The transforming parasite Theileria co-opts host cell mitotic and central spindles to persist in continuously dividing cells

The protozoan parasite Theileria inhabits the host cell cytoplasm and possesses the unique capacity to transform the cells it infects, inducing continuous proliferation and protection against apoptosis. The transforming schizont is a multinucleated syncytium that resides free in the host cell cytoplasm and is strictly intracellular. To maintain transformation, it is crucial that this syncytium is divided over the two daughter cells at each host cell cytokinesis. This process was dissected using different cell cycle synchronization methods in combination with the targeted application of specific inhibitors. We found that Theileria schizonts associate with newly formed host cell microtubules that emanate from the spindle poles, positioning the parasite at the equatorial region of the mitotic cell where host cell chromosomes assemble during metaphase. During anaphase, the schizont interacts closely with host cell central spindle. As part of this process, the schizont recruits a host cell mitotic kinase, Polo-like kinase 1, and we established that parasite association with host cell central spindles requires Polo-like kinase 1 catalytic activity. Blocking the interaction between the schizont and astral as well as central spindle microtubules prevented parasite segregation between the daughter cells during cytokinesis. Our findings provide a striking example of how an intracellular eukaryotic pathogen that evolved ways to induce the uncontrolled proliferation of the cells it infects usurps the host cell mitotic machinery, including Polo-like kinase 1, one of the pivotal mitotic kinases, to ensure its own persistence and survival.

The IKK inhibitor BMS-345541 affects multiple mitotic cell cycle transitions

The IkappaB kinase (IKK) complex controls processes such as inflammation, immune responses, cell survival and the proliferation of both normal and tumor cells. By activating NFkappaB, the IKK complex contributes to G1/S transition and first evidence has been presented that IKKalpha also regulates entry into mitosis. At what stage IKK is required and whether IKK also contributes to progression through mitosis and cytokinesis, however, has not yet been determined. In this study, we use BMS-345541, a potent allosteric small molecule inhibitor of IKK, to inhibit IKK specifically during G2 and during mitosis. We show that BMS-345541 affects several mitotic cell cycle transitions, including mitotic entry, prometaphase to anaphase progression and cytokinesis. Adding BMS-345541 to the cells released from arrest in S-phase blocked the activation of Aurora A, B and C, Cdk1 activation and histone H3 phosphorylation. Additionally, treatment of the mitotic cells with BMS-345541 resulted in precocious cyclin B1 and securin degradation, defective chromosome separation and improper cytokinesis. BMS-345541 was also found to override the spindle checkpoint in nocodazole-arrested cells. In vitro kinase assays using BMS-345541 indicate that these effects are not primarily due to a direct inhibitory effect of BMS-345541 on mitotic kinases such as Cdk1, Aurora A or B, Plk1 or NEK2. This study points towards a new potential role of IKK in cell cycle progression. Since deregulation of the cell cycle is one of the hallmarks of tumor formation and progression, the newly discovered level of BMS-345541 function could be useful for cell cycle control studies and may provide valuable clues for the design of future therapeutics.

Beta1 integrin deficiency results in multiple abnormalities of the knee joint

The lack of beta1 integrins on chondrocytes leads to severe chondrodysplasia associated with high mortality rate around birth. To assess the impact of beta1 integrin-mediated cell-matrix interactions on the function of adult knee joints, we conditionally deleted the beta1 integrin gene in early limb mesenchyme using the Prx1-cre transgene. Mutant mice developed short limbed dwarfism and had joint defects due to beta1 integrin deficiency in articular regions. The articular cartilage (AC) was structurally disorganized, accompanied by accelerated terminal differentiation, altered shape, and disrupted actin cytoskeleton of the chondrocytes. Defects in chondrocyte proliferation, cytokinesis, and survival resulted in hypocellularity. However, no significant differences in cartilage erosion, in the expression of matrix-degrading proteases, or in the exposure of aggrecan and collagen II cleavage neoepitopes were observed between control and mutant AC. We found no evidence for disturbed activation of MAPKs (ERK1/2, p38, and JNK) in vivo. Furthermore, fibronectin fragment-stimulated ERK activation and MMP-13 expression were indistinguishable in control and mutant femoral head explants. The mutant synovium was hyperplastic and frequently underwent chondrogenic differentiation. beta1-null synoviocytes showed increased proliferation and phospho-focal adhesion kinase expression. Taken together, deletion of beta1 integrins in the limb bud results in multiple abnormalities of the knee joints; however, it does not accelerate AC destruction, perturb cartilage metabolism, or influence intracellular MAPK signaling pathways.

Recruitment of EB1, a master regulator of microtubule dynamics, to the surface of the Theileria annulata schizont

The apicomplexan parasite Theileria annulata transforms infected host cells, inducing uncontrolled proliferation and clonal expansion of the parasitized cell population. Shortly after sporozoite entry into the target cell, the surrounding host cell membrane is dissolved and an array of host cell microtubules (MTs) surrounds the parasite, which develops into the transforming schizont. The latter does not egress to invade and transform other cells. Instead, it remains tethered to host cell MTs and, during mitosis and cytokinesis, engages the cell's astral and central spindle MTs to secure its distribution between the two daughter cells. The molecular mechanism by which the schizont recruits and stabilizes host cell MTs is not known. MT minus ends are mostly anchored in the MT organizing center, while the plus ends explore the cellular space, switching constantly between phases of growth and shrinkage (called dynamic instability). Assuming the plus ends of growing MTs provide the first point of contact with the parasite, we focused on the complex protein machinery associated with these structures. We now report how the schizont recruits end-binding protein 1 (EB1), a central component of the MT plus end protein interaction network and key regulator of host cell MT dynamics. Using a range of in vitro experiments, we demonstrate that T. annulata p104, a polymorphic antigen expressed on the schizont surface, functions as a genuine EB1-binding protein and can recruit EB1 in the absence of any other parasite proteins. Binding strictly depends on a consensus SxIP motif located in a highly disordered C-terminal region of p104. We further show that parasite interaction with host cell EB1 is cell cycle regulated. This is the first description of a pathogen-encoded protein to interact with EB1 via a bona-fide SxIP motif. Our findings provide important new insight into the mode of interaction between Theileria and the host cell cytoskeleton.

A NOVEL FUNCTION FOR AURORA B KINASE IN THE REGULATION OF P53 BY PHOSPHORYLATION

The mitotic kinase Aurora B plays a pivotal role in mitosis and cytokinesis and governs the spindle assembly checkpoint which ensures correct chromosome segregation and normal progression through mitosis. Aurora B is overexpressed in breast and other cancers and may be an important molecular target for chemotherapy. Tumor suppressor p53 is the guardian of the genome and an important negative regulator of the cell cycle. Previously, it was unknown whether Aurora B and p53 had mutual regulation during the cell cycle. A small molecule specific inhibitor of Aurora B, AZD1152, gave us an indication that Aurora B negatively impacted p53 during interphase and mitosis. Here, we show the antineoplastic activity of AZD1152 in six human breast cancer cell lines, three of which overexpress HER2. AZD1152 specifically inhibited Aurora B kinase activity, thereby causing mitotic catastrophe, polyploidy and apoptosis, which in turn led to apoptotic death. Further, AZD1152 administration efficiently suppressed tumor growth in orthotopic and metastatic breast cancer cell xenograft models. Notably, it was found that the protein level of Aurora B kinase declined after inhibition of Aurora B kinase activity. Investigation of the underlying mechanism suggested that AZD1152 accelerated the protein turnover of Aurora B by enhancing its ubiquitination. As a consequence of inhibition of Aurora B, p53 levels were increased in tissue culture and murine models. This hinted at a possible direct interaction between p53 and Aurora B. Indeed, it was found that p53 and Aurora B exist in complex and interact directly during interphase and at the centromere in mitosis. Further, Aurora B was shown to phosphorylate p53 at several serine/threonine residues in the DNA binding domain and these events caused downregulation of p53 levels via ubiquitination mediated by Mdm2. Importantly, phosphorylation of threonine 211 was shown to reduce p53’s transcriptional activity while other phosphorylation sites did not. On a functional level, Aurora B was shown to reduce p53’s capacity to mediate apoptosis in response to the DNA damaging agent, cisplatin. These results define a novel mechanism for p53 inactivation by Aurora B and imply that oncogenic hyperactivation or overexpression of Aurora B may compromise p53’s tumor suppressor function.

Adenine nucleotide-dependent regulation of assembly of bacterial tubulin-like FtsZ by a hypermorph of bacterial actin-like FtsA.

Cytokinesis in bacteria depends upon the contractile Z ring, which is composed of dynamic polymers of the tubulin homolog FtsZ as well as other membrane-associated proteins such as FtsA, a homolog of actin that is required for membrane attachment of the Z ring and its subsequent constriction. Here we show that a previously characterized hypermorphic mutant FtsA (FtsA*) partially disassembled FtsZ polymers in vitro. This effect was strictly dependent on ATP or ADP binding to FtsA* and occurred at substoichiometric levels relative to FtsZ, similar to cellular levels. Nucleotide-bound FtsA* did not affect FtsZ GTPase activity or the critical concentration for FtsZ assembly but was able to disassemble preformed FtsZ polymers, suggesting that FtsA* acts on FtsZ polymers. Microscopic examination of the inhibited FtsZ polymers revealed a transition from long, straight polymers and polymer bundles to mainly short, curved protofilaments. These results indicate that a bacterial actin, when activated by adenine nucleotides, can modify the length distribution of bacterial tubulin polymers, analogous to the effects of actin-depolymerizing factor/cofilin on F-actin.

c-{\it mos\/} RNA expression in somatic cells and its physiological significance in cell cycle progression

The c-mos proto-oncogene, which is expressed at relatively high levels in male and female germ cells, plays a key role in oocyte meiotic maturation. The c-mos gene product in oocytes (p39$\sp{\rm c-mos}$) is necessary and sufficient to initiate meiosis. p39$\sp{\rm c-mos}$ is also an essential component of the cytostatic factor, which is responsible for arresting vertebrate oocytes at the second meiotic metaphase by stabilizing the maturation promoting factor (MPF). MPF is a universal regulator of both meiosis and mitosis. Much less is understood about c-mos expression and function in somatic cells. In addition to gonadal tissues, c-Mos has been detected in some somatic tissues and non-germ cell lines including NIH 3T3 cells as a protein termed p43$\sp{\rm c-mos}$. Since c-mos RNA transcripts were not previously detected in this cell line by Northern blot or S1 protection analyses, a search was made for c-mos RNA in NIH 3T3 cells. c-mos transcripts were detected using the highly sensitive RNA-PCR method and RNase protection assays. Furthermore, cell cycle analyses indicated that expression of c-mos RNA is tightly controlled in a cell cycle dependent manner with highest levels of transcripts (approximately 5 copies/cell) during the G2 phase.^ In order to determine the physiological significance of c-mos RNA expression in somatic cells, antisense mos was placed under the control of an inducible promoter and introduced into either NIH 3T3 cells or C2 cells. It was found that a basal level of expression of antisense mos resulted in interference with mitotic progression and growth arrest. Several nuclear abnormalities were observed, especially the appearance of binucleated and multinucleated cells as well as the extrusion of microvesicles containing cellular material. These results indicate that antisense mos expression results in a block in cytokinesis. In summary, these results establish that c-mos expression is not restricted to germ cells, but instead indicate that c-mos RNA expression occurs during the G2 stage of the cell cycle. Furthermore, these studies demonstrate that the c-mos proto-oncogene plays an important role in cell cycle progression. As in meiosis, c-mos may have a similar but not identical function in regulating cell cycle events in somatic cells, particularly in controlling mitotic progression via activation/stabilization of MPF. ^

Temporal and spatial regulation of cell division by the nucleoid in Escherichia coli

Selection of division sites and coordination of cytokinesis with other cell cycle events are critical for every organism to proliferate. In E. coli, the nucleoid is proposed to exclude division from the site of the chromosome (nucleoid occlusion model). We studied the effect of the nucleoid on timing and placement of cell division. An early cell division protein, FtsZ, was used to follow development of the division septum. FtsZ forms a ring structure (Z ring) at potential division sites. The dynamics of Z ring was visualized in live cells by fusing FtsZ with a green fluorescent protein (GFP). Emanating FtsZ-GFP polymers from the constricted septum or aggregates in daughter cells were also observed, probably representing the FtsZ depolymerization and immature FtsZ nucleation processes. We next examined the nucleoid occlusion model. Mutants carrying abnormally positioned chromosomes were employed. In chromosomal partition mutants, replicated chromosomes cannot segregate. The Z ring was excluded from midcell to the edge of the nucleoid. This negative effect of nucleoids was further confirmed in replication deficient dnaA mutants, in which only a single chromosome is present in the cell center. These results suggest that the nucleoid, replicating or not, inhibits division in the area where the chromosome occupies. In addition, increasing the level of FtsZ does not overcome nucleoid inhibition. Interestingly in anucleate cells produced by both mutants, the Z ring was localized in the central part of the cell, which indicates that the nucleoid is not required for FtsZ assembly. Relaxation of chromosomes by reducing the gyrase activity or disruption of protein translation/translocation did not abolish the division inhibition capacity of the nucleoid. However, preventing transcription did compromise the nucleoid occlusion effect, leading to formation of multiple FtsZ rings above the nucleoid. In summary, we demonstrate that nucleoids negatively regulate the timing and position of division by inhibiting FtsZ assembly at unselected sites. Relief of this inhibition at midcell is coincident with the completion of DNA replication. On the other hand, FtsZ assembly does not require the nucleoid. ^

Regulation of Alix by an autoinhibitory intramolecular interaction

In eukaryotic cells, the ESCRTs (endosomal sorting complexes required for transport) machinery is required for cellular processes such as endosomal sorting, retroviral budding and cytokinesis. The ALG-2 interacting protein Alix is a modular adaptor protein that is critically involved in these ESCRTs-associated cellular processes and consists of an N-terminal Bro1 domain, a middle V domain and C-terminal Pro-rich domain (PRD). In these cellular processes, Alix interacts with the ESCRT-III component CHMP4 at the Bro1 domain, with HIV-1 p6 Gag or EIAV p9Gag at the V domain, and with the ESCRT-I component TSG101 at the Pro-rich domain. Here we demonstrate that the N-terminal Bro1 domain forms an intramolecular interaction with C-terminal PRD within Alix. This Bro1-PRD intramolecular interaction forms a closed conformation of Alix that autoinhibits Alix interaction with all of these partner proteins. Moreover, the binding of Ca2+-activated ALG-2 to the PRD of Alix relieves the autoinhibitory intramolecular interaction, resulting in an open conformation of Alix which is able to interact with all of these partner proteins. The partner proteins bound to Alix in turn maintain Alix in the open conformation after ALG-2 dissociation with Alix. Consistent with the effect of Ca2+-activated ALG-2 on opening/activating Alix in these ESCRTs-associated functions, ALG-2 overexpression accelerates EGF-induced degradation of EGFR in an Alix-dependent manner. These findings discover an intrinsic autoinhibitory mechanism of Alix and a two-step process to activate/open Alix and then keep Alix active/open. This study has solved long-standing issues on the regulations of Alix in ESCRTs-associated functions and the role of ALG-2-Alix interaction, and may serve as the structural basis for further studies about Alix regulations. ^

Identification and analysis of novel mitotic inhibitors of the Caenorhabditis elegans Aurora B kinase

Proper execution of mitosis requires the accurate segregation of replicated DNA into each daughter cell. The highly conserved mitotic kinase AIR-2/Aurora B is a dynamic protein that interacts with subsets of cofactors and substrates to coordinate chromosome segregation and cytokinesis in Caenorhabdiris elegans. To identify components of the AIR-2 regulatory pathway, a genome-wide RNAi-based screen for suppressors of air-2 temperature-sensitive mutant lethality was conducted. Here, I present evidence that two classes of suppressors identified in this screen are bona fide regulators of the AIR-2 kinase. The strongest suppressor cdc-48.3, encodes an Afg2/Spaf-related Cdc48-like AAA+ ATPase that regulates AIR-2 kinase activity and stability during C. elegans embryogenesis. Loss of CDC-48.3 suppresses the lethality of air-2 mutant embryos, marked by the restoration of the dynamic behavior of AIR-2 and rescue of chromosome segregation and cytokinesis defects. Loss of CDC-48.3 leads to mitotic delays and abnormal accumulation of AIR-2 during late telophase/mitotic exit. In addition, AIR-2 kinase activity is significantly upregulated from metaphase through mitotic exit in CDC-48.3 depleted embryos. Inhibition of the AIR-2 kinase is dependent on (1) a direct physical interaction between CDC-48.3 and AIR-2, and (2) CDC-48.3 ATPase activity. Importantly, the increase in AIR-2 kinase activity does not correlate with the stabilization of AIR-2 in late mitosis. Hence, CDC-48.3 is a bi-functional inhibitor of AIR-2 that is likely to act via distinct mechanisms. The second class of suppressors consists of psy-2/smk-1 and pph-4.1, which encode two components of the conserved PP4 phosphatase complex that is essential for spindle assembly, chromosome segregation, and overall mitotic progression. AIR-2 and its substrates are likely to be targets of this complex since mitotic AIR-2 kinase activity is significantly increased during mitosis when either PSY-2/SMK-1 or PPH-4.l is depleted. Altogether, this study demonstrates that during the C. elegans embryonic cell cycle, regulators including the CDC-48.3 ATPase and PP4 phosphatase complex interact with and control the kinase activity, targeting behavior and protein stability of the Aurora B kinase to ensure accurate and timely progression of mitosis. ^

Logistic regression with Markov chains as covariates

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an obvious carcinogen for lung cancer. Since CBMN (Cytokinesis-blocked micronucleus) has been found to be extremely sensitive to NNK-induced genetic damage, it is a potential important factor to predict the lung cancer risk. However, the association between lung cancer and NNK-induced genetic damage measured by CBMN assay has not been rigorously examined. ^ This research develops a methodology to model the chromosomal changes under NNK-induced genetic damage in a logistic regression framework in order to predict the occurrence of lung cancer. Since these chromosomal changes were usually not observed very long due to laboratory cost and time, a resampling technique was applied to generate the Markov chain of the normal and the damaged cell for each individual. A joint likelihood between the resampled Markov chains and the logistic regression model including transition probabilities of this chain as covariates was established. The Maximum likelihood estimation was applied to carry on the statistical test for comparison. The ability of this approach to increase discriminating power to predict lung cancer was compared to a baseline "non-genetic" model. ^ Our method offered an option to understand the association between the dynamic cell information and lung cancer. Our study indicated the extent of DNA damage/non-damage using the CBMN assay provides critical information that impacts public health studies of lung cancer risk. This novel statistical method could simultaneously estimate the process of DNA damage/non-damage and its relationship with lung cancer for each individual.^

Molecular mechanisms involved in the regulation of cell polarity and genome stability by the p21 -activated kinase, Shk1, in the fission yeast, Schizosaccharomyces pombe

The essential p21-activated kinase (PAK), Shk1, is a critical component of a Ras/Cdc42/PAK complex required for cell viability, normal cell polarity, proper regulation of cytoskeletal dynamics, and sexual differentiation in the fission yeast, Schizosaccharomyces pombe. While cellular functions of PAKs have been described in eukaryotes from yeasts to mammals, the molecular mechanisms of PAK regulation and function are poorly understood. This study has characterized a novel Shk1 inhibitor, Skb15, and, in addition, identified the cell polarity regulator, Tea1, as a potential biological substrate of Shk1 in S. pombe. Skb15 is a highly conserved WD repeat protein that was discovered from a two-hybrid screen for proteins that interact with the catalytic domain of Shk1. Molecular data indicate that Skb15 negatively regulates Shk1 kinase activity in S. pombe cells. A null mutation in the skb15 gene is lethal and results in deregulation of actin polymerization and localization, microtubule biogenesis, and the cytokinetic machinery, as well as a substantial uncoupling of these processes from the cell cycle. Loss of Skb15 function is suppressed by partial loss of Shk1, demonstrating that negative regulation of Shk1 by Skb15 is required for proper execution of cytoskeletal remodeling and cytokinetic functions. A mouse homolog of Skb15 can substitute for its counterpart in fission yeast, demonstrating that Skb15 protein function has been substantially conserved through evolution. ^ Our laboratory has recently demonstrated that Shk1, in addition to regulating actin cytoskeletal organization, is required for proper regulation of microtubule dynamics in S. pombe cells. The Shk1 protein localizes to interphase and mitotic microtubules, the septum-forming region, and cell ends. This pattern of localization overlaps with that of the cell polarity regulator, Tea1, in S. pombe cells. The tea1 gene was identified by Paul Nurse's laboratory from a screen for genes involved in the control of cell morphogenesis in S. pombe. In contrast to wild type S. pombe cells, which are rod shaped, tea1 null cells are often bent and/or branched in shape. The Tea1 protein localizes to the cell ends, like Shk1, and the growing tips of interphase microtubules. Thus, experiments were performed to investigate whether Tea1 interacts with Shk1. The tea1 null mutation strongly suppresses the loss of function of Skb15, an essential inhibitor of Shk1 function. All defects associated with the skb15 mutation, including defects in F-actin organization, septation, spindle elongation, and chromosome segregation, are suppressed by tea1Δ, suggesting that Tea1 may function in these diverse processes. Consistent with a role for Tea1 in cytokinesis, tea1Δ cells have a modest cell separation defect that is greatly exacerbated by a shk1 mutation and, like Shk1, Tea1 localizes to the septation site. Molecular analyses showed that Tea1 phosphorylation is significantly dependent on Shk1 function in vivo and that bacterially expressed Tea1 protein is directly phosphorylated by recombinant Shk1 kinase in vitro. Taken together, these results identify Tea1 as a potential biological substrate of Shk1 in S. pombe. ^ In summary, this study provides new insights into a conserved regulatory mechanism for PAKs, and also begins to uncover the molecular mechanisms by which the Ras/Cdc42/PAK complex regulates the microtubule and actin cytoskeletons and cell growth polarization in fission yeast. ^

Contraction speed of the actomyosin cytoskeleton in the absence of the cell membrane

The contraction of the actomyosin cytoskeleton, which is produced by the sliding of myosin II along actin filaments, drives important cellular activities such as cytokinesis and cell migration. To explain the contraction velocities observed in such physiological processes, we have studied the contraction of intact cytoskeletons of Dictyostelium discoideum cells after removing the plasma membrane using Triton X-100. The technique developed in this work allows for the quantitative measurement of contraction rates of individual cytoskeletons. The relationship of the contraction rates with forces was analyzed using three different myosins with different in vitro sliding velocities. The cytoskeletons containing these myosins were always contractile and the contraction rate was correlated with the sliding velocity of the myosins. However, the values of the contraction rate were two to three orders of magnitude slower than expected from the in vitro sliding velocities of the myosins, presumably due to internal and external resistive forces. The contraction process also depended on actin cross-linking proteins. The lack of α-actinin increased the contraction rate 2-fold and reduced the capacity of the cytoskeleton to retain internal materials, while the lack of filamin resulted in the ATP-dependent disruption of the cytoskeleton. Interestingly, the myosin-dependent contraction rate of intact contractile rings is also reportedly much slower than the in vitro sliding velocity of myosin, and is similar to the contraction rates of cytoskeletons (different by only 2–3 fold), suggesting that the contraction of intact cells and cytoskeletons is limited by common mechanisms.

The cluA− mutant of Dictyostelium identifies a novel class of proteins required for dispersion of mitochondria

The cluA gene of Dictyostelium discoideum encodes a novel 150-kDa protein. Disruption of cluA results in clustering of mitochondria near the cell center. This is a striking difference from normal cells, whose mitochondria are dispersed uniformly throughout the cytoplasm. The mutant cell populations also exhibit an increased frequency of multinucleated cells, suggesting an impairment in cytokinesis. Both phenotypes are reversed by transformation of cluA− cells with a plasmid carrying a constitutively expressed cluA gene. The predicted sequence of the cluA gene product is homologous to sequences encoded by open reading frames in the genomes of Saccharomyces cerevisiae and Caenorhabditis elegans, but not to any known protein. The only exception is a short region with some homology to the 42-residue imperfect repeats present in the kinesin light chain, which probably function in protein–protein interaction. These studies identify a new class of proteins that appear to be required for the proper distribution of mitochondria.

Production of identical sextuplet mice by transferring metaphase nuclei from four-cell embryos

Mouse clones were produced by serial nuclear transfer commencing with the transfer of four-cell nuclei at metaphase into unfertilized ooplasts. The donor four-cell-stage nuclei were synchronized in metaphase with nocodazole. The oocytes receiving a four-cell nucleus at metaphase formed two nuclei after artificial activation and inhibition of cytokinesis with cytochalasin B. To obtain embryos with diploid sets of chromosomes, nuclei from each reconstructed embryo were transferred individually into separate enucleated fertilized one-cell embryos, thus doubling the number of identical embryos. This procedure produced a high frequency of development of reconstructed embryos to the blastocyst stage. Of 11 sets of identical embryos produced by serial nuclear transplantation, 83% developed into blastocysts, including three sets of identical septuplet blastocysts. After transfer to recipient mice, a total of 25 (57%) live young were obtained, which included one set of identical sextuplet and two sets of identical quadruplet mice.