1988 Abstracts

Spruce BA, Jackson S, Lowry PJ, Lane DP and Glover DM

Monoclonal antibodies to a proenkephalin A fusion peptide synthesized in Escherichia coli recognize novel proenkephalin A precursor forms.

Monoclonal antibodies have been generated to a chimeric peptide comprised of Escherichia coli ß-galactosidase fused to the amino acid sequence 69-207 of human preproenkephalin A. Two monoclonal antibodies, PE-1 and PE-2, were identified by their ability to recognize the same segment of proenkephalin A fused to the cII gene product of the E. coli bacteriophage ?. The binding domains of PE-1 and PE-2 have been broadly located, with respect to the primary translation product, within the amino acid sequences 152-207 and 84-131, respectively. Immunoblot analysis of total bovine adrenomedullary chromaffin granule lysate reveals PE-1 and PE-2 immunoreactive forms of observed molecular mass 35, 33, 29, 24, 22, and 15 kDa, and an 18-kDa PE-1 immunoreactive form. Separation of granule membranes from their contents reveals differential membrane association of these high molecular weight polypeptides. There is preliminary evidence that PE-1 may be detecting a subset of polypeptides where shortening from the NH2 terminus has occurred. We postulate that the 35-kDa form represents the intact bovine enkephalin precursor of predicted molecular mass 27.3 kDa. This experimental approach should be generally applicable to the generation of antibodies which will recognize intact peptide precursors together with their post-translational cleavage products.

Journal of Biological Chemistry 263:19788-19795

Raff JW and Glover DM

Nuclear and cytoplasmic mitotic cycles continue in Drosophila embryos in which DNA synthesis is inhibited with aphidicolin.

We have microinjected aphidicolin, a specific inhibitor of DNA polymerase a, into syncytial Drosophila embryos. This treatment inhibits DNA synthesis and, as a consequence, nuclear replication. We demonstrate that under these conditions several cycles of both centrosome replication and cortical budding continue, although the cycles have a longer periodicity than is normally found. As in uninjected embryos, when the cortical buds are present, the embryos have nuclei containing decondensed chromatin surrounded by nuclear membranes as judged by bright annular staining with an anti-lamin antibody. As the buds recede, the unreplicated chromatin condenses and lamin staining becomes weak and diffuse. Thus, both cytoplasmic and nuclear aspects of the mitotic cycle continue following the inhibition of DNA replication in the Drosophila embryo.

Journal of Cell Biology 107:2009-2019

Hayward DC and Glover DM

Analysis of the Drosophila rDNA promoter by transient expression.

We have examined the expression of the bacterial gene chloramphenicol acetyl transferase (CAT) under the control of the Drosophila rDNA promoter following transfection into Drosophila tissue culture cells. Constructs having an entire NTS, corresponding to approximately 3640 base pairs of upstream rDNA sequence, or constructs with 306 base pairs of upstream sequence respectively, are transcribed at 5 fold or 2 fold higher levels than a construct with 43 base pairs of upstream DNA. In co-transfection experiments, the construct with the entire NTS competes for transcription 20 fold more effectively than the construct with 306 base pairs of upstream sequence. Constructs having either 72 base pairs or 60 base pairs of upstream rDNA sequences, on the other hand, are transcribed very much less efficiently than constructs with either 306 bp or with only 43 bp of upstream DNA. These sequences, which reduce levels of rDNA transcription in the absence of additional upstream DNA, lie in a region in which the rDNA promoter differs from its duplications within the NTS.

Nucleic Acids Research 16:4253-4268

Glover DM, Axton J, Cheshire A, Delaney S, Freeman M, Girdham C, Gonzalez C, Karess R, Leibowitz M, Llamazares S, Millar S, O'Sullivan D, Raff J, Saunders R, Sunkel C and Whitfield WGF

Mitosis in Drosophila: the centrosome cycle in early embryogenesis.

No abstract available.

In D Beach, C Basilico and J Newport (Eds), Cell Cycle Control in Eukaryotes, Cold Spring Harbor Laboratory Press, New York

Speek M, Raff JW, Harrison-Lavoie K, Little PF and Glover DM

smart2, a cosmid vector with a phage lambda origin for both systematic chromosome walking and P-element-mediated gene transfer in Drosophila.

We describe a new phage-lambda-replicon-based cosmid vector suitable for both chromosome walking and P-element-mediated transformation in Drosophila. Its unique BamW cloning site is flanked by the promoters for the SP6 and T7-encoded RNA polymerases, permitting the synthesis of probes complementary to the ends of the cloned inserts for library screening. The selectable marker is tet for bacterial cell transformation and neo for Drosophila transformation expressed under the control of the Drosophila hsp70 promoter.

Gene 64:173-177

Whitfield WG, Millar SE, Saumweber H, Frasch M and Glover DM

Cloning of a gene encoding an antigen associated with the centrosome in Drosophila.

The monoclonal antibody Bx63 recognizes a centrosomal antigen of Drosophila melanogaster by indirect immunofluorescence and identifies two proteins, with apparent molecular weights of 185 x 103 and 66 x 103, on Western blots. We have used this antibody to isolate five clones (?cs1, -2, -3, -4 and ?j63) from ?gt11 expression libraries of Drosophila DNA. Using polyclonal anti-centrosomal sera raised against both immunoaffinity-purified Bx63 antigen and electrophoretically purified fusion protein from clone ?cs3, we have demonstrated that the fusion proteins encoded by four of these clones (?cs1-4) share at least two epitopes with the 185 x 103 Mr centrosomal antigen. This indicates that clones ?cs1-4 contain DNA from the gene coding for this protein. The four clones are independent isolates from a single chromosomal site, which we show by in situ hybridization to correspond with salivary gland chromosome region 88E 4-8. A low-abundance transcript of approximately 4.0 x 103 bases corresponding to the cloned gene is detected in all stages of the Drosophila life-cycle.

Journal of Cell Science 89:467-480

Sunkel CE and Glover DM

polo, a mitotic mutant of Drosophila displaying abnormal spindle poles.

Neuroblast cells in larvae homozygous for mutant alleles of the locus polo show a high frequency of metaphases in which the chromosomes have a circular arrangement, and anaphase figures in which chromosomes appear to be randomly oriented with respect to at least one of the spindle poles. These defects appear to lead to the production of polyploid cells. Sex chromosome disjunction is affected in male meiosis, primarily in the second division, and the meiotic spindles of living cells are abnormal. One allele is a larval lethal, whereas another is semi-lethal with about 7% of homozygotes surviving as adults. Embryos from homozygous polo females have aberrant mitotic spindles that are highly branched and have broad poles. Immunofluorescence studies with an antibody that recognizes an antigen associated with the centrosome indicate that the organization of this organelle is disrupted in the mutant embryos.

Journal of Cell Science 89:25-38

BD Hames and DM Glover (Eds)

Transcription and splicing

This book gives a co-ordinated review of our present knowledge of eukaryotic RNA synthesis

Frontiers in Molecular Biology series OUP, Oxford

BD Hames and DM Glover (Eds)

Molecular Immunology

Like other volumes in the Frontiers in Molecular Biology series, this book presents an up-to-date picture of current research topics in a key area. The contributors to this book have been carefully chosen to reflect the main thrusts of research effort across a broad range within the rapidly developing field of immunology. Subjects covered include immunoglobulin gene rearrangement, the MHC antigens, B-cell activation, and the complement system.

Frontiers in Molecular Biology series OUP, Oxford

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