bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of brome mosaic virus replication and aminoacylation functions by sitespecific mutagenesis
Authors: Hall Timothy C. , Marsh Loren E. , Dreher Theo W. 
Published: May 2013

ABSTRACT
Brome Mosaic Virus (BMV) has a tripartite RNA genome; each RNA and the subgenomic RNA encoding the viral coat protein share a highly homologous region of about 200 nucleotides at the 3’ end, for which a tRNA-like structure has been proposed. Several sequences encoding functions, including replicase binding, initiation of (−) strand synthesis and tyrosine esterification are known to be nested within this region. Elongation factor EF-la binds to aminoacylated viral RNAs, but not to the uncharged forms. An additional function of the tRNA-like structure is to serve as a substrate for nucleotidyl transferase, which adds the terminal adenosine residue to the (+) sense virion RNAs. A template-dependent and template-specific replicase preparation from BMV-infected barley leaves has been characterized and extensively used for replication studies in vitro that complement studies in vivo using protoplasts. The replicase has been shown to initiate de novo both (−) strand synthesis on supplied (+) strand RNAs, and (+) strand subgenomic RNA synthesis on supplied (—) sense RNA3 templates. RNA transcripts obtained by transcription in vitro of cDNA clones containing desired base substitutions and deletions, have been supplied as templates for replication, aminoacylation and other assays. Use of such mutant RNAs has allowed the promoters for both (−) strand synthesis and for synthesis of the subgenomic (+) strand RNA to be characterized and defined. The same approach has also been used to reveal regions of the tRNA-like structure involved in the tyrosylation of the BMV RNAs. These experiments showed that, although regions important in aminoacylation and replication functions overlap, they are not identical. Some of the mutations tested in vitro have also been tested for infectivity in vivo using both barley plants and protoplasts. Mutants retaining replicase and nucleotidyl transferase template activity, but having lost aminoacylation capability are of special interest in that they should reveal the role of aminoacylation in the infection process.
The requirements for a high level of transposition of bacteriophage mu
Authors: Groenen Martien A. M. , Putte Pieter Van De 
Published: May 2013

ABSTRACT
To analyse the functional and structural requirements of Mu transposition a special mini-Mu transposon was constructed. This mini-Mu, situated on a derivative of pBR322, has an easily selectable marker gene conferring chloramphenicol resistance (CamR) cloned between the ends of the mini-Mu. The genes required for transposition are cloned outside the ends and are under control of the PL promoter of bacteriophage λ.
To obtain a high frequency of transposition the A and B products are required. Under B−conditions both cointegrate formation and simple insertions are strongly reduced. The role of B in the transposition process is unknown and is discussed.
Deletion analysis of the ends of the mini-Mu has revealed the existence of multiple sites required for transposition. The same sites were found to be strong A-binding sites. In addition two weak A-binding sites were found. Point mutations introduced into these last sites show that they are at least as important for transposition as the strong binding sites. The organization of the binding sites is asymmetric in Mu. At the left end two binding sites are situated more than 100 bp away from the end. These affect specifically the frequency of transposition. The distance between these two sites and the site close to the left end is extremely critical. At the right end also three A-binding sites are found, but only two seem to be important for transposition.
Transposition with low frequency can be obtained with a mini-Mu with only one end. Secondary att sites are selected which all show some homology with an A-binding site. An absolute requirement for a 5’T at the artificial end is observed. Based on these results a simple scheme for the evolution of a transposon is discussed.
Structure and replication of geminivirus genomes
Authors: Davies J. W. , Stanley J. , Donson J. , Mullineaux P. M. , Boulton M. I. 
Published: May 2013

ABSTRACT
The geminiviruses are a group of plant viruses containing single-stranded (ss) DNA in particles comprising two quasi-icosahedral units. Some are transmitted by whiteflies, others by leafhoppers. Comparisons were made of the genome organization and expression of cassava latent virus (CLV) and maize streak virus (MSV) and beet curly top virus (BCTV), each with distinct host range and insect vector species characteristics. From these studies, several indications as to the replication mechanism(s) are suggested.
Cell Behaviour During Active Cell Rearrangement: Evidence and Speculations
Authors: Keller Ray , Hardin Jeff 
Published: May 2013

ABSTRACT
The cell behaviour and motility underlying cell rearrangement during gastrulation in amphibian and sea-urchin embryos are discussed. In particular, the cell behaviour of deep (non-epithelial) and epithelial cell populations that undergo cell rearrangement is compared and contrasted. Deep cell rearrangement in Xenopus laevis involves both convergence of cells towards the future dorsal midline and simultaneous axial extension of the mesodermal cell mass. Time-lapse cinemicrography and scanning electron microscopy suggest that asynchronous, repetitive motions of individual deep cells, involving local extensions and retractions of their margins, may provide the motive force for rearrangement. Such protrusive activity may be guided by local differences in cell-cell contacts in the marginal zone. Epithelial cell rearrangement in the sea-urchin embryo both elongates the archenteron and simultaneously closes the blastopore. Cell rearrangement is accompanied by stage-specific changes in protrusive activity and cell shape of the basal surfaces of cells in the wall of the gut rudiment, in contrast to the apical surfaces, which show little activity. These basal protrusions may be involved in the rearrangement process.
Stem cells: a problem in asymmetry
Authors: Wolpert Lewis 
Published: May 2013

ABSTRACT
The special property of stem cells is that their development is asymmetric. They give rise both to cells that are identical to themselves and to cells that are different. The mechanism that provides this asymmetry may be intrinsic or extrinsic. Such mechanisms are considered within the context of other systems where asymmetric development occurs. The specification of mating types in yeast provides a clear example of a stem cell system generated intrinsically. In fission yeast it appears that the asymmetry is due to chromosomal differences: this is the only known mechanism for intrinsic asymmetry. While there is good evidence for intrinsic asymmetry in both plants and invertebrates - particularly the nematode - the mechanism is not known. In insects and vertebrates there is no well established example of intrinsic asymmetry if one excludes asymmetric cytoplasmic localization during cleavage of the egg. Asymmetry is thus due to environmental influences. Stem cell systems are usually well structured and the cell’s behaviour seems to be position-dependent. This is well established for the stem cells of hydra. By contrast it is claimed that the mammalian haemopoietic system is generated by an intrinsic, asymmetric, probabilistic mechanism - the validity of this view is questioned.
Marrow stromal stem cells
Authors: Owen Maureen 
Published: May 2013

ABSTRACT
Evidence for the hypothesis that there are stromal stem cells present in the soft connective tissues associated with marrow and bone surfaces that are able to give rise to a number of different cell lines is reviewed. The lines are currently designated fibroblastic, reticular, adipocytic and osteogenic. Fibroblastic colonies, each derived from a single colony-forming unit fibroblastic (CFU-F), are formed when marrow cells are cultured in vitro. In vivo assays of tissue formed by CFU-F in open transplant or in diffusion chambers, have demonstrated that some CFU-F have a high ability for self renewal and multipotentiality whereas some have more limited potential. Preliminary investigations in vitro also support the hypothesis and have shown that CFU-F are a heterogeneous population of stem and progenitor cells and that their differentiation in vitro can be modified at the colony level. The stromal cells which survive and proliferate in vitro are highly dependent on culture conditions. The number and hierarchy of cell lines belonging to the stromal fibroblastic system are not yet fully elucidated and more specific markers and better assays for the different phenotypes are required before a greater understanding can be achieved. The possibility that the marrow stromal system is part of a wider stromal cell system of the body is proposed.
Differentiation of a bipotential glial progenitor cell: what controls the timing and the choice of developmental pathway?
Authors: Raff Martin C. , Lillien Laura E. 
Published: May 2013

ABSTRACT
In the rat central nervous system (CNS) oligodendrocytes and type-2 astrocytes are thought to develop from a common precursor - the O-2A progenitor cell. Oligodendrocytes develop first and make myelin; type-2 astrocytes develop later and extend processes to nodes of Ranvier. The timing of differentiation of O-2A progenitor cells seems to depend on chemical signals secreted by another type of glial cell - the type-1 astrocyte. Type-1 astrocytes secrete platelet-derived growth factor (PDGF), which stimulates O-2A progenitor cell proliferation and drives the clock that controls the onset of oligodendrocyte differentiation, which is the constitutive pathway of progenitor cell development. Later, type-1 astrocytes are thought to secrete a CNTF-like protein that initiates type-2 astrocyte differentiation.
Epidermal stem cells in culture
Authors: Watt Fiona M. 
Published: May 2013

ABSTRACT
Cultures of human epidermal keratinocytes retain many of the characteristics of the tissue from which they are derived, and are therefore useful as an experimental model for studying stem cell properties. The cultures provide evidence that the mechanisms regulating keratinocyte proliferation are complex. The dividing cell compartment is heterogeneous, containing some cells with stem cell characteristics and others that may correspond to transit amplifying cells. The overall growth rate of the cultures is influenced by a wide range of growth stimulatory and inhibitory molecules, but it is not clear whether responsiveness to these factors differs between the different subpopulations of dividing cells. Furthermore, keratinocytes are able to express molecules that regulate their own growth. The cultures are able to achieve at least a partial homeostatic balance between proliferation and terminal differentiation and can regenerate the suprabasal cell layers following injury. Finally, a range of new experimental approaches hold promise for future studies of how keratinocyte proliferation and terminal differentiation are controlled.
Targeting of oncoproteins to membranes by fatty acylation
Authors: Magee Anthony I. , Gutierrez Lourdes , Marshall Christopher J. , Hancock John F. 
Published: May 2013

ABSTRACT
Post-translational modification of proteins with hydrophobic lipid-derived substituents is increasingly becoming recognized as a major route for targeting proteins to membranes. Glycosylphosphatidylinositol (GPI) anchors are found at the C terminus of a wide range of cell surface proteins, and may endow the cell with the ability to release them in a controlled fashion via specific phospholipases. We have concentrated on the direct attachment (acylation) of long-chain fatty acids (myristate, C14:0 and palmitate, C16:0) to proteins associated with the cytoplasmic face of cellular membranes. Two such proteins, the products of the src and ras oncogenes, require acylation respectively with myristate and palmitate for their membrane association and biological activity, including transformation. N-terminal myristoylation of p60ras seems to be a co- translational stable modification. However, our recent results show that post-translational modification of p21ras is a complex cascade of events involving proteolysis, methylation and thioesterification of palmitate. This last acylation event is dynamic in vivo and may regulate ras function. Enzymological studies of these modification events are in progress. A better understanding of acylation may provide targets for future pharmacological intervention.
Protein sorting in yeast: the role of the vacuolar protontranslocating ATPase
Authors: Kane Patricia M. , Yamashiro Carl T. , Rothman Joel H. , Stevens Tom H. 
Published: May 2013

ABSTRACT
We are investigating the physiological roles of organelle acidification in yeast by two different approaches. First, we have identified two mutants which are defective in acidification of the yeast lysosome-like vacuole from among a collection of mutants which mis-sort soluble vacuolar proteins to the cell surface. These mutants have been helpful in identifying other cellular functions linked to acidification, such as the activation of vacuolar zymogens. We have complemented this classical genetic approach to acidification with direct biochemical and reverse genetic studies on the yeast vacuolar proton-translocating ATPase (H+-ATPase), the enzyme responsible for vacuolar network acidification. Our biochemical characterization of this enzyme indicates that it is a multisubunit complex with many structural similarities to other vacuolar H+-ATPases. Like the other vacuolar H+-ATPases characterized, it also shares some structural features with the FiF0-type ATPases of mitochondria, chloroplasts, and Escherichia coli. We are currently cloning the genes for the subunits of the yeast vacuolar H+-ATPase. Mutagenesis of the cloned genes will allow us to determine the phenotype of yeast cells expressing a vacuolar H+-ATPase altered in well controlled ways. We are also beginning to investigate how the subunits of the vacuolar H+-ATPase are assembled into the enzyme complex and targeted to their proper cellular location.
Towards an in vitro system for studying clathrin-coated pit function
Authors: Moore Mary Shannon , Anderson Richard G. W. 
Published: May 2013

ABSTRACT
To elucidate the molecular basis of coated pit function, we are developing a method for reconstituting the endocytic cycle in a cell-free system. Using purified membranes attached to a solid substratum, we have successfully reconstituted coated pit assembly. We now show that the endogenous coated pits on these membranes spontaneously round up and leave the membrane at 37 °C. As measured by radioimmunoassay, clathrin loss is both pH- and temperature-dependent. The loss is also inhibited by pretreating the membranes with apyrase at 37 °C, which suggests that nucleotide triphosphates are required.
Import of proteins into the various submitochondrial compartments
Authors: Hartl Franz-Ulrich , Neupert Walter 
Published: May 2013

ABSTRACT
Import of proteins into mitochondria can be subdivided into several distinct steps. (1) Mitochondrial proteins are synthesized on free ribosomes and are released into cytosolic pools. Nucleoside triphosphates are required to keep precursors in a conformation competent for import.
(2) Precursors are directed to mitochondria by specific targeting signals (in most cases contained in N-terminal presequences) and by binding to receptors on the surface of the outer membrane.
(3) Precursors interact with a component in the outer membrane which is believed to facilitate membrane insertion (‘general insertion protein’). (4) Outer membrane proteins are then directly routed to their final location. Proteins of all other submitochondrial compartments are directed into translocation contact sites between outer and inner membranes. Transfer into contact sites is dependent on the membrane potential (ΔΨ) across the inner membrane. (5) Presequences of precursors are cleaved in the matrix by the mitochondrial processing peptidase in cooperation with the processing enhancing protein. (6) Precursors of the intermembrane space or the outer surface of the inner membrane have to be re-translocated back across the inner membrane (‘conservative sorting’).
Cytochrome c is an exception to this general import pathway. The precursor, apocytochrome c, is directly translocated across the outer membrane into the intermembrane space in a process independent of ΔΨ.
Cellular and viral control of the initiation of DNA replication
Authors: Roberts James M. , D’urso Gennaro 
Published: May 2013

ABSTRACT
Cell-free replication of SV40 DNA in extracts prepared from S phase cells is at least 20-fold more efficient than in extracts from G1 cells. The increased activity of S phase extracts correlates with the presence of an S phase-specific cellular factor that enhances DNA unwinding at the replication origin. This change in origin-DNA structure during the initiation of SV40 replication proceeds through at least three discrete steps which can be distinguished by their extent of topologic unwinding (linking differences of —1, —2 and —5). Specific DNA elements flanking the core origin enhance replication in vivo and facilitate the formation of the pre-initiation complexes, indicating that formation of these underwound conformations may be the limiting step in the initiation of DNA synthesis. In addition, the factor that activates DNA replication in extracts from S phase cells also enhances the formation of the most highly underwound —5 pre-initiation complex. These observations suggest that during SV40 replication, formation of the rate-limiting pre-initiation complex is the focus of at least three regulatory elements. Two of these are DNA sequences flanking the replication origin and the third is a cellular factor specific to the S phase cell.
DNA replication and cell cycle control in Xenopus egg extracts
Authors: Hutchison C. J. , Brill D. , Cox R. , Gilbert J. , Kill I. , Ford C. C. 
Published: May 2013

ABSTRACT
Aspects of the regulation of DNA replication and mitosis have been studied using a cell-free extract of Xenopus eggs. The extract is characterized by repeated cycles of DNA replication and mitosis, which are accompanied by periodic synthesis and degradation of cyclins as well as fluctuations in the level of Histone Hl kinase activity. DNA replication in this system is dependent upon the formation of a nucleus. However, while nuclear structures are clearly required for initiation, a complete nuclear membrane does not appear to be necessary. Indirect immunofluorescence and DIC microscopy indicate that nuclear reformation from chromosomes occurs asynchronously around individual chromatids. Lamin polymerization, biotin-11-dUTP incorporation and association of polymerases with chromatin occur before membrane formation is complete. S phase nuclei are typified by the co-distribution of both anti-DNA polymerase a and anti-PCNA antibodies as discrete spots of fluorescence which align the chromatin. However, as DNA replication is terminated, PCNA fluorescence fades and DNA polymerase " dissociates from the chromatin and is redistributed throughout the nucleoplasm. By inhibiting DNA replication with aphidicolin, both DNA polymerase a and PCNA remain associated with the chromatin throughout prolonged incubation. Under these conditions mitosis is delayed by up to 70 min, although both the general rate of protein synthesis and more importantly the rate of cyclin synthesis and histone kinase activation are unaffected. Upon nuclear envelope breakdown and lamin dispersal, cyclins degrade; however, no chromosomes are formed, and both PCNA and DNA polymerase a remain associated with the chromatin. Also, histone kinase activity is maintained at elevated levels.
NSP1, a yeast protein located at the nuclear periphery, is required for the cell cycle of Saccharomyces cerevisiae
Authors: Hurt E. C. 
Published: May 2013

ABSTRACT
Proteins located at the nuclear periphery and required for nuclear and cell division are not known in yeast. We recently identified a novel nucleoskeletal-like protein in Saccharomyces cerevisiae, which is in close proximity to the spindle pole body and the nuclear membrane. NSP1 has an essential function, since haploid yeast cells with a defective NSPJ gene cannot grow. In this report, we demonstrate that yeast cells which carry the NSP1 gene under the regulatory GALIO promoter exhibit abnormal cell morphology both under conditions where the level of NSP1 expression is greatly increased (the GALIO promoter is induced) or limited (the GALIO promoter is not induced). This result suggests that NSP1 is required for the coordinated cell cycle in S. cerevisiae.
Temporal regulation of cdc2 mitotic kinase activity and cyclin degradation in cell-free extracts of Xenopus eggs
Authors: Felix Marie-Anne , Pines Jonathan , Hunt Tim , Karsenti Eric 
Published: May 2013

ABSTRACT
In cleaving Xenopus eggs, the cell division cycle is abbreviated to a rapid succession of S and M phases. During mitosis a number of proteins show increased phosphorylation due to the activation of a histone Hl kinase, the homologue of the cdc2+ gene product of the yeast Schizosaccharomyces pombe. We have studied the regulation of the activity of this enzyme in cell-free extracts of Xenopus eggs. In extracts of activated eggs incubated at 22°C, histone Hl kinase activity shows two peaks of activation and disappearance. Activation occurs in two stages. The first stage requires protein synthesis, whereas the second does not. The second stage of activation involves post-translational activation of the kinase. Kinase activity rises to a peak and then abruptly disappears. Added sea urchin cyclin is degraded at the time of disappearance of kinase activity. The oscillation in kinase activity is then repeated, usually with lower amplitude.
Post-translational activation of the kinase requires a membrane-containing particulate cellular component, whose role has yet to be defined. The kinase can still be activated in the presence of EDTA or in the presence of the ATP analogue, 6-dimethylaminopurine, which implies that phosphorylation of the kinase complex is not required for activation. Under these conditions, however, the kinase activity does not show its normal sudden disappearance, and added cyclin is perfectly stable. These observations are consistent with the idea that post-translational activation of the kinase involves protein phosphatase activity, whereas switching off the kinase requires an ATP-Mg2+-dependent reaction, perhaps due to protein phosphorylation.