Chromosome Cycle Research Articles

Gene expression profiling of head and neck squamous cell carcinoma using cDNA microarrays

The molecular mechanisms causing the development of squamous cell carcinomas in the head and neck region are mostly unknown. Identification of molecular changes which are involved in carcinogenesis might play a key role in improving the diagnosis, therapy and prognosis of patients with carcinomas in the head and neck. The purpose of the study was to identify transcriptional alterations of apoptosis associated genes between normal mucosa and tumor tissue. We measured the mRNA expression of 408 apoptosis associated genes by microarray-technique in normal upper aerodigestive tract mucosa (n=4), and in cancer tissue (n=8) of squamous cell carcinomas of the upper aerodigestive tract. RT-PCR was performed to confirm the microarray results. A hierarchial cluster analysis, based on 22 selected marker-genes showing a separation of the two tissue types supports the hypothesis of a specific expression pattern associated with tumor development. Additionally, we found 11 genes associated with anti-apoptotic processes to be upregulated while 12 genes associated with proapoptotic functions as well as 5 DNA-replication and chromosome cycle associated genes were found to be downregulated in the tumor tissue. Furthermore 6 of 8 genes which are known to be associated with lymphocyte activation were upregulated in tumor edge tissue. These results represent a first step for the diagnostic use of microarrays in squamous cell carcinomas of the head and neck region and might improve the understanding of the molecular mechanisms of carcinogenesis in the head and neck region.

Alternative splicing and protein function

BackgroundAlternative splicing is a major mechanism of generating protein diversity in higher eukaryotes. Although at least half, and probably more, of mammalian genes are alternatively spliced, it was not clear, whether the frequency of alternative splicing is the same in different functional categories. The problem is obscured by uneven coverage of genes by ESTs and a large number of artifacts in the EST data.ResultsWe have developed a method that generates possible mRNA isoforms for human genes contained in the EDAS database, taking into account the effects of nonsense-mediated decay and translation initiation rules, and a procedure for offsetting the effects of uneven EST coverage. Then we computed the number of mRNA isoforms for genes from different functional categories. Genes encoding ribosomal proteins and genes in the category "Small GTPase-mediated signal transduction" tend to have fewer isoforms than the average, whereas the genes in the category "DNA replication and chromosome cycle" have more isoforms than the average. Genes encoding proteins involved in protein-protein interactions tend to be alternatively spliced more often than genes encoding non-interacting proteins, although there is no significant difference in the number of isoforms of alternatively spliced genes.ConclusionFiltering for functional isoforms satisfying biological constraints and accountung for uneven EST coverage allowed us to describe differences in alternative splicing of genes from different functional categories. The observations seem to be consistent with expectations based on current biological knowledge: less isoforms for ribosomal and signal transduction proteins, and more alternative splicing of interacting and cell cycle proteins.

The chromosome cycle: coordinating replication and segregation

During the cell-division cycle, chromosomal DNA must initially be precisely duplicated and then correctly segregated to daughter cells. The accuracy of these two events is maintained by two interlinked cycles: the replication licensing cycle, which ensures precise duplication of DNA, and the cohesion cycle, which ensures correct segregation. Here we provide a general overview of how these two systems are coordinated to maintain genetic stability during the cell cycle.

Cycles

Cycles

Caenorhabditis elegans Elongin BC complex is essential for cell proliferation and chromosome condensation and segregation during mitosis and meiotic division II

The ubiquitin-mediated protein degradation system is involved in a wide variety of cellular functions. The RING-H2 finger protein RBX1 is a common subunit of Cullin-based ubiquitin ligases. Caenorhabditis elegans RBX1 and CUL2 are essential for regulating chromosome condensation and segregation during mitosis and meiosis and are also critical for cell proliferation. Here, we demonstrate that Elongin B (ELB1) and C (ELC1) form a stable complex, and that depletion of either gene product by RNA-mediated interference (RNAi) causes pronounced defects in the second meiotic division. Embryos and adults that escape meiotic arrest have several irregular phenotypes. These include defects in mitotic chromosomal condensation and segregation, pronuclear rotation, and germ cell proliferation, abnormal cortical protrusion, and accumulation of the cyclin-dependent kinase inhibitor CKI1. All these defects are consistent with those found after depletion of CUL2. In addition, direct interaction between ELC1 and CUL2 is revealed by bacterial two-hybrid analysis. Thus, the RBX1/CUL2/ELC1/ELB1 complex acts as an E3 ubiquitin ligase in C. elegans and is essential for diverse functions relevant to chromosomal dynamics and cell cycle control.

Pairing, connecting, exchanging, pausing and pulling chromosomes

The British Society for Cell Biology meeting on the Cell‐Cycle Regulation of Meiosis took place in Newcastle, UK, between 13 and 15 September 2004, and was organized by M. Herbert, K. Jones, M. Whitaker and M. Levasseur ![][1] The British Society for Cell Biology autumn meeting on the Cell‐Cycle Regulation of Meiosis encompassed a unique blend of disciplines from cytology to biochemistry, and covered topics ranging from recombination to human infertility. Meiosis shares much of the same cell‐cycle machinery as mitosis, but also includes several unique features and regulatory pathways. As two consecutive rounds of chromosome segregation (meiosis I and II) follow a single round of chromosome replication, four haploid nuclei are made from a single diploid precursor cell. The unique event in meiosis is the segregation of homologous chromosomes (homologues) at meiosis I (Fig 1). Figure 1. Mitotic and meiotic chromosome cycles. In mitotic cells, connections called sister‐chromatid cohesion are formed during replication and hold the sister chromatids together until they are ready to be segregated at anaphase (i–iii). When the two kinetochores of a sister‐chromatid pair attach to microtubules that emanate from opposite poles of the cell, the pulling forces of the spindle are resisted by the cohesion between sister centromeres (iii). The resulting tension stabilizes microtubule attachments. When all chromatid pairs have achieved this biorientation on the spindle, sister‐chromatid cohesion is destroyed and the chromatids are pulled to the cell poles (iv). During meiotic prophase I, maternal and paternal homologues pair and become connected by chiasmata (v–vii). The combination of chiasmata and cohesion allows the homologue pair to be orientated on the spindle, similar to the way in which a pair of sister chromatids would be aligned during mitosis (vii). When cohesin is destroyed, the homologues are segregated (viii). A second round of segregation then occurs without an intervening … [1]: /embed/graphic-1.gif

Cytogenetic Analysis of Partial Elimination of Germ Line Limited Chromosomes from Primary Germ Cells in Acricotopus lucidus (Diptera, Chironomidae)

Unusual mitotic behavior of additional chromosomes present only in the germ line was reported for the dipteran families Cecidomyiidae, Chironomidae and Sciaridae, which are known to have complex chromosome cycles. In the chironomid Acricotopus lucidus all germ line limited chromosomes (=Ks) are eliminated from the future somatic nuclei during germ line-soma differentiation in early, still syncytial, embryonic divisions by remaining in the equatorial plane. But also in the germ line about half of the Ks are eliminated in the first mitotic division of the primary germ cells in the young gonads of first instar larvae. Until now not much is known about the cytogenetic processes in the course of germ line elimination of the Ks. In this study, the cytogenetic changes in the primary germ cells before, during and after the elimination mitosis were analysed in detail with DAPI stained gonad preparations.

Origin recognition and the chromosome cycle

Prior to the initiation of DNA replication, chromosomes must establish a biochemical mark that permits the recruitment in S phase of the DNA replication machinery that copies DNA. The process of chromosome replication in eukaryotes also must be coordinated with segregation of the duplicated chromosomes to daughter cells during mitosis. Protein complexes that utilize ATP coordinate events at origins of DNA replication and later they participate in the initiation of DNA replication. In eukaryotes, some of these proteins also play a part in later processes that ensure accurate inheritance of chromosomes in mitosis, including spindle attachment of chromosomes, accurate duplication of centrosomes and cytokinesis. A perspective of how ATP-dependent proteins accomplish this task in eukaryotes is discussed.

The centrosome cycle

The centrosome is the major microtubule-organizing center of animal cells. It influences cell shape and polarity and directs the formation of the bipolar mitotic spindle. Numerical and structural centrosome aberrations have been implicated in disease, notably cancer. In dividing cells, centrosomes need to be duplicated and segregated in synchrony with chromosomes. This centrosome cycle requires a series of structural and functional transitions that are regulated by both phosphorylation and proteolysis. Here we summarize recent information on the regulation of the centrosome cycle and its coordination with the chromosomal cell cycle.

A Prize for Proliferation

A Prize for Proliferation

Arrest of cell division and nucleoid partition by genetic alterations in the sliding clamp of the replicase and in DnaA.

In Escherichia coli, an interaction between the replication initiator DnaA and the sliding clamp protein, the beta subunit (DnaN) of DNA polymerase III, is required to regulate the chromosomal replication cycle. We report here that colony formation by, and cell division of, the temperature (42 degrees C)-sensitive dnaN59 mutant are inhibited at 34-35 degrees C when DnaA is moderately (4-to 8-fold ) overexpressed, although chromosomal replication and the beta subunit-dependent regulation of DnaA activity are not significantly inhibited. Immunoblotting analysis revealed that the beta subunit is abundant (present at a level of about 5000 dimers per cell) at 34 degrees C, and its concentration per unit cell volume was practically unaffected in the dnaN59 mutant by the overexpression of DnaA. The dnaN mutant cells that overexpress DnaA become filamentous at 34 degrees C via an sfiA-independent pathway, different from that activated by the SOS response. This filamentation is accompanied by inhibition of nucleoid partition and FtsZ ring formation. In the dnaN59 mutant, oversupply of DnaA may disturb the coordinated action of cell cycle-regulating molecules, thus leading to the inhibition of these events.

Replication-related activities establish cohesion between sister chromatids.

Replicated sister chromatids are held together from their synthesis in S phase to their separation in anaphase. The process of sister chromatid cohesion is essential for the proper segregation of chromosomes in eukaryotic cells. Recent studies in Saccharomyces cerevisiae have advanced our understanding of how sister chromatid cohesion is established, maintained, and dissolved during the cell cycle. Historical observations have suggested that establishment of cohesion is roughly coincident with replication fork passage. Emerging evidence now indicates that replication fork components, such as PCNA, a novel DNA polymerase, Trf4p/Pol sigma (formerly Trf4p/Pol kappa), and a modified clamp-loader complex, actively participate in the process of the cohesion establishment. Here, we review the molecular events in the chromosome cycle with respect to cohesion. Failure of sister chromatid cohesion results in the aneuploidy characteristic of many birth defects and tumors in humans.

TiK+ toK+: an embryonic clock?

During embryogenesis cells make appropriately timed developmental decisions. Both 'hourglass-like' and 'clock-like' mechanisms have been demonstrated to act as timers in early development. The cell cycle rhythm, using feedback circuits to drive cells unidirectionally through checkpoints, is an example of a clock-like timer, but how it operates to time developmental events is unclear. In other cell types, cyclic oscillations in K+ channel activity, which parallel cell cycle and circadian rhythms, may be part of the timing mechanism. Changes in K+ oscillations accompany key developmental transitions and oncogenic transformation. Channel blockade interferes pharmacologically with cell cycle initiation or progression, whereas channel over-expression can be oncogenic. K+ channel activity also exists in early mouse oocytes through to at least the blastocyst stage, and it oscillates in phase with the developmental cell cycles, being high in M/G1 and low in S/G2. It resembles physiologically the activity of the K+ channels of the eag- or erg-like families. Reverse transcriptase-polymerase chain reaction of mouse oocytes has revealed the presence of transcripts encoding both EAG- and ERG-like proteins throughout preimplantation development. Channel activity continues to oscillate with a cell cycle periodicity in embryos from which the nucleus has been removed, or after inhibition by puromycin of the cyclin B-cyclin-dependent kinase 1 driven component of the chromosomal cycle. Channel oscillatory activity thus appears to be able to function autonomously of the chromosomal cycle and may represent a distinct oscillatory timing activity with possible developmental significance.

Effects of SomeTrlMutations on Mitosis in Embryonic and Larval Tissues and Egg Chamber Morphology in Drosophila melanogaster

A study was made of three insertional mutations (Trl13C,Trls2325,TrlEP(3)3184) located in the second intron of the Trithorax-like(Trl) gene for the GAGA transcription factor (GAF). Their cytological effects were analyzed in oogenesis, early embryonic development, and in larval development (96–108 h) in cells of nervous ganglia and imaginal disks. Notwithstanding an interallelic difference in expression, all three P-element insertions proved to be dominant as far as the examined parameters were concerned. The most substantial defects were the formation of “granular” chromatin during the interphase and mitosis and high proportions of cells with hypercondensed chromatin (which were arrested at the G2–M boundary) and cells with abnormal chromosome segregation. A higher frequency of egg chambers with trophocytes defective in number and in chromatin condensation was observed in females carrying the mutant Trlgene. The defects were assumed to result from poor coordination of the chromosome and cell cycles and, including, the nuclear and centrosomal cycles in embryonic development and the cycles of chromosome condensation and spindle formation in cells of larval imaginal disks and nervous ganglia.

Dominant-negative mutant dynein allows spontaneous centrosome assembly, uncouples chromosome and centrosome cycles.

Cleavage cycles commence and chromosome and centrosome cycles proceed in harmony following fertilization of Drosophila eggs and completion of the meiotic divisions. The sperm-introduced centrioles replicate, separate, and while recruit pericentriolar material centrosomes (CS) form. The CS nucleate asters of microtubules (MT). Spindles form following interaction of some astral MT with kinetochores. In unfertilized eggs, chromosomes do not replicate, and CS and MT asters never form, although their components are present in the egg cytoplasm; unknown mechanisms prevent chromosome replication and CS and MT assembly. In unfertilized Laborc(D) eggs, rudimentary CS assemble spontaneously and instantaneously and nucleate small MT asters. In fertilized Laborc(D) eggs, normal CS form and organize normal asters. However, the CS replicate prior to accomplishment of the first mitosis, and spindles with multiple CS develop. In fertilized Laborc(D) eggs, while the chromosome cycles cease, CS cycles proceed as in wild type. Knowing that Laborc(D) is a dominant-negative mutation and encodes the formation of mutant cytoplasmic dynein heavy chain molecules, we show here that cytoplasmic dynein is involved in prevention of CS assembly in unfertilized eggs and establishing harmony between the chromosome and the CS cycles.

The controlling role of ATM in homologous recombinational repair of DNA damage.

The human genetic disorder ataxia telangiectasia (A-T), caused by mutation in the ATM gene, is characterized by chromosomal instability, radiosensitivity and defective cell cycle checkpoint activation. DNA double-strand breaks (dsbs) persist in A-T cells after irradiation, but the underlying defect is unclear. To investigate ATM's interactions with dsb repair pathways, we disrupted ATM along with other genes involved in the principal, complementary dsb repair pathways of homologous recombination (HR) or non-homologous end-joining (NHEJ) in chicken DT40 cells. ATM(-/-) cells show altered kinetics of radiation-induced Rad51 and Rad54 focus formation. Ku70-deficient (NHEJ(-)) ATM(-/-) chicken DT40 cells show radiosensitivity and high radiation-induced chromosomal aberration frequencies, while Rad54-defective (HR(-)) ATM(-/-) cells show only slightly elevated aberration levels after irradiation, placing ATM and HR on the same pathway. These results reveal that ATM defects impair HR-mediated dsb repair and may link cell cycle checkpoints to HR activation.

The Roles of Changes in NADPH and pH during Fertilization and Artificial Activation of the Sea Urchin Egg

Incubating unfertilized sea urchin eggs in weak bases activates nuclear centering, DNA synthesis, and chromosome cycles. These effects were initially attributed to raising the intracellular pH (pHi), but later experiments indicated that these weak bases also lead to increases in reduced pyridine nucleotides. These findings raised the question whether the activation of the nucleus was due to increased pHi or to increased NAD(P)H or possibly other effects. This report attempts to clarify how ammonia activates eggs by independently altering NADPH and pHi. To increase the pHi, unfertilized eggs were injected with zwitterionic buffers. This stimulated pronuclear centering, DNA synthesis, and nuclear envelope breakdown; there appeared to be a threshold corresponding to the fertilized pHi. However, like incubation in ammonia, injection of base also increased NAD(P)H. The NAD(P)H rise caused by directly raising the pHi occurred in the presence of intracellular calcium chelators, indicating that calcium is not required. Increasing NAD(P)H alone did not activate nuclear centering, DNA synthesis, or nuclear envelope breakdown. Although these experiments cannot eliminate a role for the NADPH increase in initiating events leading to nuclear centering and entry into mitosis, they provide additional and strong evidence that increasing the pHi may be a primary signal.

In vitro maturation of transported pig oocytes in a defined protein-free medium

The duration of the different phases of the microtubule and chromosome cycles were estimated in the native diploid cell populations of Allium cepa L root meristems proliferating undisturbed, under steady state conditions, at the physiological temperature of 15°C. The cycles were coupled by considering their fitting in relation to the short process of nuclear envelope breakdown. In the cycle related to cytoplasmic division, the preprophase band which predicts the future position of the phragmoplast made its appearance, as a wide band, 16 mm before the G2 to prophase transition, ie it was only present during the final 5% of the total G2 timing (5 h 30 mm). The band became narrow only 6 mm after prophase had started and it was present in this form for the remaining prophase time (2 h 24 mm). Its disappearance occurred strictly coinciding with nuclear envelope breakdown, at the end of prophase. No microtubules related to cytoplasmic division were apparent until 9 mm after telophase had initiated. The two initial stages of phragmoplast formation which followed occupied, respectively, 27 mm and 54.5 mm of the 2-h long telophase. On the other hand, the third and last stage in phragmoplast formation covered both the final 35 mm of mitosis and the 6 initial mm of the G1 of the next interphase. A very short (less than 4 mm) stage of microtubular nucleation around the nuclear envelope took place immediately afterwards, before the cortical array of microtubules appeared. The microtubule cycle related to nuclear division started with the apparent activation of the future spindle poles 7.4 mm before prophase was over. The mitotic spindle developed in the 5.6 mm long prometaphase. The spindle functioned in metaphase for the 42 mm it lasted, half spindles being separated for the 37 mm anaphase occupied in these cells.

Genetic mapping of Hessian fly avirulence gene vH6 using bulked segregant analysis

The Hessian fly, Mayetiola destructor (Say), an important insect pest of wheat, Triticum aestivum L., has a gene-for-gene relationship with wheat: single genes in the insect condition avirulence to specific resistance genes in wheat. We report the discovery of the first molecular genetic marker that is tightly linked to a Hessian fly avirulence gene. This dominant DNA polymorphism (OPG15-1) was identified using bulked segregant analysis and arbitrary primers in polymerase chain reactions. Bulked segregant analysis was modified to accommodate the anomalous chromosome cycle of the Hessian fly. It was used to identify DNA polymorphisms linked to the gene (vH6) that confers avirulence to the resistance gene H6 in wheat. OPG15-1 was cloned and sequenced, and a pair of site-specific primers were designed that converted it into a codominant single-stranded conformational polymorphism. Both OPG15-1 and vH6 were shown to be X-linked, and the genetic distance between the two loci was 2.5 ± 2.5 cM. In situ hybridization to polytene chromosomes of larval salivary glands indicated that OPG15-1 resides near the centromere of Hessian fly chromosome X1.Key words: Mayetiola destructor, avirulence gene, RAPD-PCR, bulked segregant analysis, single-stranded conformational polymorphism, SSCP, in situ hybridization.

A cytoplasmic cell cycle controls the activity of a K+ channel in pre-implantation mouse embryos.

We previously have reported that the activity of a 240 pS K+ channel varies during the cell cycle in pre-implantation mouse embryos. In the present study, we show that: (i) the cycling of channel activity is not prevented by inhibiting protein synthesis and hence does not involve cyclin-dependent kinase 1 (cdk1)-cyclin B; and (ii) the cycling of channel activity continues in anucleate zygote fragments with a time course similar to that observed in nucleate fragments. We further demonstrate that: (i) persistent activation of the K+ channel in one-cell embryos arrested in metaphase requires the maintenance of an active cdk1-cyclin B complex; and (ii) both DNA synthesis inhibition with aphidicolin and DNA damage produced by mitomycin C prevent the down-regulation of the channel at the start of S phase by a mechanism that requires tyrosine kinase activation. Thus, the 240 pS K+ channel in these cells is controlled by a previously unsuspected cytoplasmic clock that functions independently of the well-known clock controlling the chromosomal cell cycle, but can interact with it.