Mitochondrial association of a plus end-directed microtubule motor expressed during mitosis in Drosophila.

The kinesin superfamily is a large group of proteins (kinesin-like proteins [KLPs]) that share sequence similarity with the microtubule (MT) motor kinesin. Several members of this superfamily have been implicated in various stages of mitosis and meiosis. Here we report our studies on KLP67A of Drosophila. DNA sequence analysis of KLP67A predicts an MT motor protein with an amino-terminal motor domain. To prove this directly, KLP67A expressed in Escherichia coli was shown in an in vitro motility assay to move MTs in the plus direction. We also report expression analyses at both the mRNA and protein level, which implicate KLP67A in the localization of mitochondria in undifferentiated cell types. In situ hybridization studies of the KLP67A mRNA during embryogenesis and larval central nervous system development indicate a proliferation-specific expression pattern. Furthermore, when affinity-purified anti-KLP67A antisera are used to stain blastoderm embryos, mitochondria in the region of the spindle asters are labeled. These data suggest that KLP67A is a mitotic motor of Drosophila that may have the unique role of positioning mitochondria near the spindle.

An inverse PCR screen for the detection of P element insertions in cloned genomic intervals in Drosophila melanogaster.

We developed a screening approach that utilizes an inverse polymerase chain reaction (PCR) to detect P element insertions in or near previously cloned genes in Drosophila melanogaster. We used this approach in a large scale genetic screen in which P elements were mobilized from sites on the X chromosome to new autosomal locations. Mutagenized flies were combined in pools, and our screening approach was used to generate probes corresponding to the sequences flanking each site of insertion. These probes then were used for hybridization to cloned genomic intervals, allowing individuals carrying insertions in them to be detected. We used the same approach to perform repeated rounds of sib-selection to generate stable insertion lines. We screened 16,100 insert bearing individuals and recovered 11 insertions in five intervals containing genes encoding members of the kinesin superfamily in Drosophila melanogaster. In addition, we recovered an insertion in the region including the Larval Serum Protein-2 gene. Examination by Southern hybridization confirms that the lines we recovered represent genuine insertions in the corresponding genomic intervals. Our data indicates that this approach will be very efficient both for P element mutagenesis of new genomic regions and for detection and recovery of "local" P element transposition events. In addition, our data constitutes a survey of preferred P element insertion sites in the Drosophila genome and suggests that insertion sites that are mutable at a rate of approximately 10(-4) are distributed every 40-50 kb.

Expression of N-terminally truncated cyclin B in the Drosophila larval brain leads to mitotic delay at late anaphase.

We have introduced an N-terminally truncated form of cyclin B into the Drosophila germ-line downstream of the yeast upstream activator that responds to GAL4. When such lines of flies are crossed to lines in which GAL4 is expressed in imaginal discs and larval brain, the majority of the resulting progeny die at the late pupal stage of development. Very rarely (< 0.1% of progeny) adults emerge that have a mutant phenotype typical of flies with mutations in genes required for the cell cycle; they have rough eyes, deformed wings, abnormal bristles, and die within hours of emergence. The brains of third instar larval progeny show an abnormally high proportion of mitotic cells containing overcondensed chromatids that have undergone anaphase separation, together with cells that cannot be assigned to a particular mitotic stage. Immunostaining indicates that these anaphase cells contain moderate levels of cyclin B, suggesting that persistent p34cdc2 kinase activity can prevent progression from anaphase into telophase.

Chiropractic diagnosis and treatment of closed head trauma.

OBJECTIVE: The objective of this article is to review the current literature relating to the chiropractic diagnosis and treatment of closed head trauma. It outlines the clinical exam, offers a diagnostic protocol and describes current chiropractic management and treatment of acute and chronic closed head trauma. Particular importance is placed on the need to differentiate between mild, moderate and severe head injury. Treatment protocols are elucidated for cerebral concussion and a rationale proposed for the management and treatment of posttraumatic concussion syndrome. DATA SOURCES: Information was obtained from English language chiropractic, medical and scientific journals as well as chiropractic and medical textbooks. The CHIROLARS data retrieval system was used (year 0-1992) as was the MEDLINE data base (1988-1992). Head trauma, head injury, headache, concussion, vertigo, posttraumatic syndrome and whiplash injury were the indexing terms used. CONCLUSION: The doctor of chiropractic is often the first practitioner a patient will see following a motor vehicle accident, sports injury or other acute trauma. The chiropractor is also the practitioner a patient seeks for help after suffering for months with chronic posttraumatic concussion syndrome. It is important that we have a protocol for effectively managing both acute and chronic closed head injury.

Discrete sequence elements control posterior pole accumulation and translational repression of maternal cyclin B RNA in Drosophila.

The concentration of cyclin B transcripts at the posterior pole of the Drosophila oocyte occurs at a late stage of oogenesis and is dependent on the sequence in the 3' untranslated part of the RNA. These transcripts become incorporated into the pole cells of the developing embryo and persist through a subsequent period of embryogenesis in which these cells are not dividing. We show that RNA injected into the posterior cytoplasm of syncytial embryos accumulates in the pole cells if it contains sequences present in the 3' untranslated region of maternal cyclin B transcripts. The injected RNA is not translated until a point prior to the resumption of mitosis by these cells, once they have become incorporated into the gonads. Zygotic transcription directed from the cyclin B promoter does not begin in the pole cells until the first instar larva has hatched. Deletion of a small sequence element from the 3' untranslated region of an epitope tagged cyclin B RNA does not affect its posterior accumulation but results in its premature translation.

3' non-translated sequences in Drosophila cyclin B transcripts direct posterior pole accumulation late in oogenesis and peri-nuclear association in syncytial embryos.

We have characterised forms of the Drosophila cyclin B transcript that differ as a result of a splicing event which removes a nucleotide segment from the 3' untranslated region. In oogenesis, both cyclin A RNA and a shorter form of the cyclin B transcript are seen in the cells of the germarium that are undergoing mitosis. The shorter cyclin B transcript alone is then detectable in the presumptive oocyte until stages 7-8 of oogenesis. Both cyclin A RNA and a longer form of the cyclin B RNA are then synthesised in the nurse cells during stages 9-11, to be deposited in the oocyte during stages 11-12. These transcripts become evenly distributed throughout the oocyte cytoplasm but, in addition, those of cyclin B become concentrated at the posterior pole. Examination of the distributions of RNAs transcribed from chimeric cyclin genes indicates that sequences in the 3' untranslated region of the larger cyclin B RNA are required both for it to become concentrated at the posterior pole and to direct those transcripts in the body of the syncytial embryo to their peri-nuclear localisation. These sequences are disrupted by the splicing event which generates smaller cyclin B transcripts.

Mitosis in Drosophila development.

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.