A probe into nuclear events during the cell cycle of Saccharomyces cerevisiae: studies of folded chromosomes in cdc mutants which arrest in G1

The sedimentation behavior of folded chromosomes from celldivision-cycle (cdc) mutants which arrest in g ₁ was examined. At the restrictive temperature the folded genome of cdc 7, which arrests after spindle pole body (SPB) separation and spindle formation, cosediments with a standard g ₁ structure, indicating that by the cdc 7 step the g ₁ form of the folded genome has been assembled. In the mutant, cdc 4, which arrests before SPB separation but after SPB duplication, a standard g ₁ structure is not formed, cdc 4 cells, however, are able to enter G₀ at the restrictive temperature, and the corresponding g ₀ structure is stable. These results indicate that the cdc 4 gene product may be involved in the development of folded genome conformation which leads to the g ₁ structure. Since the cdc 4 gene product is required for SPB separation, the g ₁ structure may be defined by an association between chromosomes and spindle components. The folded chromosomes of the start mutants cdc 25 and cdc 28 are unstable at the restrictive temperature. In contrast to cdc 4, neither cdc 25 nor cdc 28 are able to enter the G₀ stage in a normal manner, i.e., the g ₀ structure is unstable at the restrictive temperature. The inference is that both the cdc 25 and cdc 28 gene products are required for the functional integrity of the folded genome at both a stage early in G₁ and in the pathway to G₀.     

Folded chromosomes in meiotic yeast cells: analysis of early meiotic events.

Analysis of folded chromosomes in cells under standard sporulation conditions shows that the g₀ form of the folded genome is used as the entry into meiosis. Premeiotic DNA replication is initiated from the g₀ structure. In contrast, mitotic DNA replication is preceded by a characteristic pre-replicative form, g₁. Nonetheless, the mitotic and meiotic replication structures are indistinguishable by sedimentation. Preliminary evidence also suggests that the meiotic equivalent of the mitotic post-replicative structure, g₂, is absent. In strains homozygous for the mating type locus, aa and αα, meiotic replicating structures are not detected, and the folded chromosomes remain in a non-cycling form. However, this non-cycling form is distinguishable from the g₀ form of aα. cells.     

Folded chromosomes of mating-factor arrested yeast cells: comparison with G0 arrest

Folded chromosomes of haploid a or diploid aa cells arrested by -factor are characterized by a heterogeneous sedimentation distribution, distinguishable from the corresponding g ₀and g ₁ structures. The extent of heterogeneity appears to depend in part on the extent of the characteristic schmoe morphology. Under conditions where a and mating-type cells are mixed together without conjugation, folded chromosomes characteristic of -factor arrested a and aa cells appear in both cell types in a synchronous manner. Attempts to order G₀ and -factor arrest suggest that these two arrest stages represent two different branch pathways out of the cell cycle. These two branch pathways may have a common branch point, or they may be characterized by two different branch points.     

Repair of DNA double-strand breaks requires two homologous DNA duplexes

DNA repair and cell survival in haploid and its diploid derivative strains ofSaccharomyces cerevisiae were studied after 100 krad X-ray irradiation. The cells were in theG ₁ stage of the cell cycle, where haploid cells had only one copy of genetic material per genome and diploid had two copies. It was found that diploid could repair double-strand breaks in its DNA after 48 hr of liquid holding which was accompanied by a four-fold rise in survival. In contrast a haploid strain failed to repair its DNA and showed no increase in survival after liquid holding. It is concluded that (1) repair of DNA double-strand breaks requires the availability of two homologous DNA duplexes, (2) restoration of cell viability during liquid holding is connected with repair of DNA double-strand breaks and (3) this repair is a slow process possibly associated with slow finding and conjugation of homologous chromosomes.     

Isolation and chromosomal distribution of a novel Ty1-copia-like sequence fromSecale, which enables identification of wheat—Secale africanum introgression lines

A repetitive sequence of 411 bp, named pSaO5₄₁₁, was identified in theSecale africanum genome (R^a) by random amplified polymorphic DNA (RAPD) analysis of wheat and wheat—S. africanum amphiploids. GenBank BLAST search revealed that the sequence of pSaO5₄₁₁ was highly homologous to a part of a Ty1-copia retrotransposon. Fluorescence in situ hybridization (FISH) analyses indicated that pSaO5₄₁₁ was significantly hybridized toS. africanum chromosomes of a wheat—S. africanum amphiploid, and it was dispersed along theSecale chromosome arms except the terminal regions. Basing on the sequence of pSaO5₄₁₁, a pair of sequence-characterized amplified region (SCAR) primers were designed, and the resultant SCAR marker was able to target both cultivated rye and the wildSecale species, which also enabled to identify effectively theS. africanum chromatin introduced into the wheat genome.     

Folded chromosomes of Saccharomyces cerevisiae

Folded chromosome phenotypes have been examined and compared in four cell-division-cycle (cdc) mutants during transitions between cycling and non-cycling states. The two start mutants, cdc 28 and cdc 25, can undergo G₀ arrest at the restrictive temperature. Arrest at start, defined by the cdc 28 and cdc 25 block points, is distinguishable from G₀ arrest. Arrest at the cdc 28 and cdc 25 block points can also be distinguished from each other: folded chromosomes appear to be destabilized at the cdc 25 block, but are stable at the cdc 28 arrest point. On the other hand, folded chromosomes from cdc 28 in sporulation medium at the restrictive temperature appear unstable, while chromosomes from cdc 25 are stable. The G₁ arrest mutants, cdc 4 and cdc 7, can undergo G₀ arrest at the restrictive temperature. In sporulation medium no meiotic replication form is detected at the restrictive temperature, although incorporation of labeled precursors into nuclear DNA does take place. A schematic model incorporating these various findings is presented.     

The role of lipid and antioxidant exchanges in cell division synchronization (mathematical model)

Cell-cycle synchronization of two diffusecoupled cells has been studied in the framework of the membrane model for the cell division cycle, proposed by Chernavskii et al. (1977). It has been shown semianalytically (using the averaging principle) and by computer stimulation that a) if the duration of theG1-phase (T _G1 ) for two identical cells is comparable with the duration of the remaining cycle (T _S+G2+M ), the lipid (L)-exchange results in a synchronization with phase difference =0. The antioxidant (A)-exchange leads to a phase-locking with =T ₀/2 (whereT ₀ is the cell cycle period; b) ifT _G1 T _S+G2+M (orT _G1 T _S+G2+M ) theL-exchange makes synchronization possible both with =0 and =T ₀/2 while theA-exchange results in phase-locking with confined to the region 0 toT ₀/2; c) for non-identical cells differing in the values of kinetic parameters, the locking band narrows as the population density increases (when some model parameters are close to the bifurcation thresholds). We expect that the cells selected artificially at a definite phase of cycle might maintain the synchronous division for a long time if the lipid exchange between cells were stimulated.     

After X-irradiation a transient arrest of L929 cells inG 2-phase coincides with a rapid elevation of the level of O6-alkylguanine-DNA alkyltransferase

Summary Following X-irradiation of exponentially growing L929 cells two major phenomena have been observed. First, there was a delay in cell division which can be ascribed to the arrest of cells in theG ₂-phase (G ₂-block), and, second, the cellular content of the O⁶-alkylguanine-DNA alkyltransferase (AGT) was markedly increased. Flow cytometrical DNA-measurements revealed that cells began to accumulate in theG ₂-phase 4 h after irradiation (p.r.) irrespective of the X-ray dose, while both the fraction of cells blocked inG ₂ and the time period the cells persisted inG ₂ increased with the radiation dose. About 24 h past release from theG ₂-block the distribution of cells in the cell cycle was similar to that of untreated control cells. In comparison with control cells the AGT content in irradiated cells (4 Gy) was highest at about 48 h p.r. (3.4-fold increase). The highest ratio of increase in AGT was, however, observed to occur between about 4 and 13 h p.r. (2.6-fold increase). As shown by flow cytometrical measurements using a BrdUrd/DNA double labeling technique, this rapid primary increase in AGT coincides very well with the entrance of cells into theG ₂-phase. This indicates that the cellular AGT content in X-irradiated (parental) cells started to exceed the basal level at the beginning of theG ₂-phase, but not before or during theS-phase. Once the AGT level was elevated it continued to increase for 2 to 3 cell doubling times.Paper given at the Workshop Molecular Radiation Biology. German Section of the DNA Repair Network, München-Neuherberg, 21.-23.3.1990     

Comparison of cytotoxic activities betweenin vitro and field grown plants ofTyphonium flagelliforme (Lodd.) blume

Anin vitro cytotoxicity screening of theTyphonium flagelliforme extracts indicated high cytotoxicity effect on human lung carcinoma NCl-H23 cells and human mammary gland carcinoma T-47D cells, but the extracts were not active on human liver carcinoma HepG2 cells. NCl-H23 cells were more susceptible toT. flagelliforme extracts than T-47D cells. EDP₅₀ values of the hexane fractions of the mature plant and thein vitro plantlet ofT. flagelliforme on NCl-H23 cells were less than 2 μg/mL Extract from the mature plant was relatively more cytotoxic than the one fromin vitro plantlet except for the hexane fraction. The chloroform and butanol fraction of the mature plant had higher cytotoxicity effect than the fraction fromin vitro plantlet on NCl-H23 cells. All the 3 fractions (hexane, chloroform, and butanol) of the mature plant exhibited higher cytotoxicity effects on human mammary gland carcinoma T-47D cells than the 3 fractions ofin vitro plantlet. However, the human liver carcinoma cells were resistant toT. flagelliforme extracts except for higher concentration of hexane fractions of both the mature and thein vitro plants and the chloroform fraction of the mature plant. Micropropagated plantlets ofT. flagelliforme could hence be used as herbal materials for the treatment of human lung and breast cancers.     

Cytological localization of thePGIP genes in the embryo suspensor cells ofPhaseolus vulgavis L

Polygalacturonase-inhibiting protein (PGIP) is a cell wall protein which inhibits fungalendopolygalacturonases. A small gene family encodesPGIP in the genome of common bean, as indicated by Southernblot experiments performed at high-stringency conditions. Southern-blot analysis of DNA extracted from different cultivars ofPhaseolus vulgaris and fromPhaseolus coccineus showed length polymorphism of the hybridizing restriction fragments. The cytological localization of thePGIP genes was determined in polytene chromosomes of theP. vulgaris embryo suspensor cells. In-situ hybridization experiments using the clonedPGIP gene revealed labelling over a single region of the pericentromeric heterochromatin of chromosome pair X, next to the euchromatin, suggesting thatPGIP gene family may be clustered in one chromosomal region.     

Genetic analysis of resistance to Type-1 Diabetes in ALR/Lt mice,a NOD-related strain with defenses against autoimmune-mediated diabetogenic stress

ALR mice are closely related to type-1 diabetes mellitus (T1DM)-prone NOD mice. The ALR genome confers systemically elevated free radical defenses, dominantly protecting their pancreatic islets from free radical generating toxins, cytotoxic cytokines, and diabetogenic T cells. The ALR major histocompatibility complex (MHC) (H2^gx haplotype) is largely, but not completely identical with the NOD H2^g7 haplotype, sharing alleles from H2-K through the class II and distally into the class III region. This same H2^gx haplotype in the related CTS strain was linked to the Idd16 resistance locus. In the present study, ALR was outcrossed to NOD to fine map the Idd16 locus and establish chromosomal regions carrying other ALR non-MHC-linked resistance loci. To this end, 120 (NOD×ALR)×NOD backcross progeny females were monitored for T1DM and genetic linkage analysis was performed on all progeny using 88 markers covering all chromosomes. Glucosuria or end-stage insulitis developed in 32 females, while 88 remained both aglucosuria and insulitis free. Three ALR-derived resistance loci segregated. As expected, one mapped to Chromosome 17, with peak linkage mapping just proximal to H2-K. A novel resistance locus mapped to Chr 8. A pairwise scan for interactions detected a significant interaction between the loci on Chr 8 and Chr 17. On Chr 3, resistance segregated with a marker between previously described Idd loci and coinciding with an independently mapped locus conferring a suppressed superoxide burst by ALR neutrophils (Susp). These results indicate that the Idd16 resistance allele, defined originally by linkage to the H2^gx haplotype of CTS, is immediately proximal to H2-K. Two additional ALR-contributed resistance loci may be ALR-specific and contribute to this strain's ability to dissipate free-radical stress.     

Autoradiographic studies on the incorporation of3H-amino acids into chromosomes during the mitotic cell cycle ofHaplopappus gracilis

The synthesis of chromosomal proteins and the incorporation of labelled proteins into chromosomes in the mitotic cell cycle ofHaplopappus gracilis, 2n=4, were traced autoradiographically with³H-arginine,³H-lysine, and³H-tryptophane. The duration of the mitotic cell cycle in the root tip cells was determined by³H-thymidine autoradiography and was measured to be 13.0 hr (G₁ 1.3 hr, S 6.5 hr, G₂ 3.8 hr and M 1.4 hr).³H-arginine labelled proteins which were synthesized at S and G₂ were found to be incorporated into chromosomes to a greater extent than proteins which were synthesized either at G₁, at the transition phase from late S to early G₂, or at the mitotic phase. Such varied incorporation was also found in³H-lysine labelled proteins, but not in³H-tryptophane labelled proteins. These findings indicate that the chromosomal proteins are synthesized mainly at S and G₂. Some of the³H-arginine labelled proteins which were synthesized during the first mitotic cell cycle, were found to be incorporated into the chromosomes of the second mitotic cell cycle. The incorporation of the proteins synthesized at one stage of the mitotic cell cycle was found to occur locally in some regions of the chromosomes, while the pattern of incorporation was observed to be similar between euchromatic and heterochromatic regions.     

A new pollen type,C-banded and fluorochrome counterstained chromosomes,and evolution inGuatteria and related genera (Annonaceae)

Guatteria, Guatteriopsis, Guatteriella andHeteropetalum share the same conspicuous pollen type which is new for theSpermatophyta. It is zonoaperturate with a folded aperture region and an extremely reduced exine. First chromosome counts and karyotype analyses forGuatteriopsis (4 species investigated) andGuatteriella (1 species) are identical with those ofGuatteria (19 species seen): 2n = 28. The genome is characterized by diploidization and partly telocentric chromosomes. Sequentially Giemsa C- and fluorochrome banded chromosomes and interphase nuclei are described. The cuticular folding pattern is distinct forHeteropetalum only. Growth forms and ecology are reported for many species. The evolutionary pattern of theGuatteria group is discussed and compared with other genera and families.     

CML typing of serologically identicalH-2 mutants

Cell-mediated lympholysis (CML) reactions were studied among four strains of C57BL/6 (B6) mice carrying mutant alleles (H-2 ^ba ,H-2 ^bd ,H-2 ^bg , andH-2 ^bh ) at thez1 locus in theK end ofH-2 ^b and the original B6 (H-2 ^b ) strain. Cross killing of target cells from lines that had not participated in the mixed lymphocyte reaction (MLR) was extensive, but usually less intense than that of target cells of stimulator cell genotype. The extent of CML crossreactivity could be limited by using cells from F₁ hybrid mice as responders in MLR. In a comprehensive analysis of the cytotoxicity exerted by 20 MLR combinations with homozygous, and 10 MLR combinations with F₁ hybrid responder cells, 19 different CML cytotoxicity patterns were identified, corresponding to at least 19 different CML target specificites. When the number of CML mismatches of each mutant with the originalH-2 ^b was calculated,H-2 ^ba was found to be most distinct fromH-2 ^b ,H-2 ^bs andH-2 ^bd were closest toH-2 ^b , andH-2 ^bh occupied an intermediate position. The validity of this sequence of relatedness is supported by published reports on skin graft survival times and on the interaction of T lymphocytes with virus-infected target cells using cells fromz1 locus mutants.     

Prolyl oligopeptidase participates in cell cycle progression in a human neuroblastoma cell line

Prolyl oligopeptidase (POP) is a post-proline cleaving enzyme, which is widely distributed in various organs, with high levels in the brain. In this study, we investigated the effects of a selective POP inhibitor, 3-({4-[2-(E)-styrylphenoxy]butanoyl}-l-4-hydroxyprolyl)-thiazolidine (SUAM-14746), on the growth of NB-1 human neuroblastoma cells. SUAM-14746 treatment for 24–72 h suppresses the growth of NB-1 cells without cell death in a dose-dependent manner (10–60 μM). Similar suppressive effects were observed with another POP inhibitor benzyloxycarbonyl-thioprolyl-thioprolinal. The SUAM-14746-induced growth inhibition in NB-1 cells was associated with pronounced G₀/G₁ arrest and reduced levels of phosphorylated retinoblastoma protein (pRb), cyclin E, and cyclin dependent kinase (CDK) 2, and increased levels of the CDK inhibitor p27^kip1 and the tumor suppressor p53. SUAM-14746 also induced transient inhibition of S and G₂/M phase progression, which was correlated with retardation of the decrease in the levels of cyclins A and B. Moreover, RNAi-mediated knockdown of POP also led to inhibition of NB-1 cell growth and the effect was accompanied by G₀/G₁ arrest. These results indicate that POP is a part of the machinery that controls the cell cycle.     

Preservation of chromosome integrity during micronucleation induced by colchicine in PtK1 cells

Colchicine induces the formation of small nuclei called micronuclei which contain limited parts of the genome. Some of them exhibit a DNA content equivalent to that of a single chromosome. Our purpose was to study the preservation of chromosome integrity during this micronucleation in PtK₁ cells. Observation of karyotypes obtained after 3 days of cell cycle restoration revealed that micronucleation did not affect chromosome integrity or the presence of each chromosome pair in the surviving cells. In early restoration cells, all the chromosomes included a centromere and were represented in the karyotype, but at variable rates. Furthermore, flow cytometry analysis of micronucleated cells, intermediate in DNA rate between control PtK₁ cells in g₁ and those in G₂/M phases, led us to consider the possibility of selective replication of some chromosomes during micronucleation. Using antibodies against the kinetochore proteins, we derived the presence of one centromeric region (1–2 spots) in the smallest micronuclei. Therefore, these data (karyotypes, number of chromosomes, DNA content and kinetochore proteins) seem to indicate that micronucleation does not induce chromosome damages or translocations. Micronuclei are a convenient tool for investigation of the role of the different chromosomes in the organization of the interphase nuclei.     

Study ofH-2 mutations in mice

The serologically defined H-2.5 specificity was tested on spleen cells and red blood cells (RBC) of theH-2 ^b haplotype and a number of its mutants. Thebm8 (bh) mutant was barely distinguishable fromb in a variety of tests made. On spleen cells ofbm1 (ba) the H-2.5 specificity seemed to be unchanged, while it was virtually absent from RBC of this mutant. Mutantsbm4 (bf),bm5 (bg1), andbm6 (bg2) were similar tobm1, with slight differences between them. The mutantbm3 (bd) retained an unchanged quantity of H-2.5 on its spleen cells, while the specificity was substantially increased on its RBC. The H-2.5 ofbm3 is not identical to that ofH-2 ^a . Possible mechanisms causing differential serology of theH-2 ^b mutants are discussed.     

High-resolution genetic and physical mapping of the cauliflower high-β-carotene gene<Emphasis Type="Italic"> Or</Emphasis> (<Emphasis Type="Italic">Orange</Emphasis>)

Mutation in the cauliflower gene Or causes high levels of -carotene to accumulate in various tissues of the plant that are normally devoid of carotenoids. To decipher the molecular basis by which Or regulates carotenoid accumulation, we have undertaken the isolation of Or by a map-based cloning strategy. Two previously isolated, locus-specific, sequence-characterized amplified region (SCAR) markers that flank Or were employed for the analysis of a large segregating population consisting of 1632 F₂ individuals, and a high-resolution genetic linkage map of the Or locus region was developed. To facilitate positional cloning, we constructed a cauliflower genomic library in a bacterial artificial chromosome (BAC) vector, using high molecular weight DNA from Or homozygotes. The BAC library comprises 60,288 clones with an average insert size of 110 kb, and represents an estimated 10-fold coverage of the genome. A BAC contig encompassing the Or locus was established by screening the library with a marker that is closely linked to Or and by identifying overlapping BAC clones by chromosome walking. Physical mapping delimited the Or locus to a 50-kb DNA fragment within a single BAC clone, which corresponds to a genetic interval of 0.3 cM.Communicated by R. Hagemann     

Optimization of α-amylase and glucoamylase production by recombinant strains ofSaccharomyces cerevisiae

Summary Replacement of the regulatory sequence of theBacillus amyloliquefaciens α-amylase gene (AMY1) by the yeast alcohol dehydrogenase gene promoter (ADC1 _p) resulted in increased levels of extracellular α-amylase production inSaccharomyces cerevisiae. Negative regulation of glucoamylase synthesis by theSTA10-encoded repressor was alleviated by replacing the nativeSTA2 gene promoter fromS. cerevisiae var.diastaticus withADC1 _p. Enhanced degradation of starch was achieved when the modified versions of theAMY1 andSTA2 genes were introduced jointly intoS. cerevisiae.