bluuebunny/crossref_metadata_2025 huggingface dataset:
Epidermal Growth Factor: Biology and Receptor Metabolism
Authors: Carpenter Graham
Published: May 2013

Epidermal growth factor (EGF) is a small (Mr 6045) protein that stimulates cell proliferation in cell culture systems and in intact animals. This growth factor has been isolated from rodents and human material and probably exists in nearly all animal species. In humans EGF has been detected in many body fluids and receptors for the growth factor are also ubiquitous. While the mitogenic activity of EGF has been most frequently reported, it clearly has other functions, such as the inhibition of gastric acid secretion, that are unrelated to mitogenic responses. Correspondingly, receptors for EGF have been localized on cells that are rapidly proliferating and cells that are essentially non-proliferating. Nevertheless, it has not been possible to define experimentally the biological function(s) of the endogenous EGF present in the intact animal.

Studies of the mechanism of action of EGF have concentrated, to date, on the plasma membrane receptor that specifically binds this ligand. The receptor is undoubtedly the first cellular component that mediates the-eventual biological response(s) of the cell to this extracellular signal. Studies of the EGF receptor have shown that this molecule, which has no subunit structure, functions not only in ligand recognition, but also may produce an intracellular ‘second message’. The receptor contains a protein kinase activity that is activated by the binding of EGF and it is this enzymic function that may yield the critical ‘second messenger’, by phosphorylation of an intracellular protein. Although intracellular targets of this EGF-sensitive protein kinase have been identified, it has not been possible to demonstrate their relevance as regulatory mediators of EGF activity.
Early response pattern analysis of the mitogenic pathway in lymphocytes and fibroblasts
Authors: Metcalfe J. C. , Hesketh T. R. , Smith G. A. , Morris J. D. H. , Corps A. N. , Moore J. P.
Published: May 2013

The early biochemical responses stimulated by the action of mitogens and growth factors on mouse thymocytes and 3T3 fibroblasts are analysed as part of a systematic attempt to define the mitogenic pathways from Go to S phase in these cells. Although the primary response to each mitogen can be distinguished by the pattern of secondary responses they initiate, there is substantial overlap in these responses. The aim is therefore to determine whether there is early convergence on a common mitogenic pathway, defined by a sequence of responses obligatory for progression from Go to 5 phase for different mitogens and cell types. The ‘dual-signal’ hypothesis for the mitogenic stimulation of thymocytes is a simple version of a common mitogenic pathway. It proposes that the T-cell receptor initiates the pathway via the breakdown of phosphatidylinositol (4,5)-bisphosphate to generate a Ca signal (from the release of inositol (l,4,5)-trisphosphate) and to activate protein kinase C (from the release of diacylglycerol). The rationale for this hypothesis lies in the co-mitogenic action of the Ca2+-ionophore, A23187, and the phorbol ester, 12-o-tetradecanoyl phorbol 13-acetate, which is assumed to activate specifically protein kinase C. However, detailed analysis of the coupling between some of the early responses, including the Ca and pH signals, phosphatidylinositol (4,5)-bisphosphate metabolism, c-myc gene activation and general metabolic stimulation, indicates clearly that the hypothesis is inadequate to account for the initiation of the normal mitogenic pathway in thymocytes.
The structure and biosynthesis of epidermal growth factor precursor
Authors: Scott J. , Patterson S. , Rall L. , Bell G. I. , Crawford R. , Penschow J. , Niall H. , Coghlan J.
Published: May 2013

The structure of mouse submaxillary gland epidermal growth factor (EGF) precursor has been deduced from complementary DNAs. The mRNA is approximately 4800 bases and predicts prepro EGF to be a protein of 1217 amino acid residues (133×10Mr). EGF (53 amino acid residues) is flanked by polypeptides of 188 and 976 residues at its carboxy and amino termini, respectively. The amino terminus of the precursor contains seven cysteine-rich peptides that resemble EGF. Towards the carboxy terminus is a 20-residue hydrophobic membrane spanning domain. The mid portion of the EGF precursor shares a 33 % homology with the low density lipoprotein receptor, which extends over 400 amino acid residues. These features suggest that EGF precursor could function as a membrane-bound receptor.

RNA dot-blot analysis and in situ hybridization show EGF mRNA to be abundant in the submaxillary gland, kidney and incisor tooth buds. Lower EGF mRNA levels were found in the lactating breast, pancreas, small intestine, ovary, spleen, lung, pituitary and liver. In the kidney EGF mRNA was most abundant in the distal convoluted tubules. Analysis of EGF precursor biosynthesis in organ culture of the submaxillary gland and kidney showed differential processing of the precursor in the two tissues. In the submaxillary gland immunoreactive low molecular weight EGF was produced, but in the kidney the high molecular weight precursor was not processed. In the distal convoluted tubule of the kidney EGF precursor may act as a receptor that is involved in ion transport.
Studies Of Tektin Filaments From Flagellar Microtubules By Immunoelectron Microscopy
Authors: Amos W. B. , Amos L. A. , Linck R. W.
Published: May 2013

Chemically resistant 2-3 nm filaments with a high α-helical content, isolated from sea-urchin sperm flagellar doublet microtubules, consist of proteins that have been named tektins. Polyclonal affinity-purified antibodies to tektins labelled sperm tails all along their lengths, as shown by indirect immunofluorescence microscopy, provided the specimens were not too well fixed. Results obtained for unfixed specimens studied by immunoelectron microscopy suggested the tektins are normally masked by tubulin. A monoclonal anti-tektin antibody labelled bare tektin filaments at longitudinal intervals of approximately 48 nm, which fits in well with the 96 nm longitudinal repeat of axonemes. We discuss a possible scheme for the regular interaction of tubulin monomers with an α-helical coiled coil.
Latent Cellular Oncogenes: The Paradox Dissolves
Authors: Duesberg Peter H.
Published: May 2013

The 20 known transforming one genes of retroviruses are defined by sequences which are transduced from cellular genes, termed proto-one genes. Based on these sequences, viral one genes have been postulated to be transduced cellular cancer genes and proto-owc genes have been postulated to be latent cancer genes that can be activated from within the cell, to cause virusnegative tumours. The hypothesis is popular because it promises direct access to cellular cancer genes. Hpwever the existence of latent cancer genes presents a paradox since such genes would be most undesirable for eukaryotes. The hypothesis predicts (i) that viral one genes and proto-owc genes are isogenic, (ii) that expression of proto-one genes induces tumours, (iii) that activated proto-one genes transform diploid cells upon transfection, like viral one genes, and (iv) it predicts diploid tumours. As yet, none of these predictions is confirmed. Instead: (i) Structural comparisons between viral one genes, essential retroviral genes, and the proto-one genes show that all viral one genes are indeed new genes, rather than transduced cellular cancer genes. They are genetic hybrids put together from truncated viral and truncated proto-owc genes, (ii) Proto-owc genes are frequently expressed in normal cells, (iii) To date, not one activated proto-owc gene has been isolated that transforms diploid cells, (iv) Above all, no diploid tumours with activated proto owc genes have been found. Moreover the probability of spontaneous transformation in vivo is at least 109 times lower than predicted from the mechanisms thought to activate proto-owc genes. Therefore the hypothesis, that proto-owc genes are latent cellular oncogenes, appears to be an overinterpretation of sequence homology to structural and functional homology with viral owe genes. Here is is proposed that only rare truncations and recombinations, that alter the germline configuration of cellular genes, generate viral and possibly cellular cancer genes. The clonal chromosome abnormalities that are consistently found in tumour cells are microscopic evidence for rearrangements that may generate cancer genes. The clonality indicates that the tumours are initiated with, and possibly by these abnormalities as predicted by Boveri in 1914.
Tobacco mosaic virus replicase and replicative structures
Authors: Young Nevin , Forney Julie , Zaitlin Milton
Published: May 2013

The RNA-dependent RNA polymerase (replicase) mediating the replication of tobacco mosaic virus (TMV) has been investigated in a number of laboratories over a period of 20 years. Cell-free enzyme preparations have been prepared which can continue the synthesis of nascent complementary RNA, initiated in vivo; however, the enzyme does not require, nor does it respond to, exogenous viral RNA as a template. The presence in plants of a virus-stimulated, host-encoded RNA-dependent RNA polymerase (RdRp) has added confusion to this field; it is now generally conceded, however, that this enzyme is not the TMV replicase.

Our recent studies have emphasized several aspects of TMV RNA replication. We have examined the nature of TMV replicative structures synthesized in vitro by utilizing a partially purified enzyme preparation isolated from TMV-infected tobacco tissue. Radiolabelled products of the reaction were analysed on agarose gels and fractions with the predicted electrophoretic migration and nuclease sensitivities of replicative form (RF) and replicative intermediate (RI) were isolated. These fractions were hybridized to a collection of bacteriophage M13 clones containing portions of the TMV genome of both plus and minus polarity. The nascent synthesis in the Rl-like molecules was restricted to the plus viral strand, while the new synthesis in the RF-like molecules was of both plus and minus polarity.

Solubilization of the membrane-bound replicase with the non-ionic detergent CHAPS has yielded complexes which remain in solution after high-speed centrifugation. The solubilized replication complexes have been utilized as starting material for enzyme purification by Sepharose 4B gel filtration chromatography.

The intracellular site of synthesis of TMV RNA has been reinvestigated in the light of reports suggesting a nuclear site of replication. The conclusion for nuclear synthesis has been based on fractionation of subcellular homogenates of virus-infected leaves or mesophyll protoplasts and identification of virus-related proteins associated with these fractions. In our studies, however, we conclude that these procedures can be misleading in that the 126 000 Mr TMV protein (and replicase activity) were found in all fractions of the homogenate analysed. Double-stranded TMV RNA, on the other hand, was barely detectable in preparations of purified nuclei; instead it was concentrated in the post-nuclear supernatant, suggesting that the nucleus is not the site of TMV RNA synthesis.
Genome interactions which influence dna palindrome mediated instability and inviability in Escherichia Coli
Authors: Leach David , Lindsey Janet , Okely Ewa
Published: May 2013

The interaction of three factors determine the detrimental effect of a palindromic DNA sequence in Escherichia coli cells. The first is the nature of the palindrome (its length, extent of central asymmetry and perhaps its base sequence), the second is the genotype of the host cell and the third is the replicon within which it is located. In this paper we extend the genetic and physical characterization of lambda bacteriophages carrying a palindrome of approximately 560 base pairs. We also show that a palindrome of approximately 110 base pairs which can be cloned in a plasmid cannot be cloned in a phage M13 derivative. These observations are relevant to the choice of vectors used in the cloning of eukaryotic DNA containing palindromic sequences.
The role of sequence-specific dna-binding proteins in adenovirus dna replication
Authors: Hay Ronald T. , Barrett Perry , Clark Lilian , Harris Mark
Published: May 2013

In prokaryotes it is well established that proteins which recognise defined DNA sequences are involved in the control of gene expression and replication. Cellular proteins in eukaryotes which may perform a similar function have been identified by their interactions with control regions of the human adenovirus genome.

Immediately after infection a small region (Ela) at the left end of the adenovirus genome is expressed. Proteins coded by the Ela region transcriptionally activate the viral early genes. The products of a number of these early genes are directly involved in replication of the viral DNA. DNA sequences which are required for efficient Ela transcription and for the initiation of DNA replication have been identified by mutational analysis. Cellular proteins which recognise these sequences were detected using a sensitive gel retention assay. The basis of this assay is that during electrophoresis DNA-protein complexes migrate more slowly through a polyacrylamide gel than free DNA. In this way a cellular protein which binds to a conserved sequence in the adenovirus enhancer has been identified and partially purified. Cellular proteins which bind to adenovirus type 2 and 4 origins of replication have also been fractionated from nuclear extracts of uninfected HeLa cells. The roles of these proteins in adenovirus replication will be discussed.
Transposable Elements in Antirrhinum Majus
Authors: Martin Cathie , Carpenter Rosemary , Robbins Tim , Hudson Andrew , Almeida Jorge , Bartlett Jeremy , MACKAY Steve , Coen Enrico
Published: May 2013

The transposable element, Tam 3, gives rise to large-scale chromosomal rearrangements at a low frequency, when it is inserted at the nivea locus of Antirrhinum majus. Some deletions that result from imprecise excision of Tam 3 provide an allelic series for functional analysis of the nivea gene promoter. Rearrangements involving deletion, dispersion and inverted duplication of flanking sequences, where Tam 3 remains in situ, have also been identified. These rearrangements have been mapped at the molecular level, and the behaviour of Tam 3 following rearrangement has been observed. It is clear that Tam 3 has enormous potential to restructure chromosomes through successive rounds of large-scale rearrangements.
Aspects of the ac/ds transposable element system in maize
Authors: Peacock W. James , Dennis Elizabeth S. , Finnegan E. Jean , Peterson Thomas A. , Taylor Brian H.
Published: May 2013

Studies of the Ac (Activator) transposable element provided the data which led Barbara McClintock to postulate that certain segments of chromosomes could transpose to different locations in the genome. McClintock also recognized the existence of Ds (Dissociation) elements which could transpose, but only in the presence of a trans-acting Ac element elsewhere in the genome. DNA sequences corresponding to Ds and Ac have now been identified, and an understanding of many of the properties of these transposable elements in the maize genome has been acquired in recent years.

It is known that cryptic Ac elements and members of at least two families of Ds elements occur in the genome of all maize lines examined. Ds elements also occur in Teosinte and the more distantly related Tripsacum. We discuss the possible origin of these elements and consider the mechanism of activation of cryptic Ac elements. A recent molecular analysis of a transition of an Ac-derived Ds-element back to an active Ac element suggests one molecular mechanism by which changes in the activity state of Ac may occur.

Distinctive phenotypes created by controlling elements within a target gene have been shown to be governed by the properties of the insertion element and the position of the insertion within the gene. Genetic effects include modulation of gene expression, alteration of gene products, instability of mutant phenotypes, deletion and duplication of chromosome segments and the production of chromosome rearrangements. We describe an example where a Ds insertion generates an additional intron in the Adhl gene which reduces gene expression through mRNA instability. We also discuss an Ac-dependent modulation of P gene activity in glume and pericarp tissues of maize which may be attributed to an alteration either in patterns of gene expression or the developmental biology of the flower.

The molecular consequences of Ac and Ds insertions and excisions are known at the DNA sequence level but little is known of the mechanism of transposition. An initial approach has been to analyse Ac transcription. Preliminary results showing transcription of a limited region of Ac are discussed. The corresponding upstream regions have been linked to the coding region of chloramphenicol acetyltransferase (CAT) and show promoter activity following electroporation into tobacco protoplasts.
Effect of Patterned Surfaces of Adhesive Islands on the Shape, Cytoskeleton, Adhesion and Behaviour of Swiss Mouse 3T3 Fibroblasts
Authors: Ireland G. W. , Dopping-Hepenstal P. , Jordan P. , O’neill C.
Published: May 2013

A pattern of circular islands of adhesive substratum can be used to control cell shape and behaviour. We have shown previously that the proportion of Swiss 3T3 cells that synthesize DNA varies with the area of the island to which they are attached, within the range 500–5000 μm2. In this paper we investigate the cytoskeleton and adhesions of cells on islands using a variety of techniques including phalloidin staining and interference reflection microscopy. Islands of area 2000 μm2 or less constrain cell shape, and cause focal contacts and actin microfilament bundles to accumulate in a circle at the margin. These changes are most obvious in islands of about 1000 gm2, in which a complete ring of adhesion is sometimes formed in the periphery of the cell. This peripheral distribution is less common in cells on even smaller islands, and the focal contacts become smaller and less numerous. It is not yet clear whether any of these structural changes are associated directly with the proliferative stimulus due to contact with the substratum. However, we expect that the use of patterned substrata will contribute to the study of how cell shape and structure regulate many cell functions.
Foetal-to-Adult Transitions in Fibroblast Phenotype: Their Possible Relevance to the Pathogenesis of Cancer
Authors: Schor S. L. , Schor A. M.
Published: May 2013

We have previously shown that the migration of foetal, adult and transformed fibroblasts into three-dimensional collagen gels is differentially affected by plating cell density. We now present data indicating that the migration of these fibroblasts is also differentially affected by local cell density in microdomains of the gel surface. In this article we discuss the possible biochemical and behavioural mechanisms that may contribute to the different migratory phenotypes expressed by foetal, adult and transformed fibroblasts; these include: (1) cell-induced alterations in the orientation and or packing density of collagen fibres in the gel; (2) deposition of specific matrix macromolecules by the fibroblasts; (3) social interactions between the cells; and (4) secretion of soluble factors affecting cell migration. We show that foetal fibroblasts secrete a migration stimulating factor (MSF) not produced by adult cells. Incubation of adult fibroblasts in the presence of MSF induces these cells to express a foetal-like migratory phenotype. Foetal fibroblasts undergo a spontaneous foetal-to-adult transition in migratory phenotype after prolonged passage in vitro ; this transition is accompanied by a cessation in MSF production. MSF appears to promote fibroblast migration at high cell density by stimulating the deposition of hyaluronic acid in the extracellular matrix.

Recent studies have indicated that skin fibroblasts from cancer, patients display certain behavioural abnormalities characteristic of transformed and/or foetal cells. In this regard, we have shown that skin fibroblasts from cancer patients commonly express a foetal-like phenotype with respect to migratory behaviour and secretion of MSF: it is of interest to note that these cancer patient fibroblasts are indistinguishable from normal adult cells in other respects, such as morphology in confluent culture. On the basis of these observations, we suggest that: (1) fibro-blasts in certain individuals fail to undergo normal foetal-to-adult transitions in a number of phenotypic characteristics; and that (2) the disruption in epithelial-mesenchymal interactions caused by the continued presence of these foetal-like fibroblasts in the adult significantly increases the risk of cancer development.
Molecular events associated with the action of haemopoietic growth factors
Authors: Farrar William L. , Evans Stuart W. , Harel-Bellan Annick , Ferris Douglas K.
Published: May 2013

Haemopoietic growth factors stimulate a number of consensus biochemical and molecular events regardless of the specificity detailed by unique ligand and receptor structures. Analysis of three distinct colony stimulating factors, CSFs (IL-3, G-CSF, GM-CSF) and the lymphocytotropic growth factor IL-2 reveal remarkable similar distal subcellular biochemical signals although initial membrane ‘signal transduction’ may differ significantly. Both early progenitor cell growth factors, such as IL-3, and late acting factors such as CSF-1, stimulate tyrosine and serine-threonine substrate phosphorylations. One substrate (p68) is phosphorylated by many CSF stimulants, including IL-2, suggesting a highly conserved role in many unique receptor(s) signal transduction processes. The proliferative CSFs and IL-2 also stimulate the expression of many of the same genes including proto-oncogenes, ornithine decarboxylase and members of the ancient family of stress response genes. Although initial membrane events may differ among the respective proliferative stimulants, biochemical and molecular convergence on highly conserved cellular substrates and the programme of gene expression is seen.
Interaction between mitochondria and the nucleus
Authors: Pon Liza A. , Vestweber Dietmar , Yang Meijia , Schatz Gottfried
Published: May 2013

The interaction between the mitochondrial and the nuclear genome is in part mediated by proteins (and possibly also RNAs) which are encoded in the nucleus and imported into mitochondria. We are beginning to understand how proteins can penetrate across both mitochondrial membranes and how some of these proteins can regulate the expression of specific mitochondrial genes.
Biochemical investigation of protein export in Escherichia coli
Authors: Hardy S. J. S. , Randall L. L.
Published: May 2013

Export of proteins from the bacterial cytoplasm to a final destination in the periplasm and outer membrane is one example of the fundamental process occurring in all cells whereby polypeptides are transferred across biological membranes. Investigations on a variety of different systems have indicated similarities in the mechanism of this process. In the cases of bacterial protein export and the transfer of polypeptides across the endoplasmic reticulum in eukaryotic cells the processes are so similar that understanding gleaned from studies of the one is usually directly applicable to the other.

The study of protein export in E. coli has two advantages over that of eukaryotic secretion. Not only is there the possibility of doing sophisticated genetic experiments, but also one can carry out biochemical investigations in vivo, a facility not so readily available with eukaryotic organisms. Such studies have, for example, shown that membrane translocation can occur both co- translationally and post-translationally, that export requires protonmotive force, that some component of the export apparatus prevents the exported protein from assuming its native structure in the cytosol, and that there are probably at least two functions for the leader sequence, one in targeting the protein to the export pathway and one in translocation across the membrane.
Genetic and biochemical analysis of protein export from Xanthomonas campestris
Authors: Dow J. M. , Daniels M. J. , Dums F. , Turner P. C. , Gough C.
Published: May 2013

Xanthomonas campestris pv. campestris, a Gram-negative phytopathogen, produces a number of extracellular enzymes which can degrade components of the host plant cell. Some non-pathogenic mutants, derived by chemical mutagenesis, were found to be defective in the export but not the synthesis of a number of these enzymes. The pathogenicity and export lesions in one such mutant, strain 8288, could be complemented by a cosmid clone pIJ3000 from the Xanthomonas library. Mutagenesis of pIJ3000 with the transposon Tn5 followed by recombination into the corresponding region of the chromosome has revealed a cluster of 6 to 8 genes whose function is required for enzyme export. Sequence analysis of part of the cluster has revealed two open reading frames that would encode proteins with extensive hydrophobic domains. Export-defective mutants retain the normally exported enzymes in the periplasmic space. These forms have the same molecular weight as the extracellular forms, suggesting that the signal sequence has been properly processed. The results are consistent with a mechanism of sequential translocation across cytoplasmic and outer membrane via the periplasm. The second translocation step may be mediated by the products of the export genes. This may be a common export mechanism amongst Gram-negative bacteria but other mechanisms do exist, sometimes in parallel in the same cell, and these are briefly reviewed.
Calcium-induced chromatin condensation and cyclin phosphorylation during chromatin condensation cycles in ammonia-activated sea urchin eggs
Authors: Patel Rajnikant , Twigg Jeremy , Crossley Ian , Golsteyn Roy , Whitaker Michael
Published: May 2013

Ammonia-activated sea urchin eggs undergo repeated cycles of DNA synthesis, nuclear envelope breakdown (NEB) and chromatin condensation. No mitotic spindle forms, nor do the eggs undergo cytokinesis. Ammonia-activated eggs exhibit a form of the cell cycle in which the nuclear cycle proceeds without segregation of the chromatin into daughter cells. We discuss here experiments that demonstrate that intracellular free calcium concentration controls the S phase-M phase transition in ammonia-activated eggs, as it does in fertilized embryos.

Cyclins are proteins that are synthesized throughout the cell cycle and destroyed abruptly during each round of chromatin condensation. We find that cycles of cyclin phosphorylation and destruction occur coincident with chromatin condensation in ammonia-activated eggs. Cyclin phosphorylation also occurs in eggs treated with the tumour promoter, phorbol myristate acetate (PMA). There is no accompanying NEB or chromatin condensation, however, and the nucleus is insensitive to exogenously-generated calcium transients. These latter data indicate that cyclin synthesis and phosphorylation is not a sufficient condition for calcium-induced NEB in sea urchin embryos. PMA must fail to induce one of the necessary cell cycle initiation signals. We suggest that the missing signal is the activation of the cell cycle control protein p34cdc2, which we have shown to be phosphorylated at fertilization and which is phosphorylated in ammonia-activated eggs.
Replication of SV40 in vitro using proteins derived from a human cell extract
Authors: Fairman M. P. , Prelich G. , Tsurimoto T. , Stillman B.
Published: May 2013

In the presence of large T antigen and plasmids containing a functional origin of replication, extracts from a human cell line will support multiple rounds of simian virus 40 (SV40) replication in vitro. Fractionation of this extract has led to the identification of several factors, some of which have been purified to homogeneity. The characterisation of these proteins has led to the separation of SV40 replication in vitro into multiple stages.

Two proteins, the cell cycle-regulated proliferating cell nuclear antigen and replication factor-C, have been shown to be essential for coordinating leading and lagging strand synthesis in this system. Another protein, replication factor-A, is a multi-subunit protein of 70, 34 and 11K (K = 103Mr) polypeptides which, because of its high affinity for DNA, is thought to function as a eukaryotic single-stranded DNA binding protein. Interactions between other cellular factors are also described that effect the initiation of DNA replication, but are not required in a more purified system. In addition a model for a hypothetical replication fork is described, which suggests a role for both a- and ô-polymerases in this system, and may be applicable to higher eukaryotes.
Nuclear structure and the control of DNA replication in the Xenopus embryo
Authors: Blow J. J. , Sheehan M. A. , Watson J. V. , Laskey R. A.
Published: May 2013

We have developed a cell-free system from frog eggs that efficiently initiates and completes a single round of semi-conservative replication. 70–100% of sperm chromatin and up to 40% of plasmid DNA molecules are completely replicated in vitro. Before DNA is replicated it is assembled into nuclei surrounded by a double unit membrane studded with nuclear pores. Flow cytometry shows that initiation events are co-ordinated within individual nuclei, although different nuclei can start to replicate at different times in the same extract. This demonstrates the importance of nuclear structure in the control of DNA replication in this system.

Only a single round of semi-conservative replication occurs in the cell-free system. This mirrors the way that only one round of DNA replication occurs in each cell cycle in vivo. When replicated nuclei are transferred to fresh extract they are unable to undergo another round of replication. However, if the nuclear envelope is permeabilised before nuclei are transferred to fresh extract, the DNA becomes capable of undergoing a further round of semi-conservative replication. These results suggest a simple model for the control of DNA replication within the cell cycle, whereby an essential initiation factor can only gain access to DNA when the nuclear envelope breaks down during mitosis.
Cis- and trans-acting factors affecting the structure of yeast centromeres
Authors: Bloom Kerry , Kenna Margaret , Saunders Michael
Published: May 2013

The isolation of yeast centromeres has provided the opportunity to describe the molecular structure of chromosome attachments to the mitotic spindle. Nucleolytic probes of chromatin structure and analysis of genetically altered centromeres have been used to determine the primary sequence and positional requirements for assembly of this chromosomal domain into a functional unit. Structural analysis of the centromere in wild-type and mutant strains provides the first indication of specific proteins associated with centromere DNA in vivo.
Fission yeast cyclin: subcellular localisation and cell cycle regulation
Authors: Alfa Caroline E. , Booher Robert , Beach David , Hyams Jeremy S.
Published: May 2013

Entry into mitosis in the fission yeast Schizosaccharomyces pombe involves the interaction of a number of genes with the major cell cycle control gene, cdc2+. One of these, cdcl3+, encodes a protein with homology to cyclin. By indirect immunofluorescence microscopy using antibodies to the appropriate bacterially-expressed protein, we have shown that both cdcl3 and cdc2 are nuclear proteins in S. pombe. Both are localised to a nuclear domain distinct from that occupied by the DAPI-staining chromatin. The immunofluorescence signals of both proteins show a progressive increase during interphase but are undetectable at mitosis. Loss of cdcl3 fluorescence at mitosis reflects the destruction of the protein. Thus, it behaves as a classic cyclin. This is not the case for cdc2, the level of which remains constant through the cell cycle. Cells carrying a disrupted copy of the cdcl3+ gene fail to accumulate either cdcl3 or cdc2 in the nucleus. Cells carrying a disrupted cdc2+ gene fail to accumulate cdc2 but reveal apparently normal levels of cdcl3. cdc13 therefore appears to be required to localise cdc2 to the nucleus but not vice versa. The destruction of cdcl3 at mitosis may allow cdc2 to redistribute to the cytoplasm.
Centrosomes and the cell cycle
Authors: Sluder Greenfield
Published: May 2013

Centrosomes are the ensemble of organelles that form the poles of the mitotic spindle. We have examined the properties of the mechanisms that control the precise doubling, or reproduction, of centrosomes during the cell cycle. A functional analysis of this event in sea urchin eggs indicates that it is limited by the reproduction of determinants that we call polar organizers, around which the centrosome is organized. Each centrosome contains two polar organizers whose splitting, physical separation, and duplication control the reproduction of the centrosome. The splitting and duplication events are distinct processes that can be experimentally put out of phase with each other for several cell cycles. A serial section ultrastructural analysis of centrosomes with altered reproduction shows that the reproductive capacity of a centrosome is correlated with the number of centrioles it contains. In other experiments we show that centrosome reproduction is cytoplasmi- cally controlled; centrosomes repeatedly double in a normal fashion in physically enucleated sea urchin eggs and eggs in which DNA synthesis has been inhibited by aphidicolin. In addition, we show that centrosomes, through the spindle microtubules they nucleate, play an important role in the mechanisms that control the timing of mitotic events and the overall duration of the cell cycle. Taken together, our observations in concert with those of others suggest that the cell cycle is a cytoplasmic phenomenon that does not require nuclear activities for cells that are not growthlimited.
Mitosis in Drosophila development
Authors: Glover D. M. , Alphey L. , Axton J. M. , Cheshire A. , Dalby B. , Freeman M. , Girdham C. , Gonzalez C. , Karess R. E. , Leibowitz M. H. , Llamazares S. , Maldonado-Codina M. G. , Raff J. W. , Saunders R. , Sunkelj C. E. , Whitfield W. G. F.
Published: May 2013

Many aspects of the mitotic cycle can take place independently in syncytial Drosophila embryos. Embryos from females homozygous for the mutation gnu undergo rounds of DNA synthesis without nuclear division to produce giant nuclei, and at the same time show many cycles of centrosome replication (Freeman et al. 1986). S phase can be inhibited in wild-type Drosophila embryos by injecting aphidicolin, in which case not only do centrosomes replicate, but chromosomes continue to condense and decondense, the nuclear envelope undergoes cycles of breakdown and reformation, and cycles of budding activity continue at the cortex of the embryo (Raff and Glover, 1988). If aphidicolin is injected when nuclei are in the interior of the embryo, centrosomes dissociate from the nuclei and can migrate to the cortex. Pole cells without nuclei then form around those centrosomes that reach the posterior pole (Raff and Glover, 1989); the centrosomes presumably must interact with polar granules, the maternally-provided determinants for pole cell formation. The pole cells form the germ-line of the developing organism, and as such may have specific requirements for mitotic cell division. This is suggested by our finding that a specific class of cyclin mRNAs, the products of the cyclin B gene, accumulate in pole cells during embryogenesis (Whitfield et al. 1989). Other genes that are essential for mitosis in early embryogenesis and in later development are discussed.
Maturation-promoting factor and the regulation of the cell cycle
Authors: Maller James L. , Gautier Jean , Langan Thomas A. , Lohka Manfred J. , Shenoy Suresh , Shalloway David , Nurse Paul
Published: May 2013

Maturation-promoting factor (MPF) is a cell cycle control element able to cause metaphase when injected into amphibian oocytes or when incubated with nuclei in a cell-free system. Highly purified MPF consists of a complex between a 34K (K = 103Mr) serineμhreonine protein kinase, identified as a Xenopus homolog of the cdc2+ gene product, p34edc2, and a 45K substrate, identified as a Xenopus B-type cyclin. p34cdc2 is also present in purified preparations of chromatin-derived growth-associated histone Hl kinase from Novikoff hepatoma cells. p34ede2 is active when dephosphorylated and inactive when phosphorylated during oocyte meiotic cell cycles and in mitotic cell cycles following egg activation. Analysis of the substrate specificity of p34cdc2 indicates a consensus sequence for phosphorylation of (K/R)S/yP(X)K/R. Among substrates identified with this consensus are histone Hl and the pp60csrc proto-oncogene, which is known to be activated and phophorylated in mitosis. MPF injection into oocytes activates ribosomal protein S6 kinase II, which is also a lamin kinase. The mechanism of activation is indirect, possibly involving the c-src proto-oncogene. Continued analysis of regulation of MPF activation/inactivation and characterization of substrates for phosphorylation will have important implications for cell cycle and cell growth control.
Cyclin synthesis and degradation and the embryonic cell cycle
Authors: Murray Andrew W.
Published: May 2013

I discuss recent advances in the study of somatic and embryonic cell cycles. In the frog embryonic cell cycle, cyclin is the only newly synthesized protein required to activate maturation-promoting factor and induce mitosis. Diminishing the rate of cyclin synthesis increases the length of interphase. Cyclin degradation is required for the progression from mitosis to interphase. Comparison of the frog embryonic cell cycle to other cell cycles suggests that all cell cycles will rely on the same closely conserved set of components. However, the component that is rate-limiting for any step in the cell cycle will vary in different cell cycles.
Retinoic acid and limb regeneration
Authors: Brockes Jeremy P.
Published: May 2013

A key problem in the study of vertebrate development is to determine the molecular basis of positional value along a developmental axis. In amphibian regeneration, retinoic acid is able to respecify positional value in a graded fashion that is dependent on its concentration. In view of the fact that retinoic acid is a naturally occurring metabolite of vitamin A, this raises the possibility that it is deployed in vivo as an endogenous morphogen. Furthermore, the recent evidence that its effects are mediated by nuclear receptors of the steroid/thyroid hormone superfamily suggests the possibility of understanding the mechanism of its graded effects on morphogenesis. Such insights would be of crucial importance for our understanding of vertebrate patterning along an axis.
Signals in limb development: STOP, GO, STAY and POSITION
Authors: Wolpert Lewis
Published: May 2013

Cell-to-cell interactions in early limb development are considered within the framework of the extracellular signals STOP, GO, STAY and POSITION, a classification which emphasises that the signals are elective rather than instructive, and that complexity arises from cells’ response. Patterning in the limb is analysed in terms of signals that specify positional values along the anteroposterior axes, and retinoic acid is thought to be a positional morphogen. There is however, evidence for patterning which does not depend on a positional signal. In the early bud the mesenchyme gives POSITION signals to the apical ridge, which in turn provides a STAY signal to the mesenchyme in the progress zone. Non-ridge ectoderm produces a STOP signal with respect to cartilage differentiation. The pattern of cartilage differentiation is specified well before cartilage condensation. Growth factors affect both cartilage and muscle differentiation in culture. Pigment patterns result from feather germs providing STOP or GO signals to the melanoblasts which enter all feather germs. The pathways for the cell-to-cell signals are not known but may involve gap junctions.
The structure of tumour necrosis factor - implications for biological function
Authors: Jones E. Y. , Stuart D. I. , Walker N. P. C.
Published: May 2013

The three-dimensional structure of TNF has been determined at 0.29 nm using the technique of X-ray crystallography. Published data on site-directed mutagenesis and antibody binding may now be assessed in the light of the structure, thus the links between structure and function for TNF may be addressed.

TNF is a compact trimer composed of three identical subunits of 157 amino acids. The main-chain topology for a single subunit is essentially a β-sandwich structure formed by two anti-parallel β-pleated sheets. This mainchain fold corresponds to the ‘jelly roll’ motif observed in viral coat proteins such as VP1, VP2 and VP3 of rhinovirus, or the hemagglutinin molecule of influenza. TNF is the first non-viral protein to contain this motif. The subunits associate tightly about a threefold axis interacting through a simple edge-to-face packing of the β-sandwich to form the solid, conical shaped trimer. A large number of the residues conserved between the amino acid sequences of TNF and lymphotoxin lie within the β-sandwich or at the threefold axis of the trimer. This implies the presence of the same β-sandwich motif in the lymphotoxin monomer and preservation of the edge-to-face mode of trimeric association.

The detailed three dimensional structure for TNF explains a wide range of observations, including data on antibody binding and site directed mutagenesis. The currently available evidence points to a region of biological importance situated at the interface between two subunits on the lower half of the trimer.
X-ray crystallographic studies on murine nerve growth factor
Authors: Mcdonald N. Q. , Lapatto R. , Murray-Rust J. , Blundell T. L.
Published: May 2013

The largest and best characterised family of neurotrophic growth factors is that of nerve growth factor (NGF) and its relatives. In order to understand the relation of structure and function, we have undertaken X-ray analyses of murine NGF. The active component β-NGF crystallises as hexagonal bipyramids that give good X-ray diffraction data using a synchrotron to 2.3Å resolution. We have prepared several heavy atom derivatives that are being used in the method of multiple isomorphous replacement to solve the phase problem and determine the three-dimensional structure. We have also prepared crystals of the precursor, 7S NGF, which is a complex of three different subunits of composition α2 β2 γ2We have collected X-ray data to 3Å resolution on two crystal forms with related cell dimensions and orthorhombic spacegroups. Detailed analyses of the structures of NGF in these crystal forms, taken together with data on sequence and biological activity, should give clues concerning the role of the precursor complex in storage and assist the identification of the surface region involved in receptor binding.
Chimeric molecules map and dissociate the potent transforming and secretory properties of PDGF A and PDGF B
Authors: Larochelle William J. , Giese Neill , May-Siroff Mary , Robbins Keith C. , Aaronson Stuart A.
Published: May 2013

Human platelet-derived growth factor (PDGF) is a connective tissue cell mitogen comprising two related chains encoded by distinct genes. The B chain is the homolog of the v-sis oncogene product. Properties that distinguish these ligands include greater transforming potency of the B chain and more efficient secretion of the A chain. By a strategy involving the generation of PDGF A and B chimeras, these properties were mapped to distinct domains of the respective molecules. Increased transforming efficiency segregated with the ability to activate both alpha and beta PDGF receptors. These findings genetically map PDGF B residues 105 to 144 as responsible for conformational alterations critical to beta PDGF receptor interaction, and provide a mechanistic basis for the greater transforming potency of the PDGF B chain.
Effect of MAP2, MAP2c, and tau on kinesin-dependent microtubule motility
Authors: Heins Susanne , Song Young-Hwa , Wille Holger , Mandelkow Eckhard , Mandelkow Eva-Maria
Published: May 2013

By making use of DIC video microscopy to monitor microtubule motility we have studied the effect of several MAPs (MAP2, MAP2c, tau) on microtubule-kinesin interactions and microtubule gliding. Of the three MAPs tested, MAP2 interferes most strongly with kinesin-dependent microtubule motility.
Studies using a fluorescent analogue of kinesin
Authors: Marya P. K. , Fraylich P. E. , Flood C. R. , Rao R. , Eagles P. A. M.
Published: May 2013

The microtubule motor protein kinesin has been conjugated with 5-iodoacetamido fluorescein (5-IAF). The analogue, AF-kinesin, supports organelle motility and the movement of microtubules.
Differential regulation of vertebrate myosins I and II
Authors: Collins Kathleen , Matsudaira Paul
Published: May 2013

Cell motility events require movement of the cytoskeleton. Actin-based movement is catalyzed by the mechanoenzyme myosin, which translocates toward the barbed end of actin filaments in an ATP-dependent fashion. There are two subclasses of myosin with different structures and functions: conventional filamentous myosin (myosin II) and monomeric myosin I. Vertebrate non-muscle myosins I and II function as similar actin motors in vitro, catalyzing virtually identical actin-activated MgATP hydrolysis and motility. The functional diversification of these two enzymes results from their differential regulation. Calcium and tropomyosin, which activate the MgATP hydrolysis and motility of vertebrate non-skeletal muscle myosin II proteins, inhibit vertebrate (brush border) myosin I. The activities and regulation of brush border myosin I provide insight into conserved and unique features of the myosin mechanoenzymes and suggest how the functions of myosins I and II are divided in vertebrate cells. Brush border myosin I as an enzyme also contributes to our understanding of the molecular mechanism of motility.
The influence of pressure on actin and myosin interactions in solution and in single muscle fibres
Authors: Geeves M. A.
Published: May 2013

Studies of the molecular mechanism of motile activity require the capacity to examine the properties of individual, isolated molecular components and the properties of these same molecular components in the organised system. Pressure perturbation is one method which can be applied to motile systems at different levels of organisation. We show here that pressure perturbs a specific interaction between actin and myosin in solution and also perturbs the cycling crossbridge in a contracting muscle.
Involvement of neurofilaments in the radial growth of axons
Authors: Cleveland Don W. , Monteiro Mervyn J. , Wong Philip C. , Gill Steven R. , Gearhart John D. , Hoffman Paul N.
Published: May 2013

The control of radial growth of axons is of functional importance because caliber is a principal determinant of conduction velocity in myelinated nerve fibers. Neurofilaments, the major cytoskeletal protein in myelinated nerves, appear to be intrinsic determinants of caliber. Evidence supporting this derives first from the linear relationship between neurofilament content and axonal diameter. Further, following distal axonal injury in a peripheral nerve, caliber is reduced in the proximal axonal stumps. This reduction in caliber is itself due to selective suppression of neurofilament gene expression, thereby leading to lower levels of newly synthesized neurofilament subunits transported into the axon and a consequent decrease in axonal neurofilament content. To demonstrate directly the physiological consequence of altering normal neurofilament accumulation, we have elevated neurofilament expression by introducing additional genes into transgenic mice. The clear result is that increases in NF-L content alone are not sufficient to increase axonal caliber. To test the consequence of disruption of normal filament accumulation, we have identified dominant assembly-disrupting mutants in NF-L and NF-M and have used these to produce transgenic animals in which neurofilament assembly should be disrupted.