Essential role of germinal vesicle material in the meiotic cell cycle of Xenopus oocytes

In almost all animal species, immature oocytes are arrested naturally in the first meiotic prophase, with a large nucleus called the germinal vesicle. A number of previous studies showed that both activation of maturation/M phase-promoting factor (MPF) (assayed by semiquantitative cytological methods) and some other maturational events occur essentially normally in enucleated oocytes from many amphibian species and mice. Hence, for nearly three decades, it has generally been believed that nuclear material is dispensable for MPF activation and the meiotic cell cycle in vertebrate oocytes. Here, we have challenged this view by examining the histone H1 kinase activities and the molecular forms of MPF in experimentally manipulated Xenopus oocytes. We show that oocytes injected with nuclear material undergo much more rapid MPF activation and maturation than uninjected control oocytes. Conversely, enucleated oocytes, unlike nucleated counterparts, undergo only weak MPF activation in meiosis I and no detectable MPF reactivation in meiosis II, the latter accompanying inhibitory tyrosine phosphorylation of cdc2 kinase, the catalytic subunit of MPF. These results argue strongly that nuclear material is indispensable for the meiotic cell cycle, particularly MPF reactivation (or cdc2 tyrosine dephosphorylation) on entry into meiosis II, in Xenopus oocytes. The classical and general view may thus need reconsideration.

Malaria’s Eve: Evidence of a recent population bottleneck throughout the world populations of Plasmodium falciparum

We have analyzed DNA sequences from world-wide geographic strains of Plasmodium falciparum and found a complete absence of synonymous DNA polymorphism at 10 gene loci. We hypothesize that all extant world populations of the parasite have recently derived (within several thousand years) from a single ancestral strain. The upper limit of the 95% confidence interval for the time when this most recent common ancestor lived is between 24,500 and 57,500 years ago (depending on different estimates of the nucleotide substitution rate); the actual time is likely to be much more recent. The recent origin of the P. falciparum populations could have resulted from either a demographic sweep (P. falciparum has only recently spread throughout the world from a small geographically confined population) or a selective sweep (one strain favored by natural selection has recently replaced all others). The selective sweep hypothesis requires that populations of P. falciparum be effectively clonal, despite the obligate sexual stage of the parasite life cycle. A demographic sweep that started several thousand years ago is consistent with worldwide climatic changes ensuing the last glaciation, increased anthropophilia of the mosquito vectors, and the spread of agriculture. P. falciparum may have rapidly spread from its African tropical origins to the tropical and subtropical regions of the world only within the last 6,000 years. The recent origin of the world-wide P. falciparum populations may account for its virulence, as the most malignant of human malarial parasites.

Antiviral agent blocks breathing of the common cold virus

A dynamic capsid is critical to the events that shape the viral life cycle; events such as cell attachment, cell entry, and nucleic acid release demand a highly mobile viral surface. Protein mass mapping of the common cold virus, human rhinovirus 14 (HRV14), revealed both viral structural dynamics and the inhibition of such dynamics with an antiviral agent, WIN 52084. Viral capsid digestion fragments resulting from proteolytic time-course experiments provided structural information in good agreement with the HRV14 three-dimensional crystal structure. As expected, initial digestion fragments included peptides from the capsid protein VP1. This observation was expected because VP1 is the most external viral protein. Initial digestion fragments also included peptides belonging to VP4, the most internal capsid protein. The mass spectral results together with x-ray crystallography data provide information consistent with a “breathing” model of the viral capsid. Whereas the crystal structure of HRV14 shows VP4 to be the most internal capsid protein, mass spectral results show VP4 fragments to be among the first digestion fragments observed. Taken together this information demonstrates that VP4 is transiently exposed to the viral surface via viral breathing. Comparative digests of HRV14 in the presence and absence of WIN 52084 revealed a dramatic inhibition of digestion. These results indicate that the binding of the antiviral agent not only causes local conformational changes in the drug binding pocket but actually stabilizes the entire viral capsid against enzymatic degradation. Viral capsid mass mapping provides a fast and sensitive method for probing viral structural dynamics as well as providing a means for investigating antiviral drug efficacy.

Abrogation of the G2 cell cycle checkpoint associated with overexpression of HSIX1: A possible mechanism of breast carcinogenesis

While conducting a search for cell cycle-regulated genes in human mammary carcinoma cells, we identified HSIX1, a recently discovered member of a new homeobox gene subfamily. HSIX1 expression was absent at the onset of and increased toward the end of S phase. Since its expression pattern is suggestive of a role after S phase, we investigated the effect of HSIX1 in the G2 cell cycle checkpoint. Overexpression of HSIX1 in MCF7 cells abrogated the G2 cell cycle checkpoint in response to x-ray irradiation. HSIX1 expression was absent or very low in normal mammary tissue, but was high in 44% of primary breast cancers and 90% of metastatic lesions. In addition, HSIX1 was expressed in a variety of cancer cell lines, suggesting an important function in multiple tumor types. These data support the role for homeobox genes in tumorigenesis/tumor progression, possibly through a cell cycle function.

Functional characterization of the C—C chemokine-like molecules encoded by molluscum contagiosum virus types 1 and 2

Many viruses have evolved mechanisms for evading the host immune system by synthesizing proteins that interfere with the normal immune response. The poxviruses are among the most accomplished at deceiving their hosts’ immune systems. The nucleotide sequence of the genome of the human cutaneous poxvirus, molluscum contagiosum virus (MCV) type 1, was recently reported to contain a region that resembles a human chemokine. We have cloned and expressed the chemokine-like genes from MCV type 1 and the closely related MCV type 2 to determine a potential role for these proteins in the viral life cycle. In monocyte chemotaxis assays, the viral proteins have no chemotactic activity but both viral proteins block the chemotactic response to the human chemokine, macrophage inflammatory protein (MIP)-1α. Like MIP-1α, both viral proteins also inhibit the growth of human hematopoietic progenitor cells, but the viral proteins are more potent in this activity than the human chemokine. These viral chemokines antagonize the chemotactic activity of human chemokines and have an inhibitory effect on human hematopoietic progenitor cells. We hypothesize that the inhibition of chemotaxis is an immune evasion function of these proteins during molluscum contagiosum virus infection. The significance of hematopoietic progenitor cell inhibition in viral pathogenesis is uncertain.

Identification and characterization of differentially expressed cDNAs of the vector mosquito, Anopheles gambiae

The isolation and study of Anopheles gambiae genes that are differentially expressed in development, notably in tissues associated with the maturation and transmission of the malaria parasite, is important for the elucidation of basic molecular mechanisms underlying vector–parasite interactions. We have used the differential display technique to screen for mRNAs specifically expressed in adult males, females, and midgut tissues of blood-fed and unfed females. We also screened for mRNAs specifically induced upon bacterial infection of larval stage mosquitoes. We have characterized 19 distinct cDNAs, most of which show developmentally regulated expression specificity during the mosquito life cycle. The most interesting are six new sequences that are midgut-specific in the adult, three of which are also modulated by blood-feeding. The gut-specific sequences encode a maltase, a V-ATPase subunit, a GTP binding protein, two different lectins, and a nontrypsin serine protease. The latter sequence is also induced in larvae subjected to bacterial challenge. With the exception of a mitochondrial DNA fragment, the other 18 sequences constitute expressed genomic sequence tags, 4 of which have been mapped cytogenetically.

The circadian clock controls the expression pattern of the circadian input photoreceptor, phytochrome B

Developmental and physiological responses are regulated by light throughout the entire life cycle of higher plants. To sense changes in the light environment, plants have developed various photoreceptors, including the red/far-red light-absorbing phytochromes and blue light-absorbing cryptochromes. A wide variety of physiological responses, including most light responses, also are modulated by circadian rhythms that are generated by an endogenous oscillator, the circadian clock. To provide information on local time, circadian clocks are synchronized and entrained by environmental time cues, of which light is among the most important. Light-driven entrainment of the Arabidopsis circadian clock has been shown to be mediated by phytochrome A (phyA), phytochrome B (phyB), and cryptochromes 1 and 2, thus affirming the roles of these photoreceptors as input regulators to the plant circadian clock. Here we show that the expression of PHYB∷LUC reporter genes containing the promoter and 5′ untranslated region of the tobacco NtPHYB1 or Arabidopsis AtPHYB genes fused to the luciferase (LUC) gene exhibit robust circadian oscillations in transgenic plants. We demonstrate that the abundance of PHYB RNA retains this circadian regulation and use a PHYB∷Luc fusion protein to show that the rate of PHYB synthesis is also rhythmic. The abundance of bulk PHYB protein, however, exhibits only weak circadian rhythmicity, if any. These data suggest that photoreceptor gene expression patterns may be significant in the daily regulation of plant physiology and indicate an unexpectedly intimate relationship between the components of the input pathway and the putative circadian clock mechanism in higher plants.

RacF1, a Novel Member of the Rho Protein Family in Dictyostelium discoideum, Associates Transiently with Cell Contact Areas, Macropinosomes, and Phagosomes

Using a PCR approach we have isolated racF1, a novel member of the Rho family in Dictyostelium. The racF1 gene encodes a protein of 193 amino acids and is constitutively expressed throughout the Dictyostelium life cycle. Highest identity (94%) was found to a RacF2 isoform, to Dictyostelium Rac1A, Rac1B, and Rac1C (70%), and to Rac proteins of animal species (64–69%). To investigate the role of RacF1 in cytoskeleton-dependent processes, we have fused it at its amino-terminus with green fluorescent protein (GFP) and studied the dynamics of subcellular redistribution using a confocal laser scanning microscope and a double-view microscope system. GFP–RacF1 was homogeneously distributed in the cytosol and accumulated at the plasma membrane, especially at regions of transient intercellular contacts. GFP–RacF1 also localized transiently to macropinosomes and phagocytic cups and was gradually released within <1 min after formation of the endocytic vesicle or the phagosome, respectively. On stimulation with cAMP, no enrichment of GFP–RacF1 was observed in leading fronts, from which it was found to be initially excluded. Cell lines were obtained using homologous recombination that expressed a truncated racF1 gene lacking sequences encoding the carboxyl-terminal region responsible for membrane targeting. These cells displayed normal phagocytosis, endocytosis, and exocytosis rates. Our results suggest that RacF1 associates with dynamic structures that are formed during pinocytosis and phagocytosis. Although RacF1 appears not to be essential, it might act in concert and/or share functions with other members of the Rho family in the regulation of a subset of cytoskeletal rearrangements that are required for these processes.

Somatic Cell Mutants Resistant to Retrovirus Replication: Intracellular Blocks during the Early Stages of Infection

To identify cellular functions involved in the early phase of the retroviral life cycle, somatic cell mutants were isolated after selection for resistance to infection. Rat2 fibroblasts were treated with chemical mutagens, and individual virus-resistant clones were recovered after selection for resistance to infection. Two clones were characterized in detail. Both mutant lines were resistant to infection by both ecotropic and amphotropic murine viruses, as well as by human immunodeficiency virus type 1 pseudotypes. One clone showed a strong block to reverse transcription of the retroviral RNA, including formation of the earliest DNA products. The second clone showed normal levels of viral DNA synthesis but did not allow formation of the circular DNAs normally found in the nucleus. Cell fractionation showed that the viral preintegration complex was present in a form that could not be extracted under conditions that readily extracted the complex from wild-type cells. The results suggest that the DNA was trapped in a nonproductive state and excluded from the nucleus of the infected cell. The properties of these two mutant lines suggest that host gene products play important roles both before and after reverse transcription.

Molecular Interaction Map of the Mammalian Cell Cycle Control and DNA Repair Systems

Eventually to understand the integrated function of the cell cycle regulatory network, we must organize the known interactions in the form of a diagram, map, and/or database. A diagram convention was designed capable of unambiguous representation of networks containing multiprotein complexes, protein modifications, and enzymes that are substrates of other enzymes. To facilitate linkage to a database, each molecular species is symbolically represented only once in each diagram. Molecular species can be located on the map by means of indexed grid coordinates. Each interaction is referenced to an annotation list where pertinent information and references can be found. Parts of the network are grouped into functional subsystems. The map shows how multiprotein complexes could assemble and function at gene promoter sites and at sites of DNA damage. It also portrays the richness of connections between the p53-Mdm2 subsystem and other parts of the network.

The RD114/simian type D retrovirus receptor is a neutral amino acid transporter

The RD114/simian type D retroviruses, which include the feline endogenous retrovirus RD114, all strains of simian immunosuppressive type D retroviruses, the avian reticuloendotheliosis group including spleen necrosis virus, and baboon endogenous virus, use a common cell-surface receptor for cell entry. We have used a retroviral cDNA library approach, involving transfer and expression of cDNAs from highly infectable HeLa cells to nonpermissive NIH 3T3 mouse cells, to clone and identify this receptor. The cloned cDNA, denoted RDR, is an allele of the previously cloned neutral amino acid transporter ATB0 (SLC1A5). Both RDR and ATB0 serve as retrovirus receptors and both show specific transport of neutral amino acids. We have localized the receptor by radiation hybrid mapping to a region of about 500-kb pairs on the long arm of human chromosome 19 at q13.3. Infection of cells with RD114/type D retroviruses results in impaired amino acid transport, suggesting a mechanism for virus toxicity and immunosuppression. The identification and functional characterization of this retrovirus receptor provide insight into the retrovirus life cycle and pathogenesis and will be an important tool for optimization of gene therapy using vectors derived from RD114/type D retroviruses.

The human papillomavirus type 16 E6 and E7 oncoproteins cooperate to induce mitotic defects and genomic instability by uncoupling centrosome duplication from the cell division cycle

Loss of genomic integrity is a defining feature of many human malignancies, including human papillomavirus (HPV)-associated preinvasive and invasive genital squamous lesions. Here we show that aberrant mitotic spindle pole formation caused by abnormal centrosome numbers represents an important mechanism in accounting for numeric chromosomal alterations in HPV-associated carcinogenesis. Similar to what we found in histopathological specimens, HPV-16 E6 and E7 oncoproteins cooperate to induce abnormal centrosome numbers, aberrant mitotic spindle pole formation, and genomic instability. The low-risk HPV-6 E6 and E7 proteins did not induce such abnormalities. Whereas the HPV-16 E6 oncoprotein has no immediate effects on centrosome numbers, HPV-16 E7 rapidly induces abnormal centrosome duplication. Thus our results suggest a model whereby HPV-16 E7 induces centrosome-related mitotic disturbances that are potentiated by HPV-16 E6.

From patterns to processes: Phase and density dependencies in the Canadian lynx cycle

Across the boreal forest of North America, lynx populations undergo 10-year cycles. Analysis of 21 time series from 1821 to the present demonstrates that these fluctuations are generated by nonlinear processes with regulatory delays. Trophic interactions between lynx and hares cause delayed density-dependent regulation of lynx population growth. The nonlinearity, in contrast, appears to arise from phase dependencies in hunting success by lynx through the cycle. Using a combined approach of empirical, statistical, and mathematical modeling, we highlight how shifts in trophic interactions between the lynx and the hare generate the nonlinear process primarily by shifting functional response curves during the increase and the decrease phases.

The HIV-1 regulatory proteins Tat and Rev are frequently targeted by cytotoxic T lymphocytes derived from HIV-1-infected individuals

The HIV-1 regulatory proteins Rev and Tat are expressed early in the virus life cycle and thus may be important targets for the immune control of HIV-1-infection and for effective vaccines. However, the extent to which these proteins are targeted in natural HIV-1 infection as well as precise epitopes targeted by human cytotoxic T lymphocytes (CTL) remain to be defined. In the present study, 57 HIV-1-infected individuals were screened for responses against Tat and Rev by using overlapping peptides spanning the entire Tat and Rev proteins. CD8+ T cell responses against Tat and Rev were found in up to 19 and 37% of HIV-1-infected individuals, respectively, indicating that these regulatory proteins are important targets for HIV-1-specific CTL. Despite the small size of these proteins, multiple CTL epitopes were identified in each. These data indicate that Tat and Rev are frequently targeted by CTL in natural HIV-1 infection and may be important targets for HIV vaccines.

Dynamic localization of a cytoplasmic signal transduction response regulator controls morphogenesis during the Caulobacter cell cycle

We present evidence that a bacterial signal transduction cascade that couples morphogenesis with cell cycle progression is regulated by dynamic localization of its components. Previous studies have implicated two histidine kinases, DivJ and PleC, and the response regulator, DivK, in the regulation of morphogenesis in the dimorphic bacterium Caulobacter crescentus. Here, we show that the cytoplasmic response regulator, DivK, exhibits a dynamic, cyclical localization that culminates in asymmetric distribution of DivK within the two cell types that are characteristic of the Caulobacter cell cycle; DivK is dispersed throughout the cytoplasm of the progeny swarmer cell and is localized to the pole of the stalked cell. The membrane-bound DivJ and PleC histidine kinases, which are asymmetrically localized at the opposite poles of the predivisional cell, control the temporal and spatial localization of DivK. DivJ mediates DivK targeting to the poles whereas PleC controls its release from one of the poles at times and places that are consistent with the activities and location of DivJ and PleC in the late predivisional cell. Thus, dynamic changes in subcellular location of multiple components of a signal transduction cascade may constitute a novel mode of prokaryotic regulation to generate and maintain cellular asymmetry.