An analysis of frequencies of chromosome configurations in wheat and wheat hybrids

Presynaptic association of homologous chromosomes is a prerequisite to the sequence of events that lead to chiasma formation. This association of homologous chromosomes, as entire units, occurs with probability a, and chiasma formation occurs independently in opposite chromosome arms with probability c. a and c have been estimated from frequencies of different chromosome configurations at metaphase I of euhexaploid wheat and several derived lines. In the euploid, a is essentially unity and c is of the order of 95%. All changes in the aneuploidy studied involved c rather than a, whereas the change induced by colchicine application primarily involved a.—Observed and expected frequencies of configurations were compared in wheat hybrids in which only homoeologues were present. The expected frequencies of configurations were estimated from the data, based on a being unity for entire groups of homoeologues and c being the probability of chiasma formation between random homoeologous arms. Observed and expected frequences of configurations were in general agreement; however, an excess of observed closed bivalents at the expense of multivalents is interpreted to mean that not all homoeologues are effectively associated in all cells.—In euhexaploid wheat, we suggest that homologues associate with almost certainty, whereas homoeologous pairs of chromosomes associate less efficiently. The aneuploidy examined in this study does not appear to affect the association of chromosomes, but rather the number of chiasmata that eventuate and, in the case of deficiency of chromosome 5B, the distribution of chiasmata within homoelogues, perhaps by way of rendering sites for chiasma formation of homoelogues more similar.     

On correcting the error due to metaphase degeneration in stathmokinetic studies

Metaphases arrested by stathmokinetic agents have a limited life span. This may have to be taken into account in drawing conclusions from experiments whose duration is of the same order as the average metaphase life span, or, in the case of a combined arrest and labelling experiment, if G2 is of long duration. The average life span of arrested metaphases in the tissue under study can be estimated by a simple experiment which isolates the G2 cohort. This enables the investigator to decide whether he can trust his metaphase count without correction for loss due to degeneration. If the proposed experiment requires an accumulation of arrested metaphases over a period longer than the average life span of an arrested metaphase there is a serious risk of under-estimating the metaphase index. We propose a simple method of assessing the magnitude of this risk, and making the necessary correction. Assuming that metaphase degeneration is a random process a corrected index of arrested metaphases can be found for expanding populations multiplying the observed metaphase arrest index by exp [(λ-β) (t-m)], where m is the minimum life span of an arrested metaphase, t is the time point from the onset of the experiment, and β and λ are rates at which metaphases are formed and degenerate, respectively. A slight modification of this formula adapts it to steady state systems.     

Heterokaryon Analysis of the Genetic Control of Pigment Synthesis in Chick Embryo Melanocytes

The genetic control of pigmentation was analyzed using five unlinked mutants, namely, c, pk, Bl, e^y and I. Each mutant blocks or reduces pigmentation. Chick melanocyte cultures of each mutant type were fused to produce all ten possible pair combinations of nondividing heterokaryons. Heterokaryons were identified autoradiographically. (One partner in each pair was labeled with ³H-thymidine.) Crosses produced comparable pairs of double heterozygotes that were analyzed in vivo and in vitro. Heterokaryon pairs were compared to their corresponding double heterozygotes.—Some combinations showed complementation and produced wild-type pigment. Others showed noncomplementation having little or no pigment. Double heterozygotes complemented each other except in the cases involving the dominant mutant, I. Four heterokaryon pairs gave different results from their corresponding double heterozygotes. The pk-Bl and pk-e^y combinations failed to complement as heterokaryons but did complement as double heterozygotes. On the other hand, the I-c and I-Bl combinations complemented as heterokaryons but not as double heterozygotes. Based on these differences it is hypothesized that the pk and I loci are nuclearly restricted regulatory elements. Examples in the literature from other systems are cited to support such a hypothesis.     

Isolation of a Tetrahymena thermophila strain which induced metaphase I meiotic arrest: new pathway of abortive conjugation

A hypodiploid strain of Tetrahymena thermophila has been obtained that shows arrest at the stage of condensed nuclei, corresponding to metaphase I of normal conjugants and induced arrest at meiotic metaphase I (i.e. at the stage of condensed, bivalent chromosomes) in its wt partner mate. The metaphase I arrested conjugants retained their old macronuclei and most of them underwent cell fusion, instead of separation of exconjugants. The doublets were viable and cortically integrated. When the arrest inducing strain was crossed to the haploid tester strain, the haploid micronuclei were arrested in the meiotic metaphase I as the diploid ones had been; the monovalent, chromosomes were condensed, the arms of sister chromatids were not separated, and they were not segregated. Separation of the arms of sister chromatids and disjunction of bivalent chromosomes were not prerequisite for the formation of microtubular spindles in those cells that were arrested in meiotic metaphase I. After re-feeding, the doublet cells resumed cell divisions, segregating two macronuclei and micronuclei at random. One macronucleus was derived from the arrest inducing strain and the other from the tester strain. Heterokaryon strains with macronuclei derived from the parental arrest inducing strain and with the micronucleus derived from the parental wt tester strain were obtained. Surprisingly, these heterokaryons did not induce meiotic arrest. Thus, the arrest in the melotic metaphase I was induced by the micronucleus and not by the macronucleus of the arrest inducing strain.     

NPP-16/Nup50 Function and CDK-1 Inactivation Are Associated with Anoxia-induced Prophase Arrest in Caenorhabditis elegans

Oxygen, an essential nutrient, is sensed by a multiple of cellular pathways that facilitate the responses to and survival of oxygen deprivation. The Caenorhabditis elegans embryo exposed to severe oxygen deprivation (anoxia) enters a state of suspended animation in which cell cycle progression reversibly arrests at specific stages. The mechanisms regulating interphase, prophase, or metaphase arrest in response to anoxia are not completely understood. Characteristics of arrested prophase blastomeres and oocytes are the alignment of condensed chromosomes at the nuclear periphery and an arrest of nuclear envelope breakdown. Notably, anoxia-induced prophase arrest is suppressed in mutant embryos lacking nucleoporin NPP-16/NUP50 function, indicating that this nucleoporin plays an important role in prophase arrest in wild-type embryos. Although the inactive form of cyclin-dependent kinase (CDK-1) is detected in wild-type–arrested prophase blastomeres, the inactive state is not detected in the anoxia exposed npp-16 mutant. Furthermore, we found that CDK-1 localizes near chromosomes in anoxia-exposed embryos. These data support the notion that NPP-16 and CDK-1 function to arrest prophase blastomeres in C. elegans embryos. The anoxia-induced shift of cells from an actively dividing state to an arrested state reveals a previously uncharacterized prophase checkpoint in the C. elegans embryo.     

A Role for Mitogen-activated Protein Kinase in the Spindle Assembly Checkpoint in XTC Cells

The spindle assembly checkpoint prevents cells whose spindles are defective or chromosomes are misaligned from initiating anaphase and leaving mitosis. Studies of Xenopus egg extracts have implicated the Erk2 mitogen-activated protein kinase (MAP kinase) in this checkpoint. Other studies have suggested that MAP kinases might be important for normal mitotic progression. Here we have investigated whether MAP kinase function is required for mitotic progression or the spindle assembly checkpoint in vivo in Xenopus tadpole cells (XTC). We determined that Erk1 and/or Erk2 are present in the mitotic spindle during prometaphase and metaphase, consistent with the idea that MAP kinase might regulate or monitor the status of the spindle. Next, we microinjected purified recombinant XCL100, a Xenopus MAP kinase phosphatase, into XTC cells in various stages of mitosis to interfere with MAP kinase activation. We found that mitotic progression was unaffected by the phosphatase. However, XCL100 rendered the cells unable to remain arrested in mitosis after treatment with nocodazole. Cells injected with phosphatase at prometaphase or metaphase exited mitosis in the presence of nocodazole—the chromosomes decondensed and the nuclear envelope re-formed—whereas cells injected with buffer or a catalytically inactive XCL100 mutant protein remained arrested in mitosis. Coinjection of constitutively active MAP kinase kinase-1, which opposes XCL100's effects on MAP kinase, antagonized the effects of XCL100. Since the only known targets of MAP kinase kinase-1 are Erk1 and Erk2, these findings argue that MAP kinase function is required for the spindle assembly checkpoint in XTC cells.     

Inhibition of Adenosine Triphosphatase Activity from a Plasma Membrane Fraction of Acer pseudoplatanus Cells by 2,2,2-Trichloroethyl 3,4-Dichlorocarbanilate

2,2,2-Trichloroethyl 3,4-dichlorocarbanilate (SW26) is toxic for Acer pseudoplatanus cell cultures. It inhibited the cellular proton extrusion and depolarized the plasmalemma. In vitro, it inhibited the plasma membrane ATPase. SW 26 was also inhibitory to membrane ATPases of other origins—plant (maize shoot), fungus (Schizosaccharomyces pombe), and animal (dog kidney)—with about the same efficiency (7.5 micromolar < I₅₀ < 22 micromolar). It did not inhibit the oligomycin-sensitive ATPase from purified plant mitochondria, nor molybdate-sensitive soluble phosphatases. SW26 was more specific for plasma membrane ATPases than diethylstilbestrol or vanadate. A Lineweaver-Burk plot analysis showed that inhibition kinetics were purely noncompetitive (K_i = 14.7 micromolar) below 20 micromolar. Above this concentration, the inhibition pattern was not consistent with Michaelis-Menten kinetics, and a Hill plot representation revealed a positive cooperativity.     

Streamlining DNA Barcoding Protocols: Automated DNA Extraction and a New cox1 Primer in Arachnid Systematics

Background

DNA barcoding is a popular tool in taxonomic and phylogenetic studies, but for most animal lineages protocols for obtaining the barcoding sequences—mitochondrial cytochrome C oxidase subunit I (cox1 AKA CO1)—are not standardized. Our aim was to explore an optimal strategy for arachnids, focusing on the species-richest lineage, spiders by (1) improving an automated DNA extraction protocol, (2) testing the performance of commonly used primer combinations, and (3) developing a new cox1 primer suitable for more efficient alignment and phylogenetic analyses.

Methodology

We used exemplars of 15 species from all major spider clades, processed a range of spider tissues of varying size and quality, optimized genomic DNA extraction using the MagMAX Express magnetic particle processor—an automated high throughput DNA extraction system—and tested cox1 amplification protocols emphasizing the standard barcoding region using ten routinely employed primer pairs.

Results

The best results were obtained with the commonly used Folmer primers (LCO1490/HCO2198) that capture the standard barcode region, and with the C1-J-2183/C1-N-2776 primer pair that amplifies its extension. However, C1-J-2183 is designed too close to HCO2198 for well-interpreted, continuous sequence data, and in practice the resulting sequences from the two primer pairs rarely overlap. We therefore designed a new forward primer C1-J-2123 60 base pairs upstream of the C1-J-2183 binding site. The success rate of this new primer (93%) matched that of C1-J-2183.

Conclusions

The use of C1-J-2123 allows full, indel-free overlap of sequences obtained with the standard Folmer primers and with C1-J-2123 primer pair. Our preliminary tests suggest that in addition to spiders, C1-J-2123 will also perform in other arachnids and several other invertebrates. We provide optimal PCR protocols for these primer sets, and recommend using them for systematic efforts beyond DNA barcoding.     

Interferon Gamma +874T/A Polymorphism Increases the Risk of Hepatitis Virus-Related Diseases: Evidence from a Meta-Analysis

BackgroundInterferon gamma (IFN-γ) is a key regulatory cytokine, which plays an important role in antiviral defense of an infected host. However, the association between the IFN-γ +874T/A gene polymorphism and hepatitis virus-related diseases is heterogeneous.MethodsBased on the Preferred Reporting Items for Systematic Reviews and Meta-analyses statement, a comprehensive literature search of eligible studies in Embase, Pubmed, and the Cochrane Library was undertaken through November 2014. Odds ratios (ORs) and the corresponding 95% confidence intervals (CIs) were used to measure the strength of the models.ResultsSeventeen case-control articles, including 24 studies with 5503 individuals, met the inclusion criteria. The results indicated a statistically significant association between the IFN-γ +874T/A polymorphism and hepatitis virus—related diseases in a recessive gene model (AA vs. TT+TA: OR=1.350, 95% CI=1.101-1.657, P=0.004, I²%=54.3, and P_Q=0.001 for heterogeneity), especially in Asians (OR=1.407, 95% CI=1.035-1.911, P=0.029, I²%=61.9, and P_Q=0.005 for heterogeneity) and hepatitis B virus (HBV)–related disease (OR=1.486, 95% CI=1.195–1.849, P=0.000, I²%=40.4, and P_Q=0.053 for heterogeneity).ConclusionsThe evidence suggests that the IFN-γ +874T/A polymorphism increases the risk of hepatitis virus—related diseases, especially in Asians and HBV—related diseases. Further studies on this topic in different ethnicities, especially genome-wide association studies, should be conducted to strengthen our results.     

Electrophoretic Variation for X Chromosome-Linked Hypoxanthine Phosphoribosyl Transferase (Hprt) in Wild-Derived Mice

An electrophoretic variation for hypoxanthine phosphoribosyltransferase, HPRT, has been identified in samples of Mus spretus, a field mouse from southern Europe and in M. m. castaneus, a house mouse from southeast Asia. These mice will interbreed with laboratory mice to produce viable, fertile F₁ progeny. The variation for HPRT segregates as an X chromosome gene in F₁ and backcross progeny. Linkage analysis involving the markers Pgk-1 and Ags indicated a gene order of centromere— Hprt—Pgk-1—Ags in crosses involving both stocks of wild mice.     

Meiosis in Male DROSOPHILA MELANOGASTER. II. Nonrandom Segregation of Compound-Second Chromosomes

The segregation of compound-second chromosomes in males from two different stocks has been examined. Segregation is random in males from the C(2L)RM4, dp; C(2R)RM4, px stock. Gametes containing only one of the two compound chromosomes comprise 50% of the gametes, and gametes containing either both elements or neither element make up the other 50% of the gametes.——In males from the C(2L)RM, b; C(2R)RM, cn stock, gametes containing either C(2L)RM, b or C(2R)RM, cn make up the majority of the gametes. Gametes containing both chromosomes or neither chromosome account for only 2-3% of the gametes. The nonrandom segregation is due to the C(2R)RM, cn chromosome.——Viability is reduced in flies carrying the C(2R)RM, cn chromosome. This includes larval lethality, delayed development and premature adult lethality. Cytologically, this chromosome contains a large duplication of 2L material, which includes material proximal to region 38 or 39. It is suggested that the viability and segregational properties associated with this chromosome are due to the duplicated 2L material.     

An ordered arrangement of chromosomes in the somatic nucleus of common wheat Triticum aestivum L.

Spatial relationships between chromosomes of the same genome, both homologous and non-homologous, were studied in root-tip cells of common wheat, Triticum aestivum (2n = 6x = 42). Mean distance between members of all the 21 homologous pairs (seven in each of the three genomes) and of 45 out of the 63 possible non-homologous combinations of two (21 in each genome) were determined. To minimize disruption of nuclear chromosomal arrangement, the cells were pretreated with cold temperature either in tap water or in a physiological medium (White solution) and distances between cytologically marked chromosomes were measured at metaphase. Comparison of distances for homologues with those for non-homologues indicated clearly that, within each genome, the homologous chromosomes were significantly closer to one another than were the non-homologues. Distances between homologues were similar in all three genomes, as were distances between non-homologues. The data are consistent with the hypothesis that the chromosomes of each genome of common wheat are arranged in the somatic nucleus in a highly specific ordered pattern. In this hypothetical arrangement, homologous chromosomes are closely associated, while the nonhomologues occupy definite positions with respect to one another. The universality of the phenomenon and its cellular mechanism and biological significance are discussed.     

The Effects of B, K10, and AR Chromosomes on the Resistance of Maize to Viral Infection

Studies were conducted to determine if accessory (B) chromosomes, the abnormal tenth (K10) chromosome or the aberrant ratio (AR) phenomenon of maize (Zea mays L.) affect the resistance of the plants to viral infection. Genetically similar stocks of maize with and without these elements were compared to determine what effect they would have on the plants response to Brome Mosaic Virus (BMV), Maize Dwarf Mosaic Virus (MDMV), Wheat Streak Mosaic Virus (WSMV) and Barley Stripe Mosaic Virus (BSMV).—The test results with BSMV were not found to be conclusive. With BMV and MDMV, neither the B orK10 chromosomes were found to alter infections; however, these chromosomes were found to affect the resistance of the plants to WSMV infection. The B chromosomes were found to delay the onset of leaf necrosis by 15%, while the K10 chromosome was found to increase the susceptibility to necrosis by 100%. The AR phenomenon was not found to alter the resistance of maize to BMV infection. However, it was found to increase the susceptibility of maize to MDMV infection by 36% and to decrease the susceptibility of maize to WSMV infection by 92%.     

The quantitative analysis of chromosome pairing and chiasma formation based on the relative frequencies of M I configurations

In autotetraploids, chromosome pairing may be in the form of quadrivalents or bivalent pairs. Whether or not the quadrivalents are maintained until first meiotic metaphase depends on the formation of chiasmata. The relative frequencies of M I configurations thus contain information both on pairing and on chiasma formation. With distal chiasma localisation six configurations can be recognised and their relative frequencies determined: ring quadrivalents, chain quadrivalents, trivalents (with univalent), ring bivalents, open (rod) bivalents, univalent pairs. These represent five degrees of freedom permitting five parameters to be estimated: the frequency (f) of quadrivalent pairing; the frequencies of chiasmate association of the two ends (arms in metacentrics), a′, b′, after quadrivalent pairing, and a, b after bivalent pairing. — The appropriate formulae have been derived and applied to observations on Tradescantia virginiana (4n=24) which has pronounced distal chiasma localisation. Slight modifications make the model applicable to autotetraploids with interstitial in addition to distal chiasmata. In T. virginiana, chromosome pairing appeared to be random between homologues (65.8% quadrivalent pairing; 55.4% observed at M I). After quadrivalent pairing chiasmate association is frequent in the “average long” arm (95.0%) and much less so in the other arm (60.5%). This is attributed to partner exchange. After bivalent pairing chiasma frequencies are still different for the two arms (93.8% and 83.5% association respectively) but much less pronounced. Various complications are discussed.     

Arrested development of the rabbit stomach worm Obeliscoides cuniculi: varied responses to cold treatment by the offspring produced throughout the course of a single infection

Arrested development of the rabbit stomach worm Obeliscoides cuniculi: varied responses to cold treatment by the offspring produced throughout the course of a single infection. International Journal for Parasitology16: 55–61. In an experimental model, rabbits were inoculated with cold treated L3s (parental generation) to establish infections with a high proportion of arrested larvae. Over 234 days, ten batches of L3s (offspring) were derived sequentially from ten pairs of rabbits. Samples of L3s (offspring) were cold treated for various periods (13–74 days) and inoculated into test-rabbits to determine cold treatment time for maximum arrest (CTTMA). As parental infections proceeded, the CTTMA varied from batch to batch of offspring. Their propensity for arrest also varied, but no correlation was found between duration of infection by parents and propensity for arrest by offspring. Three additional rabbits were treated early in their infections with thiabendazole. Offspring larvae subsequently derived from these rabbits exhibited significantly more arrest than those from untreated hosts. The model shows that, in natural infections, overwintering arrested larvae could transmit genes for arrest to their spring progeny but that such genes would remain unexpressed until acted on by the correct environmental stimulus.     

Identification of resistance-associated proteins in closely-related maize lines varying in aflatoxin accumulation

Aspergillus flavus infection of maize and subsequent contamination with carcinogenic aflatoxins poses serious health concerns, especially in developing countries. Maize lines resistant to A. flavus infection have been identified; however, the development of commercially-useful aflatoxin-resistant maize lines has been hindered due to a lack of breeding markers. To identify maize resistance-associated proteins (RAPs) as potential markers for breeding, 52 BC1S4 lines developed from crosses between five African maize inbreds and five temperate aflatoxin-resistant lines were screened using the kernel screening assay. Five pairs of closely-related lines that had 75?C94% genetic similarity within each pair and which varied within each pair in aflatoxin accumulation were selected for proteomic investigation. Kernel embryo and endosperm protein profile differences within the pair and across pairs were compared using two-dimensional polyacrylamide gel electrophoresis. Differentially expressed (??1.5-fold) RAPs were sequenced through tandem mass spectrometry and were identified as antifungal, stress-related, storage or regulatory proteins. Sequence homology analysis highlighted several proteins in maize that confer resistance to A. flavus infection and/or aflatoxin production.     

Induction of Blocks in Nuclear Divisions and Overcondensation of Meiotic Chromosomes with Cycloheximide During Conjugation of Tetrahymena thermophila

During conjugation, the micronucleus of Tetrahymena thermophila undergoes five consecutive nuclear divisions: meiosis, third prezygotic division (pregamic mitosis) and two postzygotic mitoses of the synkaryon. The four products of the synkaryon differentiate into macronuclear anlagen and new micronuclei and the old macronucleus is resorbed. The protein synthesis inhibitor cycloheximide, applied during conjugation, induced several developmental blocks. Pairs shifted to the drug during early meiotic prophase (stages I–III) were arrested at prophase. Cycloheximide applied to cells at pachytene (stages IV-VI) to metaphase arrested the conjugants at the stage of modified prometaphase/metaphase with overcondensed, swollen bivalents. In contrast to other systems, in the presence of cycloheximide, separation of chromatids, decondensation of chromosomes and exit from metaphase I were inhibited in both diploid and haploid cells. Pairs shifted to the drug after metaphase I were arrested at postmeiotic interphase after completing one nuclear cycle. The same rule applied to the subsequent cycle; then cells were arrested at the stage of pronuclei, and those pairs with functional pronuclei and synkarya were arrested at the stage of two products of the first postzygotic division (pronuclei were not arrested in nuclear transfer and karyogamy). Only pairs with two products of the first postzygotic division were arrested at the same stage after the cycloheximide treatment. Pairs shifted to cycloheximide during the second postzygotic division were arrested in development of macronuclear anlagen and resorption of old macronuclei. The postmeiotic conjugants pulse-treated with cycloheximide (2 h) yielded heterokaryons retaining parental macronuclei (i.e. they exhibited macronuclear retention).