bluuebunny/crossref_metadata_2025_split huggingface dataset:
Control of G2 delay by the RAD9 gene of Saccharomyces cerevisiae
Authors: Weinert Ted , Hartwell Leland 
Published: May 2013

ABSTRACT
In response to DNA damage, Saccharomyces cerevisiae cells arrest the cell cycle in the Gz phase. Arrest is defective in rad.9 mutants; rad9 cells divide and die without repairing the damage. Several cell cycle mutants that are defective in DNA replication arrest in G2 at the restrictive temperature; this arrest is due to the RAD? control function. Thus RAD? is responsible for the fact that mitosis is normally dependent upon DNA replication, a function we term a ‘checkpoint’. Four additional genes have been identified that are also components of the RAD9 checkpoint.
On the coupling between DNA replication and mitosis
Authors: Newport John , Dasso Mary 
Published: May 2013

ABSTRACT
The rapid, early cell divisions in Xenopus laevis embryos are driven by an inflexible oscillator that is not influenced by the state of the DNA. In contrast, mitosis in somatic cells can be prevented by blocking replication or by damaging the DNA through irradiation. We have investigated the transition from the rapid, early cell cycle to the slower, more somatic-like cell cycle that occurs after division twelve in developingXenopus embryos, a stage called the mid-blastula transition (MBT). When aphidicolin, an inhibitor of DNA synthesis, was added to embryos just post-fertilization, the embryos continued to divide despite incomplete replication. Also, embryos incubated with aphidicolin from early times did not slow their cell cycles after division twelve as control embryos did, indicating a connection between the accumulation of DNA and the post-MBT timing of the cell cycle. However, incubation with hydroxyurea, an inhibitor of ribonucleotide reductase, resulted in an S phase arrest when the pools of dNTPs became depleted after division twelve. These experiments showed that the embryos had acquired the ability to arrest in S phase some time after the early divisions and before division thirteen. The acquisition of the ability to arrest in S phase did not depend upon new transcription. These experiments suggested that the number of nuclei present could be responsible for the extension of the cell cycle observed after the MBT. To investigate this, we added increasing concentrations of nuclei to an in vitro cell cycle system. We have shown that at high concentrations of nuclei the in vitro cycle is extended.
Analysis of the Cdc28 protein kinase complex by dosage suppression
Authors: Reed Steven I. , Hadwiger Jeffrey A. , Richardson Helena E. , Wittenberg Curt 
Published: May 2013

ABSTRACT
In the interest of identifying components of the Cdc28 protein kinase complex, dosage suppression analysis was performed on temperature-sensitive and dominant negative CDC28 mutations. Dosage suppression is based on a rationale in which elevated expression of wild-type genes can rescue mutations in a target gene as a result of interaction between the respective encoded proteins. Three sequences capable of rescuing a temperature sensitive cdc28 mutation were isolated from a library of wild-type genomic DNA segments in the high copy vector YEpl3. Two of these, named CLN1 and CLN2 were found to encode closely related proteins with homology to cyclins. The third, CKSl, encodes an 18K (K=103Mr) protein that has been shown to be a component of the Cdc28 protein kinase complex and is a homolog of the sucl+ product of fission yeast. A number of dosage suppressors of the CDC28-dnl dominant negative mutation have been isolated. The one analyzed to date encodes a truncated subunit of the mitochondrial enzyme succinyl-CoA synthetase. The basis for suppression in this case remains to be elucidated.
M phase-promoting factor: its identification as the M phasespecific H1 histone kinase and its activation by dephosphorylation
Authors: Dorée Marcel , Labbé Jean-Claude , Picard André 
Published: May 2013

ABSTRACT
A major protein kinase independent of Ca2+, cyclic nucleotide or diacylglycerol, the activity of which becomes maximal when cells enter M phase, decreases at ana-telophase, and is low during interphase, has been purified to near homogeneity from starfish oocytes and its catalytic subunit identified as p34cdc2. M phase-promoting factor (MPF) was found to co-purify with the M phasespecific kinase throughout its purification. p34cdc2 does not have to be associated with any specific protein for expression of Hl histone kinase or MPF activities. When p34’vfc2 is phosphorylated its protein kinase activity is inhibited, preventing entry into M phase, but once p34ede2 becomes dephosphorylated, its protein kinase activity increases and M phase is initiated. A second peak of MPF activity was separated from p34cdc2 in the ammonium sulfate fraction treated with ATP-y-S. It induced p34cde2 dephosphorylation and the concomitant stimulation of its kinase activity when injected in Xenopus or starfish oocytes.
Imaging and manipulation of cytosolic ions and messengers during cell activation
Authors: Harootunian Alec T. , Kao Joseph P. Y. , Adams Stephen R. , Tsien Roger Y. 
Published: May 2013

ABSTRACT
Optical methods have recently become available for continuously imaging the free concentrations of important ions and second messengers such as calcium, sodium and hydrogen inside living cells. These ion levels are found to undergo remarkable changes upon stimulation of quiescent cells with growth factors known to stimulate phosphoinositide breakdown. In serum-starved REF-52 fibroblasts, growth factors such as serum, vasopressin, or PDGF (platelet-derived growth factor) cause intracellular [Na+] to increase from about 4 mM to 8 mM. If mitogen treatment is combined with pharmacological depolarization of the membrane potential, repetitive [Ca2+]i spikes result in these rat fibroblasts. The mechanism of this oscillation has been investigated by light-flash release of intracellular messengers such as inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), Ca2+, and diacylglycerol, as well as more traditional biochemical techniques. The key feedback pathway appears to be Ca2+-stimulation of phospholipase C production of Ins(1,4,5)P3.
Basic fibroblast growth factor (bFGF), a multifunctional growth factor for neuroectodermal cells
Authors: Westermann Reiner , Grothe Claudia , Unsicker Klaus 
Published: May 2013

ABSTRACT
Basic fibroblast growth factor (bFGF), a heparin-binding mitogen for mesoderm-derived cells, also acts as a mitogen, differentiation inducing and maintenance factor for many neuroectodermal cells including glial cells, neurons, paraneurons, and their tumor counterparts. The molecule is expressed in several types of neuroectodermal cells in vitro and in vivo. Furthermore, bFGF occurs in many neuronal target tissues, and can prevent ontogenetic as well as lesion-induced neuron death. Thus, in terms of its wide range of functions, bFGF is apparently more than a ‘classical’ neurotrophic factor. Some of its essential features, such as regulation of expression, local availability and transport in the nervous system remain to be studied.
Growth factors as inducing agents in early Xenopus development
Authors: Slack J. M. W. 
Published: May 2013

ABSTRACT
Factors from two growth factor families have been identified as having mesoderm inducing activity. These include activin and TGFβ from the TGFβ superfamily, and all members of the fibroblast growth factor (FGF) family.
When isolated ectoderm explants are treated with any of these factors, a proportion of their cells are caused to differentiate into mesodermal tissue types instead of epidermis. There are several differences in the biological activities which can broadly be summarized by saying that activin yields dorsal type inductions and FGF ventral type inductions.
Both bFGF and an FGF receptor have been detected in Xenopus blastulae, but it has not been shown that bFGF is normally secreted from vegetal cells. Various TGF/Mike mRNAs have also been detected and it is expected that an activin-like molecule will prove to be responsible for induction of the dorsal mesoderm in vivo.
Mechanisms of positional signalling in the developing eye of Drosophila studied by ectopic expression of Sevenless and rough
Authors: Hafen Ernst , Basler Konrad 
Published: May 2013

ABSTRACT
In the developing eye of Drosophila cell fate is controlled by a cascade of inductive interactions. Little is known about how the specificity of positional signalling is achieved such that directly adjacent progenitor cells reproducibly choose distinct developmental pathways. The determination of the R7 photoreceptor in each ommatidium depends on the presence of the sevenless protein which acts as a receptor for positional information on the R7 precursor. The rough gene encodes a homeodomain protein that plays an instructive role in the determination of the R3 and R4 photoreceptor cells. The use of ectopic expression of sevenless and rough has provided insight into the mechanisms of positional signalling and the normal function of rough. Ubiquitous expression of sevenless does not alter cell fate suggesting that the inducing signal is both spatially and temporally controlled. Conversely, ectopic expression of rough in the R7 precursor causes a transformation of R7 cells into Rl-6 type cells. This indicates that rough acts, similar to other homeobox genes, as a selector gene that determines the fate of single cells.
The biochemistry and biology of the myeloid haemopoietic cell growth factors
Authors: Heyworth C. M. , Vallance S. J. , Whetton A. D. , Dexter T. M. 
Published: May 2013

ABSTRACT
In the adult, blood cell production or haemopoiesis takes place mainly in the bone marrow. The blood cell types produced are a reflection of the needs of the organism at any moment, for example bacterial infection leads to a large increase in neutrophil production. The rate and scale of blood cell production in vivo are regulated, at least in part, by the synthesis and release of specific cytokines both within the bone marrow and also from other tissues. Here we detail the range of cytokines which act directly on haemopoietic stem cells and myeloid progenitor cells. Also cellular systems which will permit the elucidation of the specific interactions between these various cytokines which regulate stem cell self-renewal, differentiation and proliferation are described.
Growth and differentiation factors of pluripotential stem cells
Authors: Heath John K. , Smith Austin G. , Hsu Li-Wei , Rathjen Peter D. 
Published: May 2013

ABSTRACT
The mammalian embryo develops as a quasi-stem cell system whose differentiation and pluripotentiality in vitro is controlled by a single regulatory factor, Differentiation Inhibiting Activity/Leukemia Inhibitory Factor (DIA/LIF). DIA/LIF is expressed in two distinct functional forms, derived from the use of alternate transcriptional start sites, one of which is freely diffusible and the other tightly associated with the extracellular matrix. The dissemination of the DIA/LIF signal is therefore under specific molecular control. The expression of DIA/LIF in vitro is both developmentally programmed and controlled by the action of other growth factors, the most notable of which are members of the fibroblast growth factor family expressed by the stem cells themselves. This indicates that differentiation and proliferation in early development of the mouse are controlled, at least in part, by an interactive network of specific growth and differentiation regulatory factors.
Characterization of INT-2: A member of the fibroblast growth factor family
Authors: Dickson Clive , Acland Piers , Smith Rosalind , Dixon Mark , Deed Richard , Macallan David , Walther Wolfgang , Fuller-Pace Frances , Kiefer Paul , Peters Gordon 
Published: May 2013

ABSTRACT
int-2 was discovered as a proto-oncogene transcriptionally activated by MMTV proviral insertion during mammary tumorigenesis in the mouse. Sequence analysis showed int-2 to be a member of the fibroblast growth factor family of genes. In normal breast and most other adult mouse tissues, int-2 expression was not detected except for low levels in brain and testis. However, using in situ hybridization, expression was found at a number of sites during embryonic development, from day 7 until birth. An analysis of the int-2 transcripts found in embryonal carcinoma cells revealed six major classes of RNA initiating at three promoters and terminating at either of two polyadenylation sites. Despite the transcriptional complexities, all size classes of RNA encompass the same open reading frame. Using an SV40 early promoter to drive transcription of an int-2 cDNA in COS-1 cells, several proteins were observed. These were shown to be generated by initiation from either of two codons: One, a CUG, leads to a product which localizes extensively to the cell nucleus and partially to the secretory pathway. In contrast, initiation at a downstream AUG codon results in quantitative translocation across the endoplasmic reticulum and the accumulation of products ranging in size from 27.5×103Mr to 31.5×103Mr in organelles of the secretory pathway. These proteins represented glycosylated and non-glycosylated forms of the same primary product with or without the signal peptide removed. These findings suggest the potential for a dual role of int-2-, an autocrine function acting at the cell nucleus, and a possible paracrine action through a secreted product.
Phosphorylatioh of vertebrate smooth muscle and nonmuscle myosin heavy chains in vitro and in intact cells
Authors: Kelley Christine A. , Kawamoto Sachiyo , Conti Mary Anne , Adelstein Robert S. 
Published: May 2013

ABSTRACT
In this article we summarize our recent experiments studying the phosphorylation of vertebrate myosin heavy chains by protein kinase C and casein kinase II. Protein kinase C phosphorylates vertebrate nonmuscle myosin heavy chains both in vitro and in intact cells. A single serine residue near the end of the helical portion of the myosin rod is the only site phosphorylated in a variety of vertebrate nonmuscle myosin heavy chains. There does not appear to be a site for protein kinase C phosphorylation in vertebrate smooth muscle myosin heavy chains. Casein kinase II phosphorylates a single serine residue located near the carboxyl terminus of the 204×103 Mr smooth muscle myosin heavy chain in vitro as well as in cultured smooth muscle cells. It does not phosphory-late the 200×103Mr smooth muscle myosin heavy chain. However, the site is present in vertebrate nonmuscle myosin heavy chains. The 204×103Mr myosin heavy chain of embryonic chicken gizzard smooth muscle is exceptional in not containing a site for casein kinase II phosphorylation.
A myosin-like protein from smooth muscle
Authors: Kohama Kazuhiro , Lin Yuan , Takano-Ohmuro Hiromi , Ishikawa Ryoki 
Published: May 2013

ABSTRACT
A protein was purified from chicken gizzard smooth muscle. It bound ATP and actin. Actin activated the Mg2+-ATPase activity of this protein. The Ca2+-ATPase activity was lower than K+-EDTA ATPase activity. Thus, it appears that this protein is akin to myosin I rather than to conventional myosin. However, ATPase activities of the protein were much lower than those of myosin I. A protein cofactor, such as protein kinase, which would enhance these activities remains to be purified from the smooth muscle.
The use of native thick filaments in in vitro motility assays
Authors: Sellers James R. , Han Yung Jin , Kachar Bechara 
Published: May 2013

ABSTRACT
Native thick filaments from the clam, Mercinaria mercinaria translocate actin filaments both toward and away from the center of the thick filament in an in vitro motility assay. The thick filaments from the adductor muscle are about 10 gm long whereas those from the catch muscle are 30-50 gm long. These thick filaments should prove useful in understanding the mechanism of myosin-dependent movement of actin filaments.
Protein engineering and the study of muscle contraction in Drosophila flight muscles
Authors: Sparrow John , Drummond Douglas , Peckham Michelle , Hennessey Emma , White David 
Published: May 2013

ABSTRACT
We describe an experimental approach to the use of genetics to study muscle contraction in Drosophila melanogaster. Mutations induced by in vitro mutagenesis are inserted into the genome of flies using P-element mediated transformation, permitting the effects of the mutant genes to be studied in vivo in the indirect flight muscles (IFMs). Details of how mechanical experiments can be performed on skinned IFMs, despite their small size, are provided. The effects of two in vitro actin mutations, G368E and E316K, are described. The problems of performing biochemical and biophysical experiments on the IFMs and their myofibrillar proteins are described, together with indications as to how these may be overcome.
Bidirectional movement of actin filaments along tracks of heavy meromyosin and native thick filaments
Authors: Toyoshima Yoko Yano 
Published: May 2013

ABSTRACT
Flexibility of the myosin molecule was studied by an in vitro motility assay in terms of the direction of actin movement. Actin filaments can move in both directions on tracks of heavy meromyosin made on a nitrocellulose surface, and, furthermore, along the native thick filaments passing over their central bare zone. These observations indicate that the myosin molecule has a considerable flexibility in interacting with actin filaments.
Kinesin interactions with membrane bounded organelles in vivo and in vitro
Authors: Brady Scott T. , Pfister K. Kevin 
Published: May 2013

ABSTRACT
The ability of kinesin to interact with microtubules in a nucleotide-dependent manner and mediate microtubule-based motility has received the greatest amount of attention to date. Several lines of experimentation are now beginning to examine the interaction with membrane-bounded organelles. Immunochemical, biochemical and morphological approaches have shown that kinesin is associated with some, but not all, classes of membrane-bounded organelles found in cells. Similarly, evidence suggests that the distal portion of the rod and the tail portions of the kinesin heavy chain as well as the kinesin light chains may be important for the interaction with membrane surfaces. As a substantial amount of information about the molecular structure and biochemistry of kinesin has become available, the functional implications of interactions with membrane structures in vivo are being addressed.
Axonemal dynein from Tetrahymena
Authors: Crossley E. M. , Hyman S. C. , Wells C. 
Published: May 2013

ABSTRACT
Axonemal dynein from Tetrahymena cilia can be separated on a sucrose gradient into two fractions, at least one of which appears to be polymorphic. We have been using immuno-electron microscopy in order to try and locate the different types of dynein molecules within the axonemal structure.
Preparation of marked microtubules for the assay of the polarity of microtubuie-based motors by fluorescence
Authors: Hyman A. A. 
Published: May 2013

ABSTRACT
Short microtubule seeds are constructed using heavily rhodamine-labeled tubulin. Polymerisation off the ends of these seeds is initiated using a mixture of 1:10 labeled and unlabeled tubulin, so that the new polymerisation is only dimly labeled. This is done in the presence of NEM tubulin, which inhibits growth from the microtubule minus ends. The polarity-marked microtubules are fixed at a desired length by adding taxol.
Dynamin: A microtubule-associated GTP-binding protein
Authors: Obar Robert A. , Shpetner Howard S. , Vallee Richard B. 
Published: May 2013

ABSTRACT
We recently identified dynamin as a third nucleotide-sensitive microtubule-associated protein in brain tissue, in addition to kinesin and cytoplasmic dynein. Molecular cloning analysis has revealed that dynamin contains the three consensus elements characteristic of GTP-binding proteins, and biochemical results support a role for GTP in dynamin function. Dynamin is also homologous to the Mx proteins, involved in interferon-induced viral resistance, and the product of the yeast VPS1 gene, involved in vacuolar protein sorting. These results identify a novel class of GTP-utilizing proteins, with apparently diverse functions.
From genes to tensile forces: genetic dissection of contractile protein assembly and function in Drosophila melanogaster
Authors: Fyrberg Eric , Beall Clifford , Fyrberg Christine C. 
Published: May 2013

ABSTRACT
Myoflbrils, the contractile organelles of skeletal muscle, are highly ordered and precisely regulated actomyosin networks. Investigations of myofibril assembly are revealing the cellular mechanisms by which contractile components are arranged and regulated. In order to facilitate this research we have developed formal molecular genetics for myofibrillar proteins of Drosophila flight muscle. Presently, mutations can be used systematically to perturb or eliminate any of the classical myofibrillar proteins within these fibers, and the in vivo consequences can be conveniently evaluated using protein electrophoresis, electron microscopy, or by assaying flight performance. Here we review some recent progress.
Differential effects of laminin and merosin on neurite outgrowth by developing retinal ganglion cells
Authors: Cohen James , Johnson Alan R. 
Published: May 2013

ABSTRACT
In previous work, we showed that cultured avian embryonic retinal ganglion cells (RGC) extend neurites on EHS-laminin early in development, but lose this ability with maturation, as a result of a sharp decline in laminin receptor numbers. Here we show that EHS-laminin promotes neurite outgrowth also from embryonic mammalian RGC, in contrast to previous reports, and that these exhibit similar agedependent growth responses on laminin. Antibody blocking studies show that this behaviour is mediated in mouse RGC by 㯁6β1 integrin dimers. The laminin isoform merosin is also effective as a neurite outgrowth-promoting substrate for RGC but differs in its ability to elicit a response at advanced stages of development (up to hatching in the chick). Neurite outgrowth by RGC on merosin is inhibited, at all ages, by the function-blocking, anti-αβ integrin antibody, CSAT, suggesting that these neurons use alternative αβ1 dimers in their interactions with EHS-laminin and merosin. Together, these findings emphasise the generality of the responsiveness of vertebrate embryonic RGC to laminin during development, and reveal interesting differences in the effects of laminin variants on CNS axon growth and regeneration.