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J W Raff

Publications and source records attributed to J W Raff.

13 recordsLinked to original sources
PubMed2000

D-TACC: a novel centrosomal protein required for normal spindle function in the early Drosophila embryo.

We identify Drosophila TACC (D-TACC) as a novel protein that is concentrated at centrosomes and interacts with microtubules. We show that D-TACC is essential for normal spindle function in the early embryo; if D-TACC function is perturbed by mutation or antibody injection, the microtubules emanating from centrosomes in embryos are short and chromosomes often fail to segregate properly. The C-terminal region of D-TACC interacts, possibly indirectly, with microtubules, and can target a heterologous fusion protein to centrosomes and microtubules in embryos. This C-terminal region is related to the mammalian transforming, acidic, coiled-coil-containing (TACC) family of proteins. The function of the TACC proteins is unknown, but the genes encoding the known TACC proteins are all associated with genomic regions that are rearranged in certain cancers. We show that at least one of the mammalian TACC proteins appears to be associated with centrosomes and microtubules in human cells. We propose that this conserved C-terminal 'TACC domain' defines a new family of microtubule-interacting proteins.

Amino Acid Sequence↗
PubMed1999

The missing (L) UNC?

In many cells, centrosomes are required to position nuclei at specific locations in the cytoplasm. The nature of the link between centrosomes and nuclei is mysterious, but the recently characterised UNC84 protein appears to be involved.

Animals↗
PubMed1999

The disappearance of cyclin B at the end of mitosis is regulated spatially in Drosophila cells.

We have followed the behaviour of a cyclin B-green fluorescent protein (GFP) fusion protein in living Drosophila embryos in order to study how the localization and destruction of cyclin B is regulated in space and time. We show that the fusion protein accumulates at centrosomes in interphase, in the nucleus in prophase, on the mitotic spindle in prometaphase and on the microtubules that overlap in the middle of the spindle in metaphase. In cellularized embryos, toward the end of metaphase, the spindle-associated cyclin B-GFP disappears from the spindle in a wave that starts at the spindle poles and spreads to the spindle equator; when the cyclin B-GFP on the spindle is almost undetectable, the chromosomes enter anaphase, and any remaining cytoplasmic cyclin B-GFP then disappears over the next few minutes. The endogenous cyclin B protein appears to behave in a similar manner. These findings suggest that the inactivation of cyclin B is regulated spatially in Drosophila cells. We show that the anaphase-promoting complex/cyclosome (APC/C) specifically interacts with microtubules in embryo extracts, but it is not confined to the spindle in mitosis, suggesting that the spatially regulated disappearance of cyclin B may reflect the spatially regulated activation of the APC/C.

Animals↗
PubMed1997

LK6, a short lived protein kinase in Drosophila that can associate with microtubules and centrosomes.

A number of polyclonal mouse sera were raised against Drosophila proteins that bound to microtubules in vitro (Kellogg et al. (1989) J. Cell Biol. 109, 2977-2991). Some of these sera recognised centrosomes in vivo, and we have been using these to screen expression libraries to isolate cDNAs encoding these putative centrosomal microtubule-associated proteins. Here we report the cloning of one such cDNA that encodes a novel serine/threonine protein kinase called LK6. The protein appears to exist in two forms: an abundant 185 kDa form and a rarer approximately 220 kDa form that interacts with microtubules. At least some of the LK6 protein is located in centrosomes at all stages of the cell cycle in fly embryos. Interestingly, the protein contains a PEST-like sequence and is rapidly turned over in vivo. Constitutive overexpression of LK6 is deleterious to flies and causes defects in microtubule organisation in both eggs and early embryos, whereas constitutive overexpression of a mutant form containing a point mutation that severely impairs the kinase activity is without effect. These findings suggest that LK6 may play a role in regulating microtubule function.

Amino Acid Sequence↗
PubMed1995

Heterochromatin protein 1 distribution during development and during the cell cycle in Drosophila embryos.

Heterochromatin protein 1 (HP1) was initially discovered as a protein that is associated with the heterochromatin at the chromocenter of polytene chromosomes in Drosophila larval salivary glands. In this paper we investigate the localization of heterochromatin protein 1 in the diploid nuclei of Drosophila embryos. We focus on its association with the interphase heterochromatin in fixed embryos before and during cycle 14, the developmental time at which heterochromatin becomes most conspicuous, and also follow its localization during mitosis. The GAGA transcription factor was recently shown to be localized at sequences within alpha-heterochromatin in pre-cycle 14 embryos, and an antibody against this protein serves as a convenient marker for these sequences. We find an enrichment of heterochromatin protein 1 in the intensely DAPI-staining regions near the apical surface of nuclear cycle 10 embryos. At this stage GAGA factor is localized into punctate structures in this same region. This enrichment for HP1 is markedly increased during nuclear cycle 14. Surprisingly, whereas GAGA factor retains its association with the heterochromatin throughout the cell cycle, a significant fraction of HP1 is dispersed throughout the spindle around the segregating chromosomes during mitosis. This dispersed pool of heterochromatin protein 1 was observed during mitosis in both early and late Drosophila embryos and in an analysis of a bacterially produced 6x histidine-heterochromatin protein 1 fusion protein injected into living Drosophila embryos. When Drosophila tissue culture cells were prepared by a method which removes soluble protein and avoids fixation of the mitotic chromosomes, an enrichment for heterochromatin protein 1 in the heterochromatin of the chromosomes was discovered also.

Animals↗
PubMed1994

The Drosophila GAGA transcription factor is associated with specific regions of heterochromatin throughout the cell cycle.

In virtually all eukaryotes the centromeric regions of chromosomes are composed of heterochromatin, a specialized form of chromatin that is rich in repetitive DNA sequences and is transcriptionally relatively silent. The Drosophila GAGA transcription factor binds to GA/CT-rich sequences in many Drosophila promoters, where it activates transcription, apparently by locally altering chromatin structure and allowing other transcription factors access to the DNA. Here we report the paradoxical finding that GAGA factor is associated with specific regions of heterochromatin at all stages of the cell cycle. A subset of the highly repetitive DNA sequences that make up the bulk of heterochromatin in D. melanogaster are GA/CT-rich and we find a striking correlation between the distribution of GAGA factor and this class of repeat. We propose that GAGA factor binds directly to these repeats and may thereby play a role in modifying heterochromatin structure in these regions. Our observations demonstrate for the first time that a transcriptional regulator can associate with specific DNA sequences in a fully condensed mitotic chromosome. This may help explain how the distinctive character of a committed or differentiated cell can be maintained during cell proliferation.

Animals↗
PubMed1993

The centrosome.

Explore the source record for details and available documents.

Animals↗
PubMed1993

Drosophila gamma-tubulin is part of a complex containing two previously identified centrosomal MAPs.

gamma-tubulin is a minor tubulin that is localized to the microtubule organizing center of many fungi and higher eucaryotic cells (Oakley, B. R., C. E. Oakley, Y. Yoon, and M. C. Jung. 1990. Cell. 61: 1289-1301; Stearns, T., L. Evans, and M. Kirschner. 1991. Cell. 65:825-836; Zheng, Y., M. K. Jung, and B. R. Oakley. 1991. Cell. 65:817-823). Here we show that gamma-tubulin is a component of a previously isolated complex of Drosophila proteins that contains at least two centrosomal microtubule-associated proteins called DMAP190 and DMAP60. Like DMAP190 and DMAP60, the gamma-tubulin in extracts of early Drosophila embryos binds to microtubules, although this binding may be indirect. Unlike DMAP190 and DMAP60, however, only 10-50% of the gamma-tubulin in the extract is able to bind to microtubules. We show that gamma-tubulin binds to a microtubule column as part of a complex, and a substantial fraction of this gamma-tubulin is tightly associated with DMAP60. As neither alpha- nor beta-tubulin bind to microtubule columns, the gamma-tubulin cannot be binding to such columns in the form of an alpha:gamma or beta:gamma heterodimer. These observations suggest that gamma-tubulin, DMAP60, and DMAP190 are components of a centrosomal complex that can interact with microtubules.

Animals↗
PubMed1990

Two distinct mechanisms localise cyclin B transcripts in syncytial Drosophila embryos.

We demonstrate that two independent mechanisms act on maternally derived cyclin B transcripts to concentrate the transcripts at the posterior pole of the Drosophila oocyte and at the cortex of the syncytial embryo. The cortical accumulation occurs because the cyclin B transcript is concentrated around nuclei and comigrates with them to the cortex. The perinuclear localisation of the transcript is blocked by inhibitors of microtubule polymerisation and the transcript colocalises with microtubular structures during the cell cycle, suggesting that the transcript is associated either directly or indirectly with microtubules. Neither microtubules nor actin filaments are required to maintain the posterior concentration of cyclin B transcripts. Instead, this seems to depend on the association of the transcripts with a component of the posterior cytoplasm. The distribution pattern of the transcript at the posterior pole throughout embryogenesis and in a variety of mutant embryos suggests that this component is associated with polar granules.

Animals↗
PubMed1989

Centrosomes, and not nuclei, initiate pole cell formation in Drosophila embryos.

An injection of aphidicolin into early Drosophila embryos inhibits DNA synthesis and nuclear division, while centrosome replication and many other aspects of the mitotic cycle continue. If aphidicolin is injected at nuclear cycle 7-8, the normal migration of nuclei to the embryo cortex is completely inhibited. In most of these embryos, however, centrosomes continue to migrate in a coordinated manner to the cortex, where they reorganize tubulin, actin, and the overlying plasma membrane. Remarkably, the centrosomes that migrate to the posterior pole of such embryos initiate pole cell formation in the absence of nuclei. These observations demonstrate that centrosomes alone are able to direct a major reorganization of the cortical cytoskeleton when they arrive at the surface of the embryo. They also suggest that the coordinated movement of nuclei to the embryo cortex is mediated by forces acting on the centrosome rather than on the nucleus itself.

Actins↗
PubMed1988

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 BamHI 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.

Animals↗
PubMed1988

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 alpha, 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.

Animals↗