Calcium-induced conformational changes in the amino-terminal half of gelsolin

Gelsolin is an actin-binding protein that is regulated by the occupancy of multiple calcium-binding sites. We have studied calcium induced conformational changes in the G1-2 and G1-3 sub-domains, and report the binding affinities for the three type II sites. A new probe for G3 has been produced and a K(d) of 5 microM has been measured for calcium in the context of G1-3. The two halves of gelsolin, G1-3 and G4-6 bind weakly with or without calcium, suggesting that once separated by apoptotic proteolysis, G1-3 and G4-6 remain apart allowing G1-3 to sever actin in a calcium free manner.     

Gene-specific and strand-specific DNA repair in the G1 and G2 phases of the cell cycle.

We have analyzed the fine structure of DNA repair in Chinese hamster ovary (CHO) cells within the G1 and G2 phases of the cell cycle. Repair of inactive regions of the genome has been suggested to increase in the G2 phase of the cell cycle compared with other phases. However, detailed studies of DNA repair in the G2 phase of the cell cycle have been hampered by technical limitations. We have used a novel synchronization protocol (D. K. Orren, L. N. Petersen, and V. A. Bohr, Mol. Cell. Biol. 15:3722-3730, 1995) which permitted detailed studies of the fine structure of DNA repair in G2. CHO cells were synchronized and UV irradiated in G1 or early G2. The rate and extent of removal of cyclobutane pyrimidine dimers from an inactive region of the genome and from both strands of the actively transcribed dihydrofolate reductase (DHFR) gene were examined within each phase. The repair of the transcribed strand of the DHFR gene was efficient in both G1 and G2, with no major differences between the two cell cycle phases. Neither the nontranscribed strand of the DHFR gene nor an inactive region of the genome was repaired in G1 or G2. CHO cells irradiated early in G2 were more resistant to UV irradiation than cells irradiated in late G1. Since we found no major difference in repair rates in G1 and G2, we suggest that G2 resistance can be attributed to the increased time (G2 and G1) available for repair before cells commit to DNA synthesis.     

丝瓜的三聚体G蛋白的初步研究

根据拟南芥（ａｒａｂｉｄｏｐｓｉｓｔｈａｈｌｉａｎａ）ＧＰＡ１的保守区段Ａ设计一对特异引物（５′ｃｔｇｇｇｇａａｔｃｔｇｇａａａａｔｃ３′,５′ｃａｃａｇｃｔｇｔａｃａｃｃｔｃａａａｃ３′）通过ＰＣＲ从丝瓜核基因组中扩增植物的三聚体Ｇ蛋白α亚基编码基因,获得了２个片段（ＬＦＧ１,ＬＦＧ２）,并已克隆和测序（已在ＥＭＢＬ数据库中登记,登记号为：ｙ１５２７０,ｙ１５２７１）．序列分析表明ＬＦＧ１和ＬＦＧ２分别由１５１５ｂｐ和７３２ｂｐ构成,都含有三聚体Ｇ蛋白α亚基编码基因的保守区段Ａ,但也都含有内含子．根据片段的大小及ＰＣＲ的特性,ＬＦＧ１可能是丝瓜三聚体Ｇ蛋白α亚基编码基因上的片段．     

Shared and unique G alpha proteins in the zebrafish versus mammalian senses of taste and smell

Chemosensory systems in vertebrates employ G protein-coupled receptors as sensors. In mammals, several families of olfactory and gustatory receptors as well as specific G alpha proteins coupling to them have been identified, for example, gustducin for taste. Orthologous receptor families have been characterized in fish, but the corresponding G alpha genes have not been well investigated so far. We have performed a comprehensive search of several lower vertebrate genomes to establish the G alpha protein family in these taxa and to identify those genes that may be involved in chemosensory signal transduction in fish. We report that gustducin is absent from the genomes of all teleost and amphibian species analyzed, presumably due to independent gene losses in these lineages. However, 2 other G alpha genes, Gi1b and G14a, are expressed in zebrafish taste buds and 4 G proteins, Go1, Go2, Gi1b, and Golf2, were detected in the olfactory epithelium. Golf2, Gi1b, and G14a are expressed already shortly after hatching, consistent with the physiological and behavioral responses of larvae to odorants and tastants. Our results show general similarity to the mammalian situation but also clear-cut differences and as such are essential for using the zebrafish model system to study chemosensory perception.     

A single nucleotide polymorphism in the matrix metalloproteinase-1 (MMP-1) promoter influences amnion cell MMP-1 expression and risk for preterm premature rupture of the fetal membranes.

Interstitial collagen gives fetal membranes tensile strength, and membrane rupture has been attributed to collagen degradation. A polymorphism at -1607 in the matrix metalloproteinase-1 (MMP-1) promoter (an insertion of a guanine (G)) creates a core Ets binding site and increases promoter activity. We investigated whether this polymorphism is functionally significant for MMP-1 expression in amnion cells and whether it is associated with preterm premature rupture of the membranes (PPROM). The 2G promoter had >2-fold greater activity than the 1G allele in amnion mesenchymal cells and WISH amnion cells. Phorbol 12-myristate 13-acetate (PMA) increased mesenchymal cell nuclear protein binding with greater affinity to the 2G allele. Induction of MMP-1 mRNA by PMA was significantly greater in cells with a 1G/2G or 2G/2G genotype compared with cells homozygous for the 1G allele. When treated with PMA, the 1G/2G and 2G/2G cells produced greater amounts of MMP-1 protein than 1G/1G cells. A significant association was found between fetal carriage of a 2G allele and PPROM. We conclude that the 2G allele has stronger promoter activity in amnion cells, that it confers increased responsiveness of amnion cells to stimuli that induce MMP-1, and that this polymorphism contributes to the risk of PPROM.     

Changes in the chondriome during the cell cycle of a tissue culture of embryonic pig kidney

The regular cyclic changes of number, size, shape and ultrastructure of cells mitochondria during G1, S, G2-periods and mitosis have been shown by morphometric methods on nonsynchronized culture of PEK tissue. Number of mitochondria is equal in G1 and S cells. Middle size and unbranched mitochondria prevail in G1-period, middle size and large organellae of complicated shape are characteristic for S-period. In G2-period number of mitochondria increases in 1,5 times. Simultaneously the portion of middle and small unbranched mitochondria increases and number of large organellae of complicated shape decreases. Number of mitochondria in mitotic cells in comparison with cells of G2-period does not change distinctly. Most mitochondria are middle and small size, usually unbranched in this period. Considerable increase of mitochondria number in G2-period is probably due to division of the branched mitochondria characteristic for the previous S-period. The mitochondria ultrastructure does not undergo marked changes during the interphase of the cell-cycle and characterizes by prevailing of the orthodox forms of mitochondria: in late G2-period and in the process of mitosis the most mitochondria become condensed.     

Analysis of the catabolism of aggrecan in cartilage explants by quantitation of peptides from the three globular domains

A method has been developed for the production, isolation, and quantitation of 15 marker peptides from the three globular domains (G1, G2, and G3) and the interglobular domain of bovine aggrecan (aggregating cartilage proteoglycan). Three of the peptides are from G1, two are from the interglobular domain, four are from G2, and six are from G3. The method involves separation of tryptic peptides by sequential anion-exchange, cation-exchange, and reversed-phase high performance liquid chromatography and quantitation by absorbance at 220 nm. The values obtained (peak area per microgram of core protein) were a function of the molar yield and also the size and aromatic residue content of individual peptides. This procedure has been applied to aggrecan purified from fresh calf articular cartilage and to aggrecan isolated from the medium and tissue compartments of cartilage explant cultures, maintained in basal medium for 15 days without and with interleukin-1 alpha. These analyses indicate that aggrecan which is released into explant medium has a reduced content of the G1 domain, but has a normal content of the G2 domain, the COOH-terminal region of the interglobular domain, and also the G3 domain. On the other hand, aggrecan which is retained by the cartilage during 15 days of culture has a normal content of G1, interglobular domain, and G2 domains, but, in the presence of interleukin-1 alpha, it has a reduced content of the G3 domain. The percentage of medium molecules which retained the G1 domain was higher in control cultures (about 35%) than in interleukin cultures (about 20%), and this was consistent with the relative aggregability of these samples. Taken together these results suggest that catabolism of aggrecan in articular cartilage involves a specific proteolysis of the core protein at a site which is within the interglobular domain and NH2-terminal to the sequence LPGG. This process occurs in control cultures but is accelerated by the addition of interleukin-1 alpha. Degraded molecules which lack the G1 domain are released preferentially into the medium; however, these molecules carry both the G2 and G3 domains, indicating that these domains do not confer strong matrix binding properties on aggrecan. The method described here for the isolation of peptides from bovine aggrecan should have wide application to structural and biosynthetic studies on this molecule in species such as human and rat, since many of the marker peptides are from highly conserved regions of the aggrecan core protein.     

Cell Cycle Synchronization at the G2/M Phase Border by Reversible Inhibition of CDK1

Chemical agents for cell cycle synchronization have greatly facilitated the study of biochemical events driving cell cycle progression. G1, S and M phase inhibitors have been developed and used widely in cell cycle research. However, currently there are no effective G2 phase inhibitors and synchronization of cultured cells in G2 phase has been challenging. Recently, a selective CDK1 inhibitor, RO-3306, has been identified that reversibly arrests proliferating human cells at the G2/M phase border and provides a novel means for cell cycle synchronization. A single-step protocol using RO-3306 permits the synchronization of >95% of cycling cancer cells in G2 phase. RO-3306 arrested cells enter mitosis rapidly after release from the G2 block thus allowing for isolation of mitotic cells without microtubule poisons. RO-3306 represents a new molecular tool for studying CDK1 function in human cells.     

The septins function in G1 pathways that influence the pattern of cell growth in budding yeast

The septins are a conserved family of proteins that have been proposed to carry out diverse functions. In budding yeast, the septins become localized to the site of bud emergence in G1 but have not been thought to carry out important functions at this stage of the cell cycle. We show here that the septins function in redundant mechanisms that are required for formation of the bud neck and for the normal pattern of cell growth early in the cell cycle. The Shs1 septin shows strong genetic interactions with G1 cyclins and is directly phosphorylated by G1 cyclin-dependent kinases, consistent with a role in early cell cycle events. However, Shs1 phosphorylation site mutants do not show genetic interactions with the G1 cyclins or obvious defects early in the cell cycle. Rather, they cause an increased cell size and aberrant cell morphology that are dependent upon inhibitory phosphorylation of Cdk1 at the G2/M transition. Shs1 phosphorylation mutants also show defects in interaction with the Gin4 kinase, which associates with the septins during G2/M and plays a role in regulating inhibitory phosphorylation of Cdk1. Phosphorylation of Shs1 by G1 cyclin-dependent kinases plays a role in events that influence Cdk1 inhibitory phosphorylation.     

Interaction between cdc13+ and cdc2+ in the control of mitosis in fission yeast; dissociation of the G1 and G2 roles of the cdc2+ protein kinase

A cold-sensitive (cs) allele of cdc2, a gene that acts in both the G1 and G2 phases of the fission yeast cell cycle, has been isolated by classical mutagenesis. Further mutagenesis of a cdc2cs strain yielded an extragenic suppressor that rescued the cs cell cycle defect but simultaneously conferred a temperature-sensitive (ts) cdc phenotype. This suppressor mutation was shown to be an allele of cdc13, a previously identified gene. A variety of allele-specific interactions between cdc2 and cdc13 were discovered. These included suppression of cdc13ts alleles by introduction of the cdc2+ gene on a multi-copy plasmid vector. cdc13+ is required in G2 for mitotic initiation and was shown to play no role in the G1 phase of the cell cycle. cdc2+, however, is essential in G1 for DNA replication and in G2 for mitosis. The newly isolated cs allele of cdc2 that is rescued by a ts allele of cdc13 is defective only in its G2 function. cdc13+ cooperates with cdc2+ in the initiation of mitosis but not in the regulation of DNA replication. We propose that the cdc13+ gene product might be a G2-specific substrate of the cdc2+ protein kinase.     

Contribution to the determination of the chemotherapeutic drug G1 in biological fluids

A sensitive gas chromatographic method for the quantitative determination of the new antibacterial and antifungal drug G1, 1-(5-bromofuran-2-yl)-2-bromo-2-nitroethene, has been optimized. The method involves a fast and single extraction step from spiked serum and urine samples. The G1 drug was quantified using an internal standard method and by means of a nitrogen-selective detector. The results are statistically significant and show that mean levels of G1 as low as 1 μg ml⁻¹ can be measured accurately.     

Changes in the organization of chromosomes during the cell cycle: response to ultraviolet light.

Fusion between mitotic and interphase cells results in the premature condensation of the interphase chromosomes into a morphology related to the position in the cell cycle at the time of fusion. These prematurely condensed chromosomes (PCC) have been used in conjunction with u.v. irradiation to examine the interphase chromosome condensation cycle of HeLa cells. The following observations have been made: (I) There is a progressive decondensation of the chromosomes during G1 which is accentuated by u.v. irradiation: (2) The chromosomes become more resistant to u.v.-induced decondensation during G2 and mitosis. (3) There is a close correlation between the degree of chromosome decondensation and the amount of unscheduled DNA synthesis induced by u.v. irradiation during G1 and mitosis: (4) Hydroxyurea enhances the ability of u.v. irradiation to promote the decondensation of chromosomes during G1, G2 and mitosis. Hydroxyurea also potentiates the lethal action of u.v. irradiation during mitosis and G1. These data are discussed in relation to the suggestion that chromosomes undergo a progressive decondensation during G1 and condensation during G2.     

In CK2 inactivated cells the cyclin dependent kinase inhibitor Sic1 is involved in cell-cycle arrest before the onset of S phase

Protein kinase CK2 is a heterotetramer composed of two catalytic and two regulatory subunits. In Saccharomyces cerevisiae the catalytic subunits (alpha and alpha') are encoded by the CKA1, CKA2 genes. cka1Deltacka2(ts) mutants arrest cell cycle in both G1 and G2/M at 37 degrees C. Hence, it has been proposed that CK2 plays an important role in cell-cycle progression and several cell-cycle proteins have been reported to be CK2 substrates. We have previously shown that Sic1, the inhibitor of Clb5-Cdc28 complexes required for the G1/S transition, is a physiologically relevant CK2 substrate. Here we show that CK2 inactivation up-regulates Sic1 level resulting in severe down-regulation of Clb5-Cdc28 kinase activity. Concurrent inactivation of Sic1 and CK2 leads to accumulation of cells with a post-synthetic DNA content and short/elongated spindles, typical of cells arrested in mitosis. These findings indicate that Sic1 plays a major role during G1 arrest of CK2-inactivated cells.     

Multigene analysis suggests ecological speciation in the fungal pathogen Claviceps purpurea

Claviceps purpurea is an important pathogen of grasses and source of novel chemical compounds. Three groups within this species (G1, G2 and G3) have been recognized based on habitat association, sclerotia and conidia morphology, as well as alkaloid production. These groups have further been supported by Random Amplification of Polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers, suggesting this species may be more accurately described as a species complex. However, all divergent ecotypes can coexist in sympatric populations with no obvious physical barriers to prevent gene flow. In this study, we used both phylogenetic and population genetic analyses to test for speciation within C. purpurea using DNA sequences from ITS, a RAS-like locus, and a portion of beta-tubulin. The G1 types are significantly divergent from the G2/G3 types based on each of the three loci and the combined dataset, whereas the G2/G3 types are more integrated with one another. Although the G2 and G3 lineages have not diverged as much as the G1 lineage based on DNA sequence data, the use of three DNA loci does reliably separate the G2 and G3 lineages. However, the population genetic analyses strongly suggest little to no gene flow occurring between the different ecotypes, and we argue that this process is driven by adaptations to ecological habitats; G1 isolates are associated with terrestrial grasses, G2 isolates are found in wet and shady environments, and G3 isolates are found in salt marsh habitats.     

Light and dark control of the cell cycle in two marine phytoplankton species

The effect of light and dark on growth, DNA replication and cell division of two marine phytoplankters Thalassiosira weissflogii (a diatom) and Hymenomonas carterae (a coccolithophorid) was investigated using flow cytometry. The two species displayed very differing behavior. When transferred from light to prolonged darkness, all coccolithophorid cells were arrested at the beginning of the G1 stage of the cell cycle. When shifted back into light, they resumed cycling at a rate slightly slower than prior to arrest. In contrast, diatom cells were arrested either in the G1 or G2 stage of the cell cycle in the dark. Upon re-exposure to light, cells which had been dark-arrested in G1 resumed cycling at the same rate as prior to arrest, while cells arrested in G2 cycled much more slowly. These results suggest that in both species, light control of cell cycle progression is effective only over a restricted part of the cell cycle, as has been hypothesized by Spudich & Sager (J cell biol 83 (1980) 136) [38] for Chlamydomonas. In the coccolithophorid there is a single light-dependent segment located at the beginning of G1, whereas the diatom appears to have two such segments, one in G1 and the other in G2, corresponding to two different light requiring processes.     

On the existence of 'arrested G2 cells' in mouse epidermis

It has been postulated that mouse epidermis contains two populations of resting cells, one of which is blocked at the G1-S boundary and the other between G2 and mitosis. The 'arrested G2 cells' were estimated, by the labelled mitosis method, to comprise 5-10% of the epidermal population and presumed to function as a 'reserve pool' which could be activated by wounding. A comprehensive search has now been carried out for arrested G2 cells in mouse epidermis using the direct methods of single cell and flow through cytophotometry. No evidence was obtained which supports the existence of such a cell compartment. Suitable control experiments were carried out to ensure that G2 cells were not lost during the isolation of epidermal nuclei.     

Lethal response of HeLa cells to x-irradiation in the latter part of the generation cycle.

The age-response for the killing of HeLa S3 cells by X-rays during the latter part of the generation cycle has been examined in detail. As synchronous cells move from the G1/S boundary through S phase, the relatively high sensitivity of late G1 cells gradually decreases; minimum sensitivity is reached in mid-S and maintained during the remainder of that phase. The response of cells as they progress from S to the point in G2 at which they are temporarily arrested by radiation (or by inhibitors of protein synthesis) was measured in populations free of both S phase cells and late G2 cells that had passed the arrest point: cells retain their high resistance from early G2 up to the arrest point. The response of G2 cells that have passed the arrest point before being irradiated was examined by exposing randomly growing cultures to X-rays and collecting cells periodically thereafter, as they entered mitosis. Survival values very close to those of sensitive mitotic cells were found in the 2 h period after irradiation during which unarrested cells continued to reach mitosis. Values typical of lateS/early G2 were found only after cells that had been arrested began arriving at mitosis. Thus, HeLa S3 cell undergo an abrupt increase in sensitivity at or near the arrest point. The sensitivity to a second irradiation of cells arrested in G2 by a conditioning X-ray dose increases rapidly in the early part of the arrest period.     

Studies of the cell cycle of in vitro cultured skin fibroblasts in goats: work in progress

The effect of serum starvation and olomoucine treatment on the cell cycle and apoptosis of goat skin fibroblasts cultured in vitro is reported in this paper. The cells were obtained from the ear of a female goat 1.5 years of age. Analysis of cell cycle distribution by fluorescence-activated cell sorting (FACS) showed that 3.4, 60.8 and 15.1% of normally cycling cells were at G1, G0 and S phase, respectively. Serum starvation for 1, 3 and 5 days arrested 70.1, 70.2 and 83.4% cells, respectively, at G0/G1 phase. Seventy-eight percent of confluent cells were at G0/G1 stage, but in contrast to the serum starved group, this high percentage of G0/G1 cells was mainly associated with G1 cells. Of cells not deprived of serum, 73.6% were arrested at G1/G0 when treated with 100 microM olomoucine for 9 h compared to 85.5% of cells that had been starved of serum for 2 days (co-inhibition) (P<0.01). After co-inhibition, 45% of cells entered S phase when re-cultured in normal medium for 5 h, indicating that the inhibition was reversible. Under normal culture conditions, 1.2% of cells underwent apoptosis. Serum starvation for 1, 2, 3, 5 and 10 days caused apoptosis in 1.7, 3.9, 4.5, 11.7 and 90.3% of cells, respectively. Treatment with 100 microM olomoucine for 9h did not increase the number of apoptotic cells significantly (1.9%, P>0.05). When cells were co-inhibited, 4.1% of cells underwent apoptosis. In conclusion, although serum withdrawal for 5 days or more effectively arrested cells at G0/G1 stages, it increased apoptosis of cells significantly. However, co-inhibition by serum withdrawal and olomoucine treatment was found to be an appropriate treatment to obtain more healthy G0/G1 cells based on the low percentage of apoptotic cells after treatment.