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PubMed1976

Esterases in the mosquito Culex pipiens pipiens L: formal genetics and polymorphism of adult esterases.

The genetics of two esterase loci active in autogenous adults of the mosquito Culex pipiens pipiens L. has been studied by means of starch gel electrophoresis. Three alleles at the Est-1 locus and eight at the Est-2 locus are described. Both loci have a null allele. Active alleles are codominant and there is no hybrid enzyme in heterozygotes. The Est-1 locus codes esterases preferentially hydrolyzing alpha-naphthylacetate and the Est-2 locus esterases preferentially hydrolyzing beta-naphthylacetate. Strains homozygous for both loci were selected. Linkage studies of the two loci have shown that they are not sex linked but are linked to each other, the crossover frequency being 8.6%. The polymorphism of two laboratory and two natural populations is described for both loci. Phenotypic distributions are in good agreement with Hardy-Weinberg expectations.

Alleles↗
PubMed1994

The relationship between synaptonemal complex length and genome size in four vertebrate classes (Osteicthyes, Reptilia, Aves, Mammalia).

We investigated the relationship between synaptonemal complex (SC) length and genome size in 18 species of vertebrates from the classes Osteicthyes (bony fish), Reptilia (reptiles), Aves (birds), and Mammalia (mammals). When total SC length was plotted against genome size for all 18 vertebrate species, there did not appear to be a correlation between the two variables. However, when birds were excluded from the data and a linear regression analysis was performed, variation in genome size accounted for approximately 50% of the variation in total SC length (r2 = 0.47). Dividing the average total SC length for a species by its 4C DNA amount yields the species' SC/DNA ratio. SC/DNA ratios of birds were approximately twice as high as the SC/DNA ratios of reptiles and mammals. Bony fish showed intraclass divergence in SC/DNA ratios. The sunfish (Centrarchidae) had SC/DNA ratios almost as high as those of birds, while the remaining fish in the study had SC/DNA ratios similar to those of reptiles and mammals. These observations indicate that inter and intraclass divergence in the relationship between total SC length and genome size has occurred in the vertebrates. Coupled with evidence from the literature, our results also suggest that SC/DNA ratios are positively correlated with crossover frequency.

Animals↗
PubMed2000

A homologue of the yeast HOP1 gene is inactivated in the Arabidopsis meiotic mutant asy1.

Synapsis of homologous chromosomes is a key event in meiosis as it is essential for normal chromosome segregation and is implicated in the regulation of crossover frequency. We have previously reported the identification and cytological characterisation of a T-DNA-tagged asynaptic mutant of Arabidopsis thaliana. We have demonstrated that this mutant, asy1, is defective in meiosis in both males and females. Cloning and nucleotide sequencing of the ASY1 gene has revealed that it encodes a polypeptide of 596 amino acids that exhibits similarity to the HOP1 gene of Saccharomyces cerevisiae, which is known to encode a protein essential for synaptonemal complex assembly and normal synapsis. Expression studies indicate that, in common with a number of other Arabidopsis meiotic genes, ASY1 exhibits low-level expression in a range of plant tissues. Southern analysis coupled with database searching has resulted in the identification of an ASY1 homologue, ASY2. Although asy1 exhibits a strong asynaptic phenotype, a residual low level of synapsis indicates that ASY1 and ASY2 may exhibit a low degree of functional redundancy.

Arabidopsis↗
PubMed1996

Membrane changes associated with the temperature-sensitive P85gag-mos-dependent transformation of rat kidney cells as determined by dielectrophoresis and electrorotation.

Conventional dielectrophoresis (cDEP) and electrorotation (ROT) measurements have been used to determine the dielectric properties of a clone of normal rat kidney cells, designated 6m2, that exhibits a transformed phenotype at 33 degrees C and a non-transformed phenotype at 39 degrees C. cDEP measurements of the crossover frequencies at which individual 6m2 cells experienced zero cDEP force performed as a function of the conductivity of the suspension medium revealed that, in response to a temperature shift from 33 degrees C to 39 degrees C for 24 h, the mean specific cell membrane capacitance and conductance fell significantly (P < 0.01) from 42.3 (+/-1.3) to 30.3 (+/-2.9) mF/m2 and 743 (+/-422) to 567 (+/-326) S/m2, respectively. ROT analyses demonstrated a similar reduction for the membrane capacitance from 37.2 (+/-7.3) to 27.4 (+/-6.1) mF/m2, and also showed that accompanying changes in the mean internal electrical conductivity and dielectric permittivity of the cells were insignificant. Scanning electron microscopy was used to examine the surface morphology of the cells and, in agreement with our previous reports for leukemia cells, the observed membrane capacitance values correlated closely with the morphological complexity of the cell membrane surface. The observed changes in the membrane dielectric properties are discussed in terms of their biological significance and their relationship to previously-detected changes in cell surface charge.

Animals↗
PubMed1989

Alteration of the effects of caffeine by prenatal stress.

We examined the effect of prenatal stress exposure on sensitivity to caffeine using behavioral and physiological measures. Pregnant rats were handled 5 minutes daily from the 14th to 21st day of gestation. Male offspring were tested when 60 days of age in a modified open field apparatus 30 and 90 minutes after injection with caffeine (0, 10, 30 mg/kg). Caffeine increased crossover frequency and duration at the 10 mg/kg dose. Rearing frequency and duration were increased by the 10 mg/kg dose while the 30 mg/kg dose was ineffective. Gnawing was increased by caffeine, especially 90 minutes postinjection. Headpoke activity was decreased by caffeine treatment. Caffeine had no effect on defecation and urination. Gnawing activity was increased by caffeine in prenatally nonstressed animals, but was depressed in prenatally stressed animals. Prenatal stress increased sensitivity to caffeine on corner activity and rearing. The other measures were not affected differentially by prenatal stress exposure. Rectal temperature was depressed 0.75 degrees C in both prenatally stressed and nonstressed animals, by the 30 mg/kg dose of caffeine. Thus, our results indicate that prenatal stress affects sensitivity to caffeine in the adult offspring. However, the long-term effects of prenatal stress exposure are dependent on the measures employed.

Animals↗
PubMedDate not supplied

Low level lead exposure during lactation increases rough and tumble play fighting of juvenile rats.

Lactating rats were given distilled water or distilled water containing 0.067% lead chloride (500 ppm lead) as their sole source of drinking fluid from Days 1-21 of lactation. Activity, social investigation and rough and tumble play fighting behaviors of the offspring were observed on Day 26 and activity and play solicitation behaviors on Day 36. Although lead treatment reduced the mothers' fluid intake, there were no effects on pup growth and activity or on maternal behaviors. When paired with a group-housed stimulus animal on Day 26, lead-treated subjects had increases in the two measures of play fighting (crossover and pinning), and in social investigation, relative to controls. When tested with a scopolamine-treated, non-playful stimulus on Day 36, increased crossover frequencies were observed in lead-treated subjects when compared with controls. Two-min activity levels on Days 26 and 35 were unaffected by lead exposure. These results indicate that social interactive behaviors of juvenile rats are effective tools in the assessment of exposure to toxic substances early in development.

Aggression↗
PubMed2002

Membrane dielectric changes indicate induced apoptosis in HL-60 cells more sensitively than surface phosphatidylserine expression or DNA fragmentation.

The specific membrane capacitance and conductivity of mammalian cells, which reflect their surface morphological complexities and membrane barrier functions, respectively, have been shown to respond to cell physiologic and pathologic changes. Here, the effects of induced apoptosis on these membrane properties of cultured human promyelocytic HL-60 cells are reported. Changes in membrane capacitance and conductivity were deduced from measurements of cellular dielectrophoretic crossover frequencies following treatment with genistein (GEN). The apparent specific cell membrane capacitance of HL-60 cells fell from an initial value of 17.6+/-0.9 to 9.1+/-0.5 mF/m(2) 4 h after treatment. Changes began within minutes of treatment and preceded both the externalization of phosphatidylserine (PS), as gauged by the Annexin V assay, and the appearance of a sub-G1 cell subpopulation, as determined through ethidium bromide staining of DNA. Treatment by the broad spectrum caspase inhibitor N-benzyloxycarbony-Val-Ala-Asp(O-methyl)-fluoromethyketone (zVAD-fmk) did not prevent these early cell membrane dielectric responses, suggesting that the caspase system was not involved. Although membrane conductivity did not alter during the first 4 h of GEN treatment, it rose significantly and progressively thereafter. Finally, as the barrier function failed and the cells became necrotic, it increased by many orders of magnitude. The effective membrane capacitance and conductivity findings serve to focus attention on the membrane as a site for early participation in apoptosis. In conjunction with our prior reports of the use of dielectric methods for cell manipulation and separation, these results demonstrate that dielectrophoretic technologies should be applicable to the rapid detection, separation, and quantification of normal, apoptotic, and necrotic cells from cell mixtures.

Apoptosis↗
PubMed1992

The evolution of meiosis.

Meiosis is too complex to have arisen at once full blown and a stepwise scheme is proposed for its evolution, where each step is believed to have provided an immediate selective advantage: (1) The first step in this tentative sequence is the development of a haploidization process by means of a rapid series of mitotic non-disjunctions, turned on under conditions where haploidy is favored. The non-disjunctions may have resulted from a conditional mutation which caused sister centromere cohesiveness in the past mitotic metaphase. (2) Next probably came the formation of rudimentary synaptonemal complex type structures, first at Holliday-type configurations and later extending from these along chromosome pairs. These structures between homologues, though costly to produce and maintain, may have directly served the disjunctive function by setting the stage for the production of haploidy in one division, under conditions where it was advantageous. (3) Then secondarily acquired functions of the synaptonemal complex or structures associated with it may have promoted greatly increased crossover frequency, in part at least by increasing the frequency of the isomerization-type reaction. The resulting recombination of linked genes could have been advantageous under some conditions. (4) Finally, it is proposed that the capability was acquired for enhanced association of sister chromatids during the period between pachytene and anaphase I to give rise to chiasma-mediated disjunction, so that the relatively costly synaptonemal complex maintenance until anaphase I could be abandoned without losing disjunctive capability. It is implied that the modern synaptonemal complex is a structure which embodies a number of separately encoded proteins and that secondary structures and functions are associated with close homologue pairing. This scheme is based upon observable cytological and molecular characteristics of modern organisms.

Animals↗
PubMed2002

Dielectrophoretic cell separation and gene expression profiling on microelectronic chip arrays.

Cell membrane dielectric properties of five different cultivated cell lines and human peripheral blood mononuclear cells (PBMC) were determined from dielectrophoretic crossover frequency measurements on a 5 x 5 microelectronic chip array. Based on distinct dielectric property differences between individual cell types, efficient cell separations were achieved by dielectrophoresis on this 5 x 5 array, which included separation of monocytic cells (U937) or human T cell leukemia virus type 1 (HTLV-1) tax-transformed cells (Ind-2) from PBMC, as well as separation of neuroblastoma cells (SH-SY5Y) from glioma cells (HTB). The purity of dielectrophoretically separated cells can be greater than 95%. Expression profiles of IL-1, TNF-alpha, and TGF-beta genes for U937 cells mixed with PBMC before and after the separation were determined by a means of electric field-facilitated hybridization on a 10 x 10 microelectronic chip array. By using the expression levels of pure U937 cells as a control, it was shown that the gene expression profiles of the postseparation cells were significantly different from those of the preseparation cell mixtures. The increase in gene expression levels for U937 cells upon lipopolysaccharide induction could be accurately determined only in the postseparation cells, while the preseparation samples masked these changes. Furthermore, by cultivating the separated HTB and SH-SY5Y cells and measuring expression of the stress-related gene c-fos, dielectrophoretic forces were shown to have little effect on cell survival and stress. The presented approach of using microelectronic chip arrays for both cell separation and gene expression profiling provides a great potential for accurate genetic analysis of specific cell subpopulations in heterogeneous samples.

Cell Line↗
PubMed2006

Dielectrophoresis of surface conductance modulated single-walled carbon nanotubes using catanionic surfactants.

Dielectrophoresis has received considerable attention for separating nanotubes according to electronic types. Here we examine the effects of surface conductivity of semiconducting single walled carbon nanotubes (SWNT), induced by ionic surfactants, on the sign of dielectrophoretic force. The crossover frequency of semiconducting SWNT increases rapidly as the conductivity ratio between the particle and medium increases, leading to an incomplete separation of ionic surfactant suspended SWNT at an electric field frequency of 10 MHz. To reduce the conductivity ratio, the surface charge of SWNT is neutralized by an equimolar mixture of anionic surfactant sodium dodecyl sulfate (SDS) and cationic surfactant cetyltrimethylammonium bromide (CTAB), resulting in negative dielectrophoresis of semiconducting species at 10 MHz. A comparative Raman spectroscopy study shows a nearly complete separation of metallic SWNT.

Journal Article↗
PubMed2005

Human recombination hot spots hidden in regions of strong marker association.

The fine-scale distribution of meiotic recombination events in the human genome can be inferred from patterns of haplotype diversity in human populations but directly studied only by high-resolution sperm typing. Both approaches indicate that crossovers are heavily clustered into narrow recombination hot spots. But our direct understanding of hot-spot properties and distributions is largely limited to sperm typing in the major histocompatibility complex (MHC). We now describe the analysis of an unremarkable 206-kb region on human chromosome 1, which identified localized regions of linkage disequilibrium breakdown that mark the locations of sperm crossover hot spots. The distribution, intensity and morphology of these hot spots are markedly similar to those in the MHC. But we also accidentally detected additional hot spots in regions of strong association. Coalescent analysis of genotype data detected most of the hot spots but showed significant differences between sperm crossover frequencies and historical recombination rates. This raises the possibility that some hot spots, particularly those in regions of strong association, may have evolved very recently and not left their full imprint on haplotype diversity. These results suggest that hot spots could be very abundant and possibly fluid features of the human genome.

Chromosomes, Human, Pair 1↗
PubMed2003

Computational and experimental analysis of DNA shuffling.

We describe a computational model of DNA shuffling based on the thermodynamics and kinetics of this process. The model independently tracks a representative ensemble of DNA molecules and records their states at every stage of a shuffling reaction. These data can subsequently be analyzed to yield information on any relevant metric, including reassembly efficiency, crossover number, type and distribution, and DNA sequence length distributions. The predictive ability of the model was validated by comparison to three independent sets of experimental data, and analysis of the simulation results led to several unique insights into the DNA shuffling process. We examine a tradeoff between crossover frequency and reassembly efficiency and illustrate the effects of experimental parameters on this relationship. Furthermore, we discuss conditions that promote the formation of useless "junk" DNA sequences or multimeric sequences containing multiple copies of the reassembled product. This model will therefore aid in the design of optimal shuffling reaction conditions.

Base Sequence↗
PubMed1997

The hotspot conversion paradox and the evolution of meiotic recombination.

Studies of meiotic recombination have revealed an evolutionary paradox. Molecular and genetic analysis has shown that crossing over initiates at specific sites called hotspots, by a recombinational-repair mechanism in which the initiating hotspot is replaced by a copy of its homolog. We have used computer simulations of large populations to show that this mechanism causes active hotspot alleles to be rapidly replaced by inactive alleles, which arise by rare mutation and increase by recombination-associated conversion. Additional simulations solidified the paradox by showing that the known benefits of recombination appear inadequate to maintain its mechanism. Neither the benefits of accurate segregation nor those of recombining flanking genes were sufficient to preserve active alleles in the face of conversion. A partial resolution to this paradox was obtained by introducing into the model an additional, nonmeiotic function for the sites that initiate recombination, consistent with the observed association of hotspots with functional sites in chromatin. Provided selection for this function was sufficiently strong, active hotspots were able to persist in spite of frequent conversion to inactive alleles. However, this explanation is unsatisfactory for two reasons. First, it is unlikely to apply to obligately sexual species, because observed crossover frequencies imply maintenance of many hotspots per genome, and the viability selection needed to preserve these would drive the species to extinction. Second, it fails to explain why such a genetically costly mechanism of recombination has been maintained over evolutionary time. Thus the paradox persists and is likely to be resolved only by significant changes to the commonly accepted mechanism of crossing over.

Computer Simulation↗
PubMed2006

SII and fit-to-target analysis of compression system performance as a function of number of compression channels.

This work was undertaken to answer the question, 'How does the speech audibility/fit-to-gain-target provided by compression change with number of channels?' For each of 957 audiograms and a given number of compression channels, the channel crossover frequencies were set either to maximize the SII (speech intelligibility index) for low- and high-level speech spectra, or to optimize the fit-to-gain targets from the Cambridge method for loudness equalization (CAMEQ). The audiograms comprised all common configurations, and losses ranged from mild to severe. Use of these computational procedures allowed the predicted, channel-number-based performance to be determined separately from the effects of other compression parameters. From one to five channels were sufficient to yield predicted speech recognition performance within 5% of maximum for 90% of the 'mild' and 'moderate' audiograms. Three to nine channels were necessary for the same level of predicted performance for 90% of the 'severe' audiograms. Four channels or fewer were sufficient to produce less than 5 dB rms error in fit to CAMEQ targets for 90% of all audiograms.

Hearing Aids↗
PubMed1984

The genetic analysis of a chromosome-specific meiotic mutant that permits a premature separation of sister chromatids in Drosophila melanogaster.

mei- G87 is a recessive meiotic mutant that increases second chromosome nondisjunction in both males and females. A significant proportion of the diplo -2 exceptions are equational . In females, diplo -2 reductional exceptions are usually noncrossovers , but, in equational exceptions, crossover frequency and distribution are the same as that found in the haplo-2 controls. The frequencies of nondisjunction are relatively low: 0.6% in females and 1.3% in males. Nondisjunction frequency is affected by environmental conditions (possibly humidity). The defect in mei- G87 , as in other "second division" mutants, appears to be a failure to maintain sister-chromatid cohesion. mei- G87 increases nondisjunction of only the second chromosome. This may indicate either a weak mutant with only the second chromosome being sensitive enough to misbehave or it may indicate that chromosome-specific regions responsible for sister-chromatid cohesion exist.

Animals↗
PubMed1991

The effect of DNA sequence polymorphisms on intragenic recombination in the rosy locus of Drosophila melanogaster.

The effect of simple DNA sequence polymorphisms on intragenic recombination in the rosy locus of Drosophila melanogaster was assayed. Two crosses were performed involving nearly identical molecular distances between selective ry null mutations (3778 nucleotides and 3972 nucleotides). In one heterozygote (ry606/ry531), in addition to the nucleotide substitution ry- mutations, there were 11 simple nucleotide polymorphisms between the selective markers as well as additional flanking simple nucleotide polymorphisms within the rosy locus. In the other heterozygote (ry606/ry609), there were no additional polymorphisms because the two rosy nucleotide substitution mutations were induced on the same rosy isoallele (ry+6). From ry606/ry531 heterozygous females, 27 intragenic crossovers and five marker conversions were seen among 4.53 x 10(5) progeny. From ry606/ry609 heterozygous females, 23 intragenic crossovers and eight marker conversions were seen among 4.18 x 10(5) progeny. The intragenic crossover frequencies per kilobase of DNA were very similar, 1.6 x 10(-5) for ry606/ry531 and 1.4 x 10(-5) for ry606/ry609. Thus, simple DNA sequence polymorphisms neither inhibit nor promote intragenic recombination in D. melanogaster.

Animals↗
PubMed1993

Meiotic behavior and linkage relationships in the secondarily homothallic fungus Agaricus bisporus.

This study followed the transmission of 64 segregating genetic markers to 52 haploid offspring, obtained from both homokaryotic and heterokaryotic meiospores, of a cross (AG 93b) of Agaricus bisporus, the commonly cultivated "button mushroom." The electrophoretic karyotypes of the AG 93b component nuclei were determined concurrently (n = 13). Eleven distinct linkage groups were identified by two-point analysis. DNA-DNA hybridization showed that nine of these corresponded to unique chromosome-sized DNAs. Two other chromosomal DNAs were marked with nonsegregating markers, including the rDNA repeat. Two remaining chromosomes remained unmarked but hybridized to repeated-sequence probes. Cross 93b had an essentially conventional meiosis in which both independent assortment and joint segregation of markers occurred, but in which crossing over was infrequent over much of the mapped genome. The 48 homokaryotic spore-offspring had overall crossover frequencies that were similar to, but possibly slightly less than, those of three homokaryon constituents of heterokaryotic spore-offspring. These daa provide support for our earlier cytogenetic model of sporogenesis in A. bisporus, that explains why heterokaryotic spore-offspring usually appear to exhibit no recombination. No evidence favoring an alternative, mitotic model of sporogenesis was found. The resulting genetic map appears to survey the genome extensively and for the first time permits localization of loci determining economically important traits in this fungal crop species. Large differences in the vigor of homokaryotic offspring were correlated with the inheritance of certain chromosome segments and were also often associated with significant departures from Mendelian segregation ratios.

Agaricus↗
PubMed1995

Hot spots of recombination in fission yeast: inactivation of the M26 hot spot by deletion of the ade6 promoter and the novel hotspot ura4-aim.

The M26 mutation in the ade6 gene of Schizosaccharomyces pombe creates a hot spot of meiotic recombination. A single base substitution, the M26 mutation is situated within the open reading frame, near the 5' end. It has previously been shown that the heptanucleotide sequence 5' ATGACGT 3', which includes the M26 mutation, is required for hot spot activity. The 510-bp ade6-delXB deletion encompasses the promoter and the first 23 bp of the open reading frame, ending 112 bp upstream of M26. Deletion of the promoter in cis to M26 abolishes hot spot activity, while deletion in trans to M26 has no effect. Homozygous deletion of the promoter also eliminates M26 hot spot activity, indicating that the heterology created through deletion of the promoter per se is not responsible for the loss of hot spot activity. Thus, DNA sequences other than the heptanucleotide 5' ATGACGT 3', which must be located at the 5' end of the ade6 gene, appear to be required for hot spot activity. While the M26 hotspot stimulates crossovers associated with M26 conversion, it does not affect the crossover frequency in the intervals adjacent to ade6. The flanking marker ura4-aim, a heterology created by insertion of the ura4+ gene upstream of ade6, turned out to be a hot spot itself. It shows disparity of conversion with preferential loss of the insertion. The frequency of conversion at ura4-aim is reduced when the M26 hot spot is active 15 kb away, indicating competition for recombination factors by hot spots in close proximity.

Base Sequence↗