and have been reported to be arranged in a head-to-head fashion. Here we report the 5' flanking sequences of COL4A5 and COL4A6 and that COL4A6 is transcribed from two alternative promoters in a tissue-specific fashion. Analysis of the sequence immediately upstream of the transcription start sites revealed some features of housekeeping genes--i.e., the lack of a TATA motif and the presence of CCAAT and CTC boxes. Further analysis revealed that COL4A6 contains two alternative promoters that control the generation of two different transcripts. One transcription start site (from exon 1') is 442 bp away from the transcription start site of COL4A5, while an alternative transcription start site (from exon 1) is located 1050 bp from the first one and drives the expression of a second transcript that encodes an alpha 6(IV) chain with a different signal peptide. Reverse transcription-PCR experiments revealed that the transcript from exon 1' is abundant in placenta, whereas the transcript from exon 1 is more frequently found in kidney and lung. These results provide additional clues to answering the general question of what mechanisms are used to generate unique basement membrane structures in different tissues.
Identification of genes that interact with the sina gene in Drosophila eye development.
Authors: Carthew R W , Neufeld T P , Rubin G M
Published: May 2006

The sina gene encodes a nuclear protein that is required for the correct development of R7 photoreceptor cells in the Drosophila eye. We conducted a genetic screen for mutations that reduce the activity of sina and found mutations that define nine genes whose products may be required for normal sina activity. Three of these genes also appear to be essential for signaling by the Sevenless-Ras pathway in R7 cells, of which one gene corresponds to the rolled locus (rl). The rl gene is known to encode a mitogen-activated protein kinase necessary for signaling by Ras. These results suggest that the products of these three genes may participate in a signaling pathway involving both Ras and Sina, possibly by functionally linking these two proteins.
Voltage window for sustained elevation of cytosolic calcium in smooth muscle cells.
Authors: Fleischmann B K , Murray R K , Kotlikoff M I
Published: May 2006

Action potentials activate voltage-dependent calcium channels and attendant increases in cytosolic calcium concentration ([Ca2+]i) in many excitable cells. The role of these channels in the regulation of [Ca2+]i in nonspiking cells that do not depolarize to membrane potentials sufficient to activate a substantial fraction of the available current is less clear. Measurements of the peak activation and steady-state inactivation of L-type calcium currents have predicted the existence of a noninactivating current window over a voltage range where channel inactivation is incomplete. The degree to which such small currents might regulate [Ca2+]i, however, has not been established. Here we demonstrate a "calcium window" in nondialyzed, quiescent smooth muscle cells over a small voltage range near the resting membrane potential. Sustained depolarizations in this voltage range, but not to more positive potentials, resulted in sustained rises in calcium, despite the fact that macroscopic inward currents were < 2 pA. The calcium window corresponded well with the predicted window current determined under the same conditions; the peak of the calcium window occurred at -30 mV, with steady-state rises in [Ca2+]i in some cells at -50 mV. Steady-state rises in [Ca2+]i following depolarization were completely blocked by nisoldipine and were augmented and shifted to more negative potentials by BAY K8644. Voltage-dependent calcium channels thus regulate steady-state calcium levels in nonspiking cells over a voltage range where macroscopic currents are only barely detectable. This voltage range is bounded at negative potentials by calcium channel activation and at more positive potentials by channel inactivation.
Three-dimensional structure of the platelet integrin recognition segment of the fibrinogen gamma chain obtained by carrier protein-driven crystallization.
Authors: Donahue J P , Patel H , Anderson W F , Hawiger J
Published: May 2006

We have developed a method for crystallizing small functional protein segments so that their three-dimensional structure can be determined by x-ray diffraction analysis. This method consists of linking a small protein segment of unknown tertiary structure to either the amino or carboxyl terminus of a larger carrier protein of known tertiary structure. Crystallization of the small segment is then driven by crystallization of the carrier protein. Using this approach, we have obtained crystals of the human fibrinogen gamma-chain carboxyl-terminal segment linked to the carboxyl terminus of chicken egg white lysozyme. The three-dimensional structure of the carboxyl-terminal segment of the fibrinogen gamma chain was determined by x-ray diffraction analysis at a resolution of 2.4 A. This segment encompasses the recognition site for the integrin alpha IIb beta 3 receptor on activated platelets and for the clumping receptor on pathogenic staphylococci and also bears donor and acceptor sites for factor XIIIa-catalyzed crosslinking of fibrin. Therefore, the structural information derived from our analysis will provide a rational basis for the design of inhibitors of these important functions of fibrinogen. Moreover, carrier protein-driven crystallization will facilitate the determination of the three-dimensional structure of functional segments of other proteins that are, like fibrinogen, difficult to crystallize in toto.
In vivo transfer and expression of a human epidermal growth factor gene accelerates wound repair.
Authors: Andree C , Swain W F , Page C P , Macklin M D , Slama J , Hatzis D , Eriksson E
Published: May 2006

This report details the transfer of a human epidermal growth factor (hEGF) expression plasmid to porcine partial-thickness wound keratinocytes by particle-mediated DNA transfer (Accell). After gene transfer an external sealed fluid-filled wound chamber was used to protect the wound, provide containment of the exogenous DNA and expressed peptide, and permit sampling of the wound fluid. Analysis of wound fluid for hEGF and total protein, an indicator of reformation of the epithelial barrier, showed that wounds bombarded with the hEGF plasmid exhibited a 190-fold increase in EGF concentration and healed 20% (2.1 days) earlier than the controls. EGF concentrations in wound fluid persisted over the entire 10-day monitored period, decreasing from 200 pg/ml to 25 pg/ml over the first 5 days. Polymerase chain reaction results showed that plasmid DNA was present in the wound for at least 30 days. These findings demonstrate the possible utility of in vivo gene transfer to enhance epidermal repair.
Motility voltage sensor of the outer hair cell resides within the lateral plasma membrane.
Authors: Huang G , Santos-Sacchi J
Published: May 2006

The outer hair cell (OHC) from the organ of Corti is believed to be responsible for the mammal's exquisite sense of hearing. A membrane-based motile response of this cell underlies the initial processing of acoustic energy. The voltage-dependent capacitance of the OHC, possibly reflecting charge movement of the motility voltage sensor, was measured in cells during intracellular dialysis of trypsin under whole cell voltage clamp. Within 10 min after dialysis, light and electron microscopic examination revealed that the subplasmalemmal structures, including the cytoskeletal framework and subsurface cisternae, were disrupted and/or detached from adjacent plasma membrane. Dialysis of heat-inactivated trypsin produced no changes in cell structure. Simultaneous measures of linear and nonlinear membrane capacitance revealed minimal changes, indicating that contributions by subsurface structures to the generation of the nonlinear capacitance are unlikely. This study strongly suggests that voltage-dependent charge movement in the OHC reflects properties of the force generator's voltage sensor and that the sensor/motor resides solely within the lateral plasma membrane.
Replication of the genomic RNA of a positive-strand RNA animal virus from negative-sense transcripts.
Authors: Ball L A
Published: May 2006

Studies of RNA replication among the positive-strand RNA animal viruses have been hindered by the apparent inability of their RNA-dependent RNA polymerases to initiate replication on the corresponding negative-sense RNAs. However, here I report that in the case of the nodavirus flock house virus (FHV), which has a bipartite positive-sense RNA genome, the viral RNA replicase can replicate a negative-sense transcript of the genome segment that encodes the viral capsid proteins. For this work, the FHV replication cycle was experimentally reconstructed in baby hamster kidney cells that were transfected with specialized transcription plasmids designed to direct the synthesis of RNAs which corresponded closely to the two genome segments of FHV. The RNA replicase encoded by the larger genome segment could utilize either the positive or the negative strand of the smaller segment as a template, and it catalyzed RNA replication to produce similar RNA products in the two situations. Surprisingly, studies of the nucleotide sequences that were required for replication showed that the 3' end of the negative-strand RNA contained only a minimal cis-acting signal. The success of these experiments will facilitate further studies of the cis- and trans-acting factors involved in the recognition and replication of negative-sense RNA in this system.
Nuclear assembly is independent of linker histones.
Authors: Dasso M , Dimitrov S , Wolffe A P
Published: May 2006

The role of linker histones in the assembly of functional nuclei was examined with the use of a cell-free extract of Xenopus eggs that transforms condensed sperm chromatin into DNA-replication-competent pronuclei. When linker histones were removed from the extract, the resultant pronuclei were indistinguishable from those formed in the complete extract. The assembly of functional nuclear membrane, nuclear lamina, and prereplication centers allowed identical DNA replication efficiencies. Thus, linker histones are not required for the assembly of morphologically normal nuclei capable of DNA replication.
Hereditary eosinophil peroxidase deficiency: immunochemical and spectroscopic studies and evidence for a compound heterozygosity of the defect.
Authors: Romano M , Patriarca P , Melo C , Baralle F E , Dri P
Published: May 2006

Hereditary eosinophil peroxidase (EPO; EC 1.11.1.7) deficiency is a rare abnormality of eosinophil granulocytes characterized by decreased or absent peroxidase activity and decreased volume of the granule matrix. The molecular basis of the defect is not known. We report here its molecular characterization in an EPO-deficient subject and his family members. The EPO-deficient eosinophils contained EPO-related material as determined immunochemically using either monoclonal or polyclonal anti-EPO antibodies but had no spectroscopic evidence of EPO. Eosinophil precursors from the EPO-deficient subject contained normally sized EPO mRNA, which was reverse transcribed into the corresponding cDNA clones encompassing the whole gene. Sequencing of these clones disclosed two mutations, a G-->A transition causing a nonconservative replacement of an arginine residue with a histidine and an insertion causing a shift in the reading frame with the appearance of a premature stop codon. The two mutations were located on different chromosomes indicating a compound heterozygosity for the defect. Both the son and the daughter of the proband inherited the G-->A transition, and their eosinophils contained a peroxidase activity intermediate between that of control subjects and the proband, suggesting that the transition is a deficiency-causing mutation. Eosinophil precursors from the EPO-deficient subject were found to actively synthesize an EPO that was apparently normal in terms of cytochemical reaction for peroxidase and immunoreactivity with monoclonal and polyclonal anti-EPO antibodies, but spectroscopically abnormal. The cytochemical reaction for peroxidase tended to decrease or disappear in the eosinophil precursors of the EPO-deficient subject but not of a normal subject as differentiation went on, suggesting that the Arg-->His substitution causes the production of an unstable EPO that undergoes progressive degradation as the cells mature.
One subspecies of the red junglefowl (Gallus gallus gallus) suffices as the matriarchic ancestor of all domestic breeds.
Authors: Fumihito A , Miyake T , Sumi S , Takada M , Ohno S , Kondo N
Published: May 2006

The noncoding control region of the mitochondrial DNA of various gallinaceous birds was studied with regard to its restriction fragment length polymorphism (RFLP) and sequences of the first 400 bases. Tandem duplication of the 60-base unit was established as a trait unique to the genus Gallus, which is shared neither by pheasants nor by quails. Unlike its close ally Gallus varius (green junglefowl), the red junglefowl Gallus gallus is a genetically very diverse species; the 7.0% sequence divergence was seen between those from Thailand (G. g. gallus and G. g. spadiceus) and the other from the Indonesian island of Java (G. g. Bankiva). Furthermore, the divergence increased to 27.83% if each transversion is regarded as an equivalent of 10 transitions. On the other hand, a mere 0.5-3.0% difference (all transitions) separated various domestic breeds of the chicken from two G. g. gallus of Thailand, thus indicating a single domestication event in the area inhabited by this subspecies of the red junglefowl as the origin of all domestic breeds. Only transitions separated six diverse domesticated breeds. Nevertheless, a 2.75% difference was seen between RFLP type I breeds (White Leghorn and Nagoya) and a RFLP type VIII breed (Ayam Pelung). The above data suggested that although the mitochondrion of RFLP type V was the main contributor to domestication, hens of other RFLP types also contributed to this event.
Subtiligase: a tool for semisynthesis of proteins.
Authors: Chang T K , Jackson D Y , Burnier J P , Wells J A
Published: May 2006

A variant of subtilisin BPN', which we call subtiligase, has been used to ligate esterified peptides site-specifically onto the N termini of proteins or peptides in aqueous solution and in high yield. We have produced biotinylated or heavy-atom derivatives of methionyl-extended human growth hormone (Met-hGH) by ligating it onto synthetic peptides containing biotin or mercury. Polyethylene glycol (PEG)-modified atrial natriuretic peptide (ANP) was produced by ligating ANP onto peptides containing sites for PEG modification. We have established the N-terminal sequence requirements for efficient ligation onto proteins, using either synthetic substrates or pools of filamentous phage containing Met-hGH with random N-terminal sequences (substrate phage). To facilitate ligations involving proteins with highly structured or buried N termini, a more stable subtiligase was designed that effectively ligates peptides onto Met-hGH even in 4 M guanidine hydrochloride. The use of subtiligase should expand the possibilities for protein semisynthesis and rational protein design.
Growth-phase-dependent synthesis of histones in the archaeon Methanothermus fervidus.
Authors: Sandman K , Grayling R A , Dobrinski B , Lurz R , Reeve J N
Published: May 2006

Histone preparations from Methanothermus fervidus (HMf) contain two small polypeptides, HMfA and HMfB, which in solution are dimers and compact DNA to form nucleosome-like structures. These archaeal nucleosome-like structures constrain positive DNA supercoils, in contrast to the negatively supercoiled DNA in eukaryal nucleosomes. HMfA has been found to make up as much as 80% of HMf preparations synthesized by M. fervidus cells during the exponential growth phase of batch cultures but to decrease to approximately 50% as cultures enter the stationary phase. By using a nondenaturing polyacrylamide gel system at pH 6.1, we have demonstrated that HMf preparations contain HMfA homodimers, HMfB homodimers, and HMfA-HMfB heterodimers and that heating a mixture of recombinant HMfA and HMfB homodimers at 95 degrees C for 5 min generates HMfA-HMfB heterodimers. Circular dichroism spectroscopy indicates that HMfA and HMfB have very similar secondary structures, but based on agarose gel electrophoretic mobility shifts, DNA topology assays, and electron microscopy, they have different DNA binding properties. HMfA binding to DNA could be detected at lower protein/DNA ratios than HMfB, but HMfB binding resulted in more extensive DNA compaction. The increased HMfB synthesized in cells approaching the stationary phase and the highly compacted state of HMfB-bound DNA are consistent with preparations for the impending period of limited genome activity.
Propensity for a leucine zipper-like domain of human immunodeficiency virus type 1 gp41 to form oligomers correlates with a role in virus-induced fusion rather than assembly of the glycoprotein complex.
Authors: Wild C , Dubay J W , Greenwell T , Baird T , Oas T G , McDanal C , Hunter E , Matthews T
Published: May 2006

For a number of viruses, oligomerization is a critical component of envelope processing and surface expression. Previously, we reported that a synthetic peptide (DP-107) corresponding to the putative leucine zipper region (aa 553-590) of the transmembrane protein (gp41) of human immunodeficiency virus type 1 (HIV-1) exhibited alpha-helical secondary structure and self-associated as a coiled coil. In view of the tendency of this type of structure to mediate protein association, we speculated that this region of gp41 might play a role in HIV-1 envelope oligomerization. However, later it was shown that mutations which should disrupt the structural elements of this region of gp41 did not affect envelope processing, transport, or surface expression (assembly oligomerization). In this report we compare the effects of amino acid substitutions within this coiled-coil region on structure and function of both viral envelope proteins and the corresponding synthetic peptides. Our results establish a correlation between the destabilizing effects of amino acid substitutions on coiled-coil structure in the peptide model and phenotype of virus entry. These biological and physical biochemical studies do not support a role for the coiled-coil structure in mediating the assembly oligomerization of HIV-1 envelope but do imply that this region of gp41 plays a key role in the sequence of events associated with viral entry. We propose a functional role for the coiled-coil domain of HIV-1 gp41.
Transcriptional induction of Ty recombination in yeast.
Authors: Nevo-Caspi Y , Kupiec M
Published: May 2006

Families of repeated sequences are present in the genomes of all eukaryotes. Little is known about the mechanism(s) that prevents recombination between repeated sequences. In the yeast Saccharomyces cerevisiae, recombination between homologous sequences placed at nonhomologous locations in the genome (ectopic recombination) has been shown to occur at high frequencies for artificially created repeats, but at relatively low frequencies for a natural family of repeated sequences, the Ty family. We have previously shown that a high level of Ty cDNA in the cell causes an increase in the rate of nonreciprocal recombination (gene conversion) of a marked Ty element. In the present study, we show that it is also possible to elevate the rate of recombination of a marked Ty by increasing its transcription. This induction is different from, and acts synergistically to, the one seen upon increased levels of donor Ty cDNA. We show that the induction by transcription does not require the products of the RAD50, RAD51, and RAD57 genes. In contrast, cDNA-mediated recombination is dependent on the product of the RAD51 gene but not on products of the genes RAD50 or RAD57.
Identification of the apparent carrier in mycolic acid synthesis.
Authors: Besra G S , Sievert T , Lee R E , Slayden R A , Brennan P J , Takayama K
Published: May 2006

The mycolic acids are large (C70-90) alpha-alkyl, beta-hydroxy fatty acids and are the major determinants of the mycobacterial cell wall's impermeable barrier. The biosynthesis of mycolic acids is barely understood (they are probably the products of specialized elongation and Claisen-type condensation), and yet their synthesis is the site of action of several mainline antituberculosis drugs. We describe the isolation from Mycobacterium smegmatis and the full characterization of a 6-O-mycolyl-beta-D-mannopyranosyl-1-monophosphoryl-3,7,11,15,19,23 ,27- heptamethyl-(2Z,6E,10E)-octacosatrien-1-ol . The identification of a mycolyl-mannosylphosphopolyprenol supported by cell-free labeling experiments and earlier literature suggests unusual biochemical pathways in which mature mycolic acids are formed from beta-oxo precursors while attached to a mannosyl-P-polyprenol, in which form they are transported through the membrane prior to final deposition as arabinan-bound mycolates.
Targeting of the polyhydroxybutyrate biosynthetic pathway to the plastids of Arabidopsis thaliana results in high levels of polymer accumulation.
Authors: Nawrath C , Poirier Y , Somerville C
Published: May 2006

In the bacterium Alcaligenes eutrophus, three genes encode the enzymes necessary to catalyze the synthesis of poly[(R)-(-)-3-hydroxybutyrate] (PHB) from acetyl-CoA. In order to target these enzymes into the plastids of higher plants, the genes were modified by addition of DNA fragments encoding a pea chloroplast transit peptide, a constitutive plant promoter, and a poly(A) addition sequence. Each of the modified bacterial genes was introduced into Arabidopsis thaliana by Agrobacterium-mediated transformation, and plants containing all three genes were obtained by sexual crosses. These plants accumulated PHB up to 14% of the dry weight as 0.2- to 0.7-micron granules within plastids. In contrast to earlier experiments in which expression of the PHB biosynthetic pathway in the cytoplasm led to a deleterious effect on growth, expression of the PHB biosynthetic pathway in plastids had no obvious effect on the growth or fertility of the transgenic plants and resulted in a 100-fold increase in the amount of PHB that accumulated. We conclude that there does not appear to be any biological barrier to high-level production of PHB in higher plants. The high level of PHB accumulation also suggests that the synthesis of plastid acetyl-CoA is regulated by a mechanism which responds to metabolic demand.
The tsA58 simian virus 40 large tumor antigen disrupts megakaryocyte differentiation in transgenic mice.
Authors: Robinson M O , Zhou W , Hokom M , Danilenko D M , Hsu R Y , Atherton R E , Xu W , Mu S , Saris C J , Swift S
Published: May 2006

Thrombocytopenia is a condition of multiple etiologies affecting the megakaryocyte lineage. To perturb this lineage in transgenic mice, the tsA58 mutation of the simian virus 40 large tumor antigen was targeted to megakaryocytes using the platelet factor 4 promoter. Ten of 17 transgenic lines generated exhibited low platelet levels, each line displaying a distinct, heritable level of thrombocytopenia. Within a line, the degree of the platelet reduction correlated directly with transgene zygosity. The platelet level could be further reduced by the inactivation of one copy of the endogenous retinoblastoma gene. Western blot analysis detected large tumor antigen protein in the most severely affected lines; less affected lines were below the level of detection. Platelets and megakaryocytes from thrombocytopenic mice exhibited morphological abnormalities. Mice with either normal or reduced platelet levels developed megakaryocytic malignancies with a mean age of onset of about 8 months. There was no correlation between severity of thrombocytopenia and onset of malignancy. These mice provide a defined genetic model for thrombocytopenia, and for megakaryocytic neoplasia, and implicate the retinoblastoma protein in the process of megakaryocyte differentiation.
In vivo occupancy of the vitamin D responsive element in the osteocalcin gene supports vitamin D-dependent transcriptional upregulation in intact cells.
Authors: Breen E C , van Wijnen A J , Lian J B , Stein G S , Stein J L
Published: May 2006

The steroid hormone vitamin D is a principal mediator of skeletal homeostasis. 1,25-Dihydroxyvitamin D3 treatment of ROS 17/2.8 osteoblast-like cells results in a ligand-dependent increase in transcription of the bone-specific osteocalcin gene. This transcriptional upregulation requires the positive cis-acting vitamin D responsive element (VDRE). We have used the ligation-mediated polymerase chain reaction to demonstrate that protein occupancy of the VDRE within the intact cell correlates with increased synthesis of osteocalcin transcripts. These protein-DNA contacts were not present in the absence of vitamin D or in osteosarcoma cells (ROS 24.1) lacking the vitamin D receptor. Our results establish in intact cells the requirement for both ligand- and receptor-dependent occupancy of the VDRE for vitamin D responsive enhancement of osteocalcin gene transcription.
All four known cyclic adducts formed in DNA by the vinyl chloride metabolite chloroacetaldehyde are released by a human DNA glycosylase.
Authors: Dosanjh M K , Chenna A , Kim E , Fraenkel-Conrat H , Samson L , Singer B
Published: May 2006

We have previously reported that human cells and tissues contain a 1,N6-ethenoadenine (epsilon A) binding protein, which, through glycosylase activity, releases both 3-methyladenine (m3A) and epsilon A from DNA treated with methylating agents or the vinyl chloride metabolite chloroacetaldehyde, respectively. We now find that both the partially purified human epsilon A-binding protein and cell-free extracts containing the cloned human m3A-DNA glycosylase release all four cyclic etheno adducts--namely epsilon A, 3,N4-ethenocytosine (epsilon C), N2,3-ethenoguanine (N2,3-epsilon G), and 1,N2-ethenoguanine (1,N2-epsilon G). Base release was both time and protein concentration dependent. Both epsilon A and epsilon C were excised at similar rates, while 1,N2-epsilon G and N2,3-epsilon G were released much more slowly under identical conditions. The cleavage of glycosyl bonds of several heterocyclic adducts as well as those of simple methylated adducts by the same human glycosylase appears unusual in enzymology. This raises the question of how such a multiple, divergent activity evolved in humans and what may be its primary substrate.
Human immunodeficiency virus type 1 mRNA expression in peripheral blood cells predicts disease progression independently of the numbers of CD4+ lymphocytes.
Authors: Saksela K , Stevens C , Rubinstein P , Baltimore D
Published: May 2006

To address the significance of human immunodeficiency virus (HIV) replication in peripheral blood mononuclear cells (PBMCs), we have used reverse transcriptase-initiated PCR to measure HIV-1 mRNA expression in PBMC specimens collected from a cohort of HIV-infected individuals during a long-term prospective study. We found dramatic differences in HIV mRNA expression among individuals with very similar clinical and laboratory indices, and this variation strongly correlated with the future course of the disease. No evidence of viral replication was detected in PBMCs from asymptomatic individuals who, thereafter, had normal levels of CD4+ cells for at least 5 years. Irrespective of whether the CD4+ cell numbers were normal at the time of sampling, abundant expression of HIV-1 mRNA in PBMCs predicted accelerated disease progression within the next 2 years. Thus, independently of what may be the rate of HIV replication in other viral reservoirs, such as lymphatic tissue, the amount of HIV mRNA in PBMCs appears to reflect the subsequent development of HIV disease. We have also used the reverse transcriptase-initiated PCR assay to demonstrate a transient response to 3'-azido-3'-deoxythymidine treatment. Determination of HIV-1 mRNA expression in the PBMCs of infected individuals could, therefore, have significant clinical utility as a prognostic indicator and as a means to guiding and monitoring antiviral therapies.
Arabidopsis thaliana contains two differentially expressed 3-hydroxy-3-methylglutaryl-CoA reductase genes, which encode microsomal forms of the enzyme.
Authors: Enjuto M , Balcells L , Campos N , Caelles C , Arró M , Boronat A
Published: May 2006

The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR; EC 1.1.1.34) catalyzes the first rate-limiting step in plant isoprenoid biosynthesis. Arabidopsis thaliana contains two genes, HMG1 and HMG2, that encode HMGR. We have cloned these two genes and analyzed their structure and expression. HMG1 and HMG2 consist of four exons and three small introns that interrupt the coding sequence at equivalent positions. The two genes share sequence similarity in the coding regions but not in the 5'- or 3'-flanking regions. HMG1 mRNA is detected in all tissues, whereas the presence of HMG2 mRNA is restricted to young seedlings, roots, and inflorescences. The similarity between the two encoded proteins (HMGR1 and HMGR2) is restricted to the regions corresponding to the membrane and the catalytic domains. Arabidopsis HMGR2 represents a divergent form of the enzyme that has no counterpart among plant HMGRs characterized so far. By using a coupled in vitro transcription-translation assay, we show that both HMGR1 and HMGR2 are cotranslationally inserted into endoplasmic reticulum-derived microsomal membranes. Our results suggest that the endoplasmic reticulum is the only cell compartment for the targeting of HMGR in Arabidopsis and support the hypothesis that in higher plants the formation of mevalonate occurs solely in the cytosol.
Positive and negative selection of T cells in T-cellreceptor transgenic mice expressing a bcl-2 transgene.
Authors: Strasser A , Harris A W , von Boehmer H , Cory S
Published: May 2006

To explore the role of bcl-2 in T-cell
development, a bcl-2 transgene was introduced into mice expressing a T-cell
receptor (TCR) transgene encoding reactivity for the mouse male antigen HY
presented by the H-2Db class I antigen of the major histocompatibility complex
(MHC). Normal thymic development is contingent on the ability of immature
thymocytes to interact with self-MHC molecules presented by thymic stroma
(positive selection). Thus, thymocyte numbers are low in female anti-HY TCR
transgenic mice with a nonselecting (H-2Dd) background. Expression of bcl-2
inhibited the death of nonselectable thymocytes since, strikingly, female H-2Dd
bcl-2/TCR transgenic mice developed normal numbers of CD4+CD8+ thymocytes,
although these did not mature further into functional T cells. Hence, TCR-MHC
interaction may induce positive selection through two signals, one which saves
cells from death by increasing Bcl-2 synthesis and another which promotes
maturation. Male H-2Db anti-HY TCR transgenic mice normally have a very small
thymus, due to deletion of the self-reactive T cells. Expression of bcl-2
reduced the efficiency of deletion, since bcl-2/TCR transgenic male mice
accumulated 4- to 6-fold more thymocytes than did TCR transgenic male
littermates. Anti-HY TCR-expressing cells were also more numerous in the
peripheral lymphoid tissues, but these cells expressed abnormally low levels of
CD8 co-receptor and were not responsive to the HY antigen. Thus, although bcl-2
expression hampers the deletion of immature self-reactive cells in the thymus,
self-tolerance is maintained.
Growth hormone (GH) induces tyrosine-phosphorylatedproteins in mouse L cells that express recombinant GH receptors.
Authors: Wang X , Xu B , Souza S C , Kopchick J J
Published: May 2006

Porcine and bovine GH receptor (GHR) cDNAs were
stably expressed in mouse L cells, which normally do not possess detectable
levels of mouse GHR. Expression of the GHR cDNAs resulted in specific binding of
125I-labeled GH by these cell lines. To study GHR-related signaling events in
these cells, protein tyrosine phosphorylation was examined. In GH-treated cells,
a tyrosine-phosphorylated protein with a molecular mass of approximately 95 kDa
(pp95) was increased dramatically (approximately 100-fold) relative to
non-GH-treated cells. The amount of pp95 within the cells after GH treatment was
positively correlated with the number of GHRs on the cells. Tyrosine
phosphorylation of pp95 could not be induced by prolactin, insulin, insulin-like
growth factor I, interleukin 2, epidermal growth factor, platelet-derived growth
factor, or fibroblast growth factor. Phosphorylation of pp95 was found to be a
rapid event that could be observed 60 sec after GH treatment. Also, pp95 appears
to exist as a complex of two proteins, i.e., pp95 and pp96. The GH-induced
response by these cells may be of use in screening GH analogs for biological
activity.
A second gene for gonadotropin-releasing hormone: cDNAand expression pattern in the brain.
Authors: White S A , Bond C T , Francis R C , Kasten T L , Fernald R D , Adelman J P
Published: May 2006

In vertebrates, the gonadotropin-releasing hormone
(GnRH) decapeptide is secreted from hypothalamic nerve terminals to regulate
reproduction via control of synthesis and release of pituitary gonadotropins.
Only one GnRH peptide has been found in mammals, with one exception, although
numerous other vertebrate species express more than one of the eight known
decapeptide forms as shown by immunocytochemical labeling of distinct cell
groups in the brain. However, neither the functional nor the evolutionary
relationships among these GnRH forms are clear, because only one preprohormone
gene sequence from any species has been reported. The most ubiquitous
alternative form of GnRH is [His5,Trp7,Tyr8]GnRH (also referred to as
chicken-II), which differs from the mammalian sequence at amino acids 5, 7, and
8. This peptide has been shown to have the most potent releasing-hormone
activity, although immunocytochemical staining has suggested it is synthesized
only in the mesencephalon. Here we report the cloning and expression pattern of
the gene for the precursor of this form from the teleost fish Haplochromis
burtoni. This is the second GnRH-encoding gene to be characterized in this
species. The newly discovered preprohormone gene differs from that previously
reported in two ways. First, whereas the original gene predicts only a single
associated peptide, this one predicts two associated peptides, both of which
appear to be unique. Second, the gene for [His5,Trp7,Tyr8]GnRH is expressed in
only one cell group in the mesencephalon. In contrast, the previously reported
gene is expressed only in the terminal nerve. The striking differences between
the preprohormone structure and localization suggest that the genes coding for
the two known GnRH forms in H. burtoni did not arise from a recent duplication
event. Interestingly, neither of the two genes found to date in this species is
expressed in cells which project from the hypothalamus to the pituitary,
suggesting that yet a third gene coding for GnRH may exist.
Crystal structure of an idiotype-anti-idiotype Fabcomplex.
Authors: Ban N , Escobar C , Garcia R , Hasel K , Day J , Greenwood A , McPherson A
Published: May 2006

Anti-idiotypic monoclonal antibody 409.5.3 is
raised against an antibody that neutralizes feline infectious peritonitis virus.
This antibody, used as an immunogen, elicits the production of
anti-anti-idiotypic antibodies that in turn neutralize the virus. The crystal
structure of the complex between anti-idiotypic Fab 409.5.3 and idiotypic Fab
fragment of virus-neutralizing antibody has been solved by molecular replacement
using real-space Patterson search and filtering by Patterson
correlation-coefficient refinement. The structure has been refined to an R value
of 0.21 based on 21,310 unique reflections between 40.0 and 2.9 A. The
three-dimensional structure reveals extensive, specific interactions that
involve 118 van der Waals contacts and at least 9 probable hydrogen bonds. The
two Fabs are rotated 61 degrees with respect to each other around the
approximate long axis of the complex and are within 26 degrees being aligned
along their major axes.
Dissecting the catalytic mechanism of protein-tyrosinephosphatases.
Authors: Zhang Z Y , Wang Y , Dixon J E
Published: May 2006

Protein-tyrosine phosphatases (PTPases) contain an
evolutionarily conserved segment of 250 amino acids referred to as the PTPase
catalytic domain. The recombinant PTPase domain from Yersinia enterocolitica
enhances the rate of hydrolysis of p-nitrophenyl phosphate, a phosphate
monoester, by approximately 10(11) over the non-enzyme-catalyzed rate by water.
Specific amino acid residues responsible for the catalytic rate acceleration
have been examined by site-directed mutagenesis. Our results suggest that
Asp-356 (D356) and Glu-290 (E290) are the general acid and the general base
catalysts responsible for Yersinia PTPase-catalyzed phosphate ester hydrolysis.
The PTPase with both E290Q and D356N mutations shows no pH dependence for
catalysis but displays a rate enhancement of 2.6 x 10(6), compared to the
noncatalyzed hydrolysis of p-nitrophenyl phosphate by water. This rate
enhancement probably occurs via transition-state stabilization. Our results
suggest that all PTP-ases use a common mechanism that depends upon formation of
a thiol-phosphate intermediate and general acid-general base
catalysis.
One of two growth hormone genes in coho salmon issex-linked.
Authors: Forbes S H , Knudsen K L , North T W , Allendorf F W
Published: May 2006

Salmonid fishes have two growth hormone genes
resulting from their polyploid ancestry. We used the polymerase chain reaction
to examine genetic variation in the third intron (C) of both of these genes in
coho salmon (Oncorhynchus kisutch). A polymorphism in the length of intron C in
GH-1 is due to a variable number of copies of a 31-nt repeat that is absent from
GH-1 of the closely related chinook salmon (Oncorhynchus tshawytscha) and
rainbow trout (Oncorhynchus mykiss). Thus, this tandem repeat sequence has
become established in the genome of coho salmon since the separation of this
species from its closest relatives. All male coho salmon examined have an allele
at the second growth hormone gene, GH-2, that is not found in females. GH-2 is
thus on the sex chromosome and there is no recombination between GH-2 and the
sex-determining locus (SEX). Sequences of intron C indicate much greater
divergence between the X chromosome-specific allele and the Y
chromosome-specific allele within coho salmon than between the X
chromosome-specific alleles of coho and the closely related chinook salmon.
Thus, absence of recombination between GH-2 and SEX apparently predates
separation of these two species.