1995 Abstracts
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DNA Cloning: A Practical Approach, Volume 3 - Complex Genomes (Second edition)
This book is one of four which together are considerably updated and totally revised texts that arose out of the very successful "DNA Cloning, Volume I-III" books in the Practical Approach series, edited by David Glover. The contents of this third volume are diverse and cover a variety of techniques for cloning and expressing DNA molecules. This book is intended for PhD students, research assistants, postdocs, academic staff, in the general areas of molecular biology, genetics, biochemistry, and medical research.
Practical Approach series OUP, Oxford Whitfield WG, Chaplin MA, Oegema K, Parry H and Glover DM
The 190 kDa centrosome-associated protein of Drosophila melanogaster contains four zinc finger motifs and binds to specific sites on polytene chromosomes.
Microinjection of a bacterially expressed, TRITC labelled fragment of the centrosome-associated protein CP190 of Drosophila melanogaster, into syncytial Drosophila embryos, shows it to associate with the centrosomes during mitosis, and to relocate to chromatin during interphase. Indirect immunofluorescence staining of salivary gland chromosomes of third instar Drosophila larvae, with antibodies specific to CP190, indicate that the protein is associated with a large number of loci on these interphase polytene chromosomes. The 190 kDa CP190 protein is encoded by a 4.1 kb transcript with a single, long open reading frame specifying a polypeptide of 1,096 amino acids, with a molecular mass of 120 kDa, and an isoelectric point of 4.5. The central region of the predicted amino acid sequence of the CP190 protein contains four CysX2CysX12HisX4His zinc-finger motifs which are similar to those described for several well characterised DNA binding proteins. The data suggest that the function of CP190 involves cell cycle dependent associations with both the centrosome, and with specific chromosomal loci.
Journal of Cell Science 108:3377-3387Hutchison C and Glover DM (Eds)
Cell Cycle Control
What makes a cell begin the complicated process of cell division? How does it stop? What happens when things go wrong? The use of developing technologies has revealed the extraordinary degree to which cell cycle control mechanisms have been conserved through eukaryotic evolution. This book presents a detailed overview of current research in cell cycle control.
Frontiers in Molecular Biology series OUP, Oxford-
Regulation of the cell cycle during Drosophila development.
No abstract available.
In C Hutchison and DM Glover (Eds), Cell Cycle Control, OUP, Oxford Hao XF, Alphey L, Bandara LR, Lam EW, Glover D and La Thangue NB
Functional conservation of the cell cycle-regulating transcription factor DRTF1/E2F and its pathway of control in Drosophila melanogaster.
The cellular transcription factor DRTF1/E2F is implicated in the control of early cell cycle progression due to its interaction with important regulators of cellular proliferation, such as pocket proteins (for example, the retinoblastoma tumour suppressor gene product), cyclins and cyclin-dependent kinase subunits. In mammalian cells DRTF1/E2F is a heterodimeric DNA binding activity which arises when a DP protein interacts with an E2F protein. Here, we report an analysis of DRTF1/E2F in Drosophila cells, and show that many features of the pathway which regulate its transcriptional activity are conserved in mammalian cells, such as the interaction with pocket proteins, binding to cyclin A and cdk2, and its modulation by viral oncoproteins. We show that a Drosophila DP protein which can interact co-operatively with E2F proteins is a physiological DNA binding component of Drosophila DRTF1/E2F. An analysis of the expression patterns of a Drosophila DP and E2F protein indicated that DmDP is developmentally regulated and in later embryonic stages preferentially expressed in proliferating cells. In contrast, the expression of DmE2F-1 in late stage embryos occurs in a restricted group of neural cells, whereas in early embryos it is widely expressed, but in a segmentally restricted fashion. Some aspects of the mechanisms which integrate early cell cycle progression with the transcription apparatus are thus conserved between Drosophila and mammalian cells. The distinct expression patterns of DmDP and DmE2F-1 suggest that the formation of DP/E2F heterodimers, and hence DRTF1/E2F, is subject to complex regulatory cues.
Journal of Cell Science 108:2945-2954-
DNA Cloning: A Practical Approach, Volume 2 - Expression Systems (Second edition)
Expression systems are in widespread use for cloning specific genes, for synthesising the encoded proteins for structural and functional analysis, and for large-scale preparation of proteins of commercial interest. This book covers all the main expression systems in current use with background information and advice on the merits of each, step-by-step practical protocols, troubleshooting, and details of the latest applications.
Practical Approach series OUP, Oxford -
DNA Cloning: A Practical Approach, Volume 1 - Core Techniques (Second edition)
This volume provides detailed laboratory protocols, advice, hints and tips, example data, key literature citations, background information, and troubleshooting comments for the steps used in most molecular biology laboratories. It covers transformation, construction of cDNA and genomic libraries, probe preparation, analysis of gene organisation and expression, in vitro mutagenesis, and DNA sequencing. The information provided is a completely up to date account of each topic by established researchers.
Practical Approach series OUP, Oxford Ohkura H, Hagan IM and Glover DM
The conserved Schizosaccharomyces pombe kinase plo1, required to form a bipolar spindle, the actin ring, and septum, can drive septum formation in G1 and G2 cells.
We have identified a Schizosaccharomyces pombe gene with homology to the budding yeast gene CDC5, the Drosophila gene polo, and the mammalian family of genes encoding polo-like kinases. Disruption of this gene, plo1+, indicates that it is essential. Loss of plo1+ function leads to a mitotic arrest in which condensed chromosomes are associated with a monopolar spindle or to the failure of septation following the completion of nuclear division. In the latter case, cells show a failure both in the formation of an F-actin ring and in the deposition of septal material, suggesting that plo1+ function is required high in the regulatory cascade that controls septation. The overexpression of plo1+ in wild-type cells also results in the formation of monopolar spindles but also induces the formation of multiple septa without nuclear division. Septation can also be induced in the absence of mitotic commitment and concomitant spindle formation by the overexpression of plo1+ in cdc25-22 or cdc2-33 cells arrested in G2; in G1 cells arrested at Start by the cdc10-V50 mutation, or in cells lacking the cyclin B homolog cdc13 that undergo repeated S phases in the absence of mitosis.
Genes and Development 9:1059-1073Glover DM, Leibowitz MH, McLean DA and Parry H
Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles.
We show that female sterile mutations of aurora (aur) are allelic to mutations in the lethal complementation group ck10. This lies in a cytogenetic interval, 87A7-A9, that contains eight transcription units. A 250 by region upstream of both aur and a divergent transcription unit corresponds to the site of a specific chromatin structure (scs') previously proposed to be a barrier to insulate enhancers of the major hsp70 gene at 87A7. Syncytial embryos derived from aur mothers display closely paired centrosomes at inappropriate mitotic stages and develop interconnected spindles in which the poles are shared. Amorphic alleles result in pupal lethality and in mitotic arrest in which condensed chromosomes are arranged on circular monopolar spindles. The size of the single centrosomal body in these circular figures suggests that loss of function of the serine-threonine protein kinase encoded by aur leads to a failure of the centrosomes to separate and form a bipolar spindle.
Cell 81:95-105-
A physical map of the X chromosome of Drosophila melanogaster: cosmid contigs and sequence tagged sites.
A physical map of the euchromatic X chromosome of Drosophila melanogaster has been constructed by assembling contiguous arrays of cosmids that were selected by screening a library with DNA isolated from microamplified chromosomal divisions. This map, consisting of 893 cosmids, covers approximately 64% of the euchromatic part of the chromosome. In addition, 568 sequence tagged sites (STS), in aggregate representing 120 kb of sequenced DNA, were derived from selected cosmids. Most of these STSs, spaced at an average distance of approximately 35 kb along the euchromatic region of the chromosome, represent DNA tags that can be used as entry points to the fruitfly genome. Furthermore, 42 genes have been placed on the physical map, either through the hybridization of specific probes to the cosmids or through the fact that they were represented among the STSs. These provide a link between the physical and the genetic maps of D. melanogaster. Nine novel genes have been tentatively identified in Drosophila on the basis of matches between STS sequences and sequences from other species.
Genetics 139:1631-1647 Warbrick E, Lane DP, Glover DM and Cox LS
A small peptide inhibitor of DNA replication defines the site of interaction between the cyclin-dependent kinase inhibitor p21WAF1 and proliferating cell nuclear antigen.
Background: p21WAF1 is a potent inhibitor of the cell-cycle regulatory cyclin-dependent kinases (Cdks). It acts on Cdks in the G1 and S phases of the cell cycle, and also binds to proliferating cell nuclear antigen (PCNA), blocking DNA replication in vitro. Transcription of p21WAF1 can be induced by the human tumour suppressor protein p53, suggesting that the action of p21WAF1 may be important in cancer prevention. We have investigated the interaction between p21WAF1 and PCNA using a genetic two-hybrid screen and with arrays of synthetic peptides derived from the p21WAF1 protein sequence.
Current Biology 5:275-82
Results: We have established that the carboxy-terminal region of p21WAF1 interacts with PCNA in a yeast two-hybrid screen. Interaction with p21WAF1 involves the central loop of PCNA, which connects the two domains of the PCNA monomer. The interaction was finely mapped using peptides derived from the entire sequence of the p21WAF1 protein, and the critical residues were found to be QTSMTDFY (amino acids 144-151 of p21WAF1). Remarkably, a 20-residue peptide containing this sequence inhibited replication of simian virus 40 (SV40) DNA in vitro and could capture PCNA from whole cell extracts, demonstrating that small molecules can retain the biological activity characteristic of the whole protein. Sequential alanine-scan mutations of the peptide demonstrated that its ability to block replication correlates with its affinity for binding PCNA.
Conclusion: We have shown that PCNA and the cell-cycle regulator p21WAF1 interact in vivo, and that this interaction requires the central loop of PCNA and an eight amino-acid motif from the carboxyl terminus of p21WAF1. Peptides of p21WAF1 that interact with PCNA can inhibit DNA replication; such peptides or mimetics may thus prove useful in the treatment of hyper-proliferative diseases, including cancer.-
polo kinase.
No abstract available.
In DG Hardie and SK Hanks (Eds), Factsbook on Protein Kinases, Academic Press, London
