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Biochemical and morphological methods used to observe nuclear membranes of HeLa cells undergoing mitosis reveal that the cells are separated according to their domains, namely the integral membrane proteins which indicate the inner nuclear membrane, and the nuclear pore membrane which is classified into separate populations of mitotic vesicles. The inner nuclear membrane-derived vesicle initiated in anaphase associate with chromatin at the termination of mitosis, and the pore membranes of the lamina assemble during telephase and cytokinesis. The ordered reassembly of the nuclear envelope is caused by the early attachment of the inner nuclear membrane-derived vesicles to the chromatin.

The function of nim1/cdr 1 gene product (Nim1) in enhancing the mitotic process by inhibting the action of mitosis inhibtor Wee1 tyrosine kinase was studied by studying the impact of direct phosphorylation. Results showed that overproduction of Nim1 led to the hyperphosphorylation of Wee1. Inhibition of the Wee1 kinase also occurred with its phosphorylation by purified Nim1 kinase.

Twenty-three mutants that were hypersensitive to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 4-nitroquinoline-1-oxide (4-NQO) were isolated and studied for their effects on spontaneous intragenic recombination. Ten of these had no significant effects on recombination. Reactions to different concentrations of MNNG and 4-NQO were varied. Most of the nuv mutations were fully recessive and only a few were semidominant. Six nonallelic loci were identified in trans complementation analysis of seven of these mutations.

Errors in meiosis I were studied to detect diploids that give rise to disomes. A characterization of dominant allele SID1-1 was presented. Quantitation of meiotic segregation was done using the tetrad plating technique. Results showed that the SID1-1 effect does not cause any alteration in chromosome structure but decreased the incidence of complementary choromosome segregation in mitosis. Three types of segregational errors occurred in SID1-1 diploids and incidence increased in the wild types. SID1-1 errors were related to defective sister-chromatid association.

The factors responsible for cleavage asynchrony in the Tubifex embryo, in which the AB-cell divides much later than the CD-cell, were examined. Chromosome cycle studies revealed that AB-cells underwent M-phase five minutes after the CD-cell and that its prometaphase portion lasted 25 minutes longer. The length of the AB nucleus' prometaphase was reduced significantly only after the mitotic spindles of AB and CD nuclei, in a common cytoplasm, were united at one pole. Asters did not play any role in AB-cell mitotic spindle assembly. Results suggest that cleavage asynchrony in the embryo is caused by both cytoplasmic and nucleus-associated factors.

A study analyzes the in vivo distribution of DNA topoisomerase II (topo II) during the cell cycle of living Drosophila melanogaster embryos through time-lapse, three-dimensional, wide-field sequences. The nucleus is revealed to have three separate topo II populations of a spatially and temporally regulated localization. Topo II's exodus from the nucleus during mitosis and its ability to localize on sites which are precisely needed by the chromosome indicate the impact of separate pools on chromosome condensation and topo II's use in forecasting structural dynamics in chromosomes.

The disassembly of the extended microtubular cytoskeleton of an interphase cell into tubulin units andtheir reassembly into an operating mitotic apparatus is discussed. Two multiprotein complex sets were derived from the high-speed centrifugation of nocodazole-arrested Chinese hamster ovary fibroblast cells and subjected to immunoblot analysis. The possible role of these multiproteins to mitotic spindle nucleation was established.

The role of the wee 1 protein kinase n the regulation of entry into mitosis was investigated. The wee 1 protein suppresses entry into mitosis by mediating the tyrosine phosphorylation of cdc2 protein kinase (p34cdc), which triggers mitosis in eukaryotic cells. The study protocol involved the overexpression in bacteria and insect cells of the nim 1 protein, which induces mitosis in yeast cells by downregulating the wee 1 pathway. The results showed that the wee 1 protein is under negative regulation by phosphorylation, and that the nim 1 protein is a wee 1-specific kinase.

In the genus Laccaria, basidial formation, dikaryotic basidia, karyogamy, and meiosis were generally similar to structures and phenomena reported for other Agaricales. The haploid nuclei of dikaryotic basidia resided side by side in the basidium prior to karyogamy. Following karyogamy a single nucleolus was observed in L. montana (four-spored species); several nucleoli remained in the nucleus of L. tortilis (two-spored species). The haploid number of chromosomes for L. montana appeared to be n = 9. Postmeiotic mitosis typically occurred in the basidiospores resulting in binucleate basidiospores for four-spored species and quadrinucleate basidiospores for two-spored species. Postmeiotic mitosis sometimes occurred in the sterigmata and basidia proper. In instances where postmeiotic mitosis occurred in basidia, mature basidiospores were not formed and the basidia were collapsed, and contained up to eight nuclei.

The homolgy requirements for recombinational DNA repair in Drosophila melanogaster were studied using the P element-induced gap repair model in premeiotic germ cells. The results showed that recombinational repair can be achieved even with only a single copy of a sequence representing a short stretch of homology. However, the frequency of repair is highly sensitive to single-base mismatches within the stretch of homology.

A characterization of the bimA protein (BIMA) encoded by the bimAgene, which belongs to the Tetratrico Peptide Repeat (TPR) gene family, is presented. The 90-kD protein colocalizes with nuclei when overexpressed, as revealed by immunocytochemical studies. The nuclear BIMA staining pattern was granular and did not completely coincide with DNA staining by DAPI. In addition, BIMA localized at the spindle pole body when expressed at normal levels. The findings indicate that BIMA, and perhaps other mitotic TPR proteins, may play a role in spindle pole function during transition from mitotic metaphase to anaphase.

The role of the 236-kD intranuclear protein NuMA either in mitosis or in nuclear organization was examined directly. This was accomplished by expressing varioussegments of the protein in tissue culture cells. Defects in mitosis were causedby NuMA lacking either its amino terminal head or carboxy-terminal domains. Moreover, forcing the expression of wild-type human NuMA in temperature-sensitive hamster cell line that spontaneously generates post-mitotic micronuclei after degradation of the endogenous NuMA resulted in the restoration of assembly of a single, normal sized nucleus. Thus, NuMA is essential for mitotic spindle function and post-mitotic nuclear assembly.

The formation of small nuclear ribonucleoprotein-containing coiled bodies (CBs) is a kinetic process regulatedduring interphase and mitosis. p80 coilin, the coiled body antigen, is demonstrated to be a phosphoprotein that is specifically phosphorylated on at least two additional sites during mitosis. p80 coilin was found in all cell types studied, even in those in which CBs are not prominent but under specific metabolic conditions.

Proteasomes are nonlysosomal proteinase complexes present in all eukaryotic cells. Despite their ubiquity, their physiological role remains unclear. An investigation was conducted to test whether proteosomes have a rolein cyclin degradation. The procedure involved the determination of the intracellular distribution of proteosomes during the cell cycle and differentiation of the granulosa cell system. The results showed that proteosomes assume a distribution pattern resembling that of cyclins during division. It is proposed that proteosomes function as mitosis terminators by degrading cyclins.

The mitotic spindles of Schizosaccharomyces pombe were reconstructed from electron-microscopic cross sections and the distribution of microtubules (MTs) analyzed by computer. Chromosome attachment to the spindle is by way of kinetochore MTs, which were observed to decrease in number as mitosis progressed. Spindle elongation is thought to result from a combination of MT interdigitation and tubulin polymerization. Separation of daughter nuclei was caused by anti-parallel sliding of adjacent MTs. These results indicate that mitosis in S. pombe is similar to mitosis in other eukaryotic cells.

The involvement of intermediate filament-associated protein (IFAP) 300 and protein kinases in the reorganization of the intermediate filament (IF) network during mitosis in BHK-21 baby hamster kidney cells were investigated. The results showed that during mitosis, IFAP 300 reorganizes into a punctate, nonfilamentous pattern, similar to that assumed by vimentin. In addition, the same two protein kinases, one of which is p34(super cdc2), which phosphorylates vimentin during mitosis also phosphorylates IFAP 300. These results elucidate the roles of IFAP 300 during IF assembly and protein kinases during disassembly at mitosis.

The role of spectrin in the morphological and cell surface property changes during mitosis was investigated. Spectrin is a membrane-associated actin-binding protein which helps in the assembly of the membrane skeleton of postmitotic cells. The study protocol compared the distribution and solubility of spectrin in culture cells during interphase and mitosis. The results indicated that spectrin is phosphorylated during mitosis and is redistributed to the cytosol. This was correlated with the restructuringof the actin filament networks in the plasma membrane during mitosis.

A 62-kD protein substrate of a calcium/calmodulin-dependent protein kinase shows distinct properties from that of cyclin. It is constantly present in the cellular protein pool in the first two cycles in sea urchin and surf clam embryos which was very different from cyclin. Furthermore, the synthesis of this protein is not cyclical in process. Electron microscopy shows interactions of this protein with microtubules of the mitotic apparatus during cell division.

Fluorescence photoactivation demonstrated the movements of kinetochore fiber microtubules during metaphase and anaphase. Kinetochore microtubules were suggested to altertheir length through polymerization and depolymerization at the kinetochores during the two particular phases of mitosis. This occurence transpired as the kinetochores migrated toward and backward the poles. Only depolymerization was believed to be possible at the pole during the two mitotic stages. The results implied that two molecular processes influences the poleward force production for migration of chromosomes.

The dynamic self-organization of spindle poles in mitosis may require kinesin-related motor proteins such as Eg5 derived from Xenopus eggs. In vitro assays for spindle assembly involving Eg5 found that the immunodepletion of this protein influenced spindle formation. In addition, Eg5 was identified as a plus-end-directed microtubule motor protein. These results clarify the role of microtubule motor proteins in cell division and mitosis.

A new kinesin protein has been discovered that is also a microtubule-dependent motor enzyme participating in mitosis. The new protein was first described as a spindle antigen necessary for mitotic progression. The cloning of this protein's gene revealed a resemblance to the motor domain of kinesin-like proteins. The protein is able to cross-bridge antiparallel microtubules in vitro and to assist the microtubules in sliding over each other as must occur for spindles to elongate.

CENP-E, a kinesin-like motor protein that builds up during the G2 portion of the cell cycle, has been linked to the centromere-kinetochore protein complex that regulates several phases of cell division. These phases include the movement and congression of chromosome pairs. CENP-E also assists in moving the chromosomes to spindle midzone at anaphase but is removed when cell division is completed.

The role of motor proteins in the chromosome segregation that takes place during cell division is now known in greater detail. The proteins assemble into a structure called the kinetochore, which controls the linking of chromosomes to the mitotic spindle microtubules. The kinetochore allows the CBF3 protein complex to attach the DNA to the microtubules. Moreover, the CBF3 complex includes a minus-end-directed microtubule-based motor protein. These motors probably join microtubules and chromosomes together during mitosis.

A series of Saccharomyces cerevisiae mutant strains were generated by mutating centromere DNA (CEN) elements which did not markedly affect chromosome segregation. Characterization of these mutants showed that they could undergo DNA synthesis in similar rates as wild-type kinetics, but were delayed near the metaphase/anaphase transition. It is proposed that such a delay constitutes a mechanism to stabilize chromosomes with defective centromeres. This mechanism may be mediated by a surveillance system that monitors the completion of chromosomal attachment to the spindle fibers.

Analysis of rapid mitotic cycles in the Drosophila embryo show that these cycles constrain genetically-determined development during short 'phenocritical' periods by curtailing the length of genetic transcripts. The analysis concerned two Drosophila genes, knirps gene and knirps-related gene, which help determine abdomen segmentation. The necessary coordination of mitotic cycle duration and gene size probably works against rapid evolutionary change.

The mechanism of H1 histone kinase activity potentiation by cyclin A was investigated. Human cyclin A produced in Escherichia coli was added to extracts of parthenogenetically activated Xenopus eggs. The results showed that cyclin A forms a complex with cdc2, and this complex accelerates the conversion of inactive maturation-promoting factor into its active form by promoting dephosphorylation of cdc2 on tyrosine 15.

The mechanism by which cyclin A and cyclin B regulate mitosis by microtubule dynamics was investigated. Studies using Xenopus egg extracts showed that both cyclins act on the same kinase catalytic subunit, the cdc2 protein family member p34. Cyclin B-dependent kinase caused dramatic shortening of centrosome-nucleated microtubules, whereas cyclin A did not. Fluorescence video microscopy showed that cyclin B-dependent kinase induced microtubule shortening by increasing catastrophe frequency. A dynamical model based on four parameters and defining a threshold between unlimited microtubule growth and the establishment of steady-state dynamics was presented.

The Thr-34, Thr-46 and Ser-72 residues of pp60(super c-src) are the phosphorylation sites which are activated by p34(super cdc2)-mediated phosphorylation during mitosis. Site directed mutagenesis on these mitosis-specific phosphorylation (MSP) sites were conducted to elucidate the signaling pathway linking p34(super cdc2) and pp60(super c-src). The results showed that pp60(super c-src) is activated in a two-step process which involves MSP-mediated partial dephosphorylation of Tyr-527 of p60(super c-src) which then stimulates c-Src kinase activity.

The cdc25 protein is a specific protein tyrosine phosphatase involved in the activation of the p34 protein encoded by cdc2. The expression and cellular distribution of cdc25 throughout the cell cycle of nontransformed mammalian firbroblasts was investigated. The result showed that at the boundary between prophase and metaphase, the cdc protein is predominantly distributed in the cytoplasm. In contrast, at interphase and early prophase, the protein was localized within the nucleus. The compartmentalization of cdc25 during the cell cycle may form part of a mechanism regulating its activity.

Mutations in the yeast MDM1 gene results in a defective transfer of nuclei and mitochondria into developing daughter buds. The yeast MDM1 gene was isolated by complementation experiments and further characterized. The results showed that MDM1 codes for a protein associated with novel punctate structures distributed throughout the yeast cytoplasm. Gene disruption experiments demonstrated that MDM1 is required for mitotic growth. The results suggest that the MDM1 proteins are involved in organelle inheritance.

Saccharomyces cerevisiae genes KIP1 and CIN8 are kinesin domain-containing genes which are involved in mitotic spindle assembly. The genes were expressed and their proteins, Kip1p and Cin8p, were further characterized. The results showed that Kip1p and Cin8p perform essential but overlapping functions. Cin8p is required for pole separation during mitotic spindle assembly, and is a component of the mitotic spindle. Furthermore, at least one of the two proteins are required to separate duplicated spindle poles during the assembly of the mitotic spindle. Both proteins appear to interact with the spindle microtubules to generate the force required for separation.

Little is known about the physiological role of protein-tyrosine-phosphatases (PTPs), which act specifically on phosphotyrosyl residues. Using a BHK cell line overexpressing wild type T-cell PTP (TC.PTP) and a C-terminal truncated version of the enzyme, its physiological role was elucidated. The results showed that cells overexpressing the truncated enzyme became highly multinucleate, through an apparent failure of cytokinesis. Furthermore, the nuclear division among these cells occured asynchronously. These results suggest that PTPs are involved in furrow development during cytokinesis and in the signaling events that coordinate this process.

Three distinct regions of KAR1 protein that functions in the protein's mitotic and karyogamy activities were identified. Deletion analysis showed segregation and independent functions of the three sites. One region was required for mitosis and takes part in protein association with spindle pole body. The second domain was made of hydrophobic fragments and possessed both mitotic and karyogamy functions while the activity of the third region was restricted to nuclear fusion. These data suggested a KAR1 protein model with several protein-binding sites fastened to the nuclear membranes through its hydrophobic tail.

A method for studying sister chromatid separation in vitro was developed in a study of sister chromatid interlinkages. Sperm nuclei in frog egg extracts underwent DNA replication and cytostactic factor-arrested extracts induced metaphase spindle formation. Addition of calcium initiated anaphase and inactivated maturation promoting factor which resulted in sister chromatid separation. The presence of type II topoisomerases was needed for the separation of sister chromatids at the metaphase-anaphase stage, but chromosome segregation was prevented by either incomplete DNA replication or the presence of DNA topoisomerase II inhibitors.

The uneven postmitotic apportionment of oncogene-encoded proteins were studied in computer-assisted image analysis. Results show uneven mitotic apportionment of proteins with different subcellular localization, distinct biochemical properties and dissimilar functions. There is also unequal partitioning of DNA (26, 27), total RNA (19,28) and total protein (29) contentsin cultured cells. These results suggest that postmitotic cells expressing higher levels may be closer to the G1 to S transition and that cells with low levels would have a longer time of residency in G1.

The roles of intracellular andextracellular calcium ions (Ca2+) during the meiotic and mitotic events in early development were investigated using mouse oocytes. Metaphase II oocytes proceeded to anaphase and subsequent developmental events in response to intracellular Ca2+, but not to extracellular Ca2+. In contrast, meiosis I was arrested in the absence of extracellular Ca2+. Germinal vesicle breakdown was shown to be Ca2+ independent, whereas nuclear envelope breakdown is only partially dependent on Ca2+.

Cylins are a class of proteins invloved in the regulation of the cell cycle in various eukaryotes. A cyclin cDNA clone, called cycAt, was isolated from an Arabidopsis thaliana DNA library and characterized. Sequence analysis revealed the presence of a domain homologous to the mitotic cyclin box. Northern blot analysis showed that cytAt is expressed predominantly in the G2-phase nuclei of actively dividing cells. Microinjection of its transcripts in Xenopus oocytes led to meiotic maturation. Further analysis revealed the presence of at least three classes of cyclins in Arabidopsis.

The effects of eight mutations of the mus locus of Aspergillus nidulans on mitotic recombination were studied. Two of the mutations increased, while another two decreased inter- and intragenic recombination. The musO mutation increased only intragenic recombination. The product of the musL mutation appeared necessary for recombination and its absence resulted in chromosome loss. The behavior of some mutants suggested differential effects on the related events of mitotic gene conversion and crossing over.

Characterization of the kinase activityof the mitotic inhibitor, p107(super wee1) reveal that it effects its inhibitory function by phosphorylating the serine/threonine kinase p34(super cdc2) on the tyr-15 residue. Studies show that this phosphorylation occurs onlyif the substrate is p34(super cdc2) complexed with cyclin, as both monomeric p34(super cdc2) and tyr-15 containing peptides remained unaffected. Furthermore results from gel filtration studies show that p107(super wee1) contains both serine and tyrosine kinase activity and is capable of functioning as a dual- specificity enzyme.

Recombinant DNA techniques were used to clone and sequence avian cyclin B2 proteins and to study its subcellular distribution. It was observed that cyclinB2 proteins are predominantly cytoplasmic at interphase, but abruptly translocate to the nucleus at prophase. In a separate study, the expression of non-destructible cyclin B2 mutant in HeLa cells caused cell cycle arrest at a state characterized by condensed chromatin, disassembled nuclear envelope and multiple mitotic spindles. The multiple spindles indicate that the centrosome cycle had continued regardless of cell cycle arrest.

The purine analogue 2-aminopurine (2-AP) is a protein kinase inhibitor which can cause S-phase-arrested cells to enter mitosis. Experiments were conducted to test the ability of 2-AP to advance the cell cycle after several stage-specific blockages. The results showed that 2-AP could override cell cycle blocks regardless of the stage inhibited. Therefore, a common factor is involved in regulating most of the stages of the cell cycle. This factor may be2-AP-sensitive protein kinase. The results may have implications in the treatment of tumors.

The phosphorylation of the retinoblastoma-susceptibility gene product (RB) was monitored at different stages in the cell cycle of human T lymphocytes. The initial cell population was arrested at G(sub 0) and induced to divide synchronously after mitogenic treatment. The results showed that RB phosphorylation occurs first, at mid- G(sub 1) phase. The second phosphorylation occurs at the S phase, and the thirdat G(sub 2)/M. These events occur on different residues of RB. These results suggest that the different phosphorylated forms of RB may be involved in growthregulation at different stages in the cell cycle.

The role of reversible phosphorylation in mammalian cell division cycle was investigated by studying the function and distribution of protein phosphatase type 1 (PP1), one of the most highly conserved enzymes in mammals. Immunolocalization studies revealed that PP1 is mainly cytoplasmic at G1 and S phases of the cell cycle, but accumulates in the nucleus at G2 and tightly associates with condensed chromosomes during mitosis. Microinjection of anti-PP1 antibodies into anaphasecells prevented exit from mitosis. These results show that PP1 may have a role in mitotic exit of mammalian cells.

Laser photobleaching experiments demonstrate that kinetochore disassembly causes the shortening of stationary kinetochore fiber microtubules during mitotic anaphase, afterwhich chromosomes move toward the spindle equator in metaphase. These kinetochore movements are mediated by cytoplasmic dynein located in their fibrous corona, and kinesin which causes minus and plus-end directed movements, respectively. Microtubule assembly and disassembly processes also contribute to chromosomal movement force generation.

The protein synthesis and degradation requirements during the meiotic and mitotic events after fertilization of oocytes of the clam Spisula solidissima was characterized by treating fertilized oocytes with protein synthesis inhibitors at various stages. The main findings showed that germinal vesicle breakdown, meiosis I and progressioninto meiosis II are unaffected by protein synthesis. Protein inhibition at the mitotic cell cycle leads to arrest at the G2-M transition, whereas destruction of cyclins appeared necessary for oocytes to leave M phase.

Mitotic activity in cells involves a transient increase in intracellular free calcium (Ca2+) secondary to phosphatidylinositol 4,5-biphosphate (PIP2) hydrolysis. Following injection of PIP2 antibody, a significant decrease in PIP2 hydrolysis products and CA2+ were observed. The same effect was seen with increasing doses of heparin and Ca2+ chelators. Consequently, nuclear envelope breakdown is suppressed and mitosis is arrested.

Purification techniques on rat neurons revealed the presence of a 55 kilodalton protein capable of regulating Schwann cell (SC) proliferation despite the presence of mitogenic stimuli in vivo and in vitro. Like stromelysin, this Neural Antiproliferative Protein is capable of cleaving fibronectin which on proteolysis generates another SC antiproliferative protein. Quiescent SCs appear to down-regulate growth factor receptors despite the presence of these antiproliferative fragments.