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Disease-induced natural selection in a diploid host.

Using for each genotype an SIR-type model of disease transmission dynamics, we describe natural selection in a continuously breeding diploid host whose disease susceptibility and resistance are carried at one locus with two alleles. The system is transformed into variables that for each disease class describe the number of individuals, the gene frequency, and the deviation from Hardy-Weinberg proportions as measured by Wright's fixation index. An assumption of small variation in disease response among genotypes (slow selection) separates the system to first-order into three blocks. One block describes the population-wide disease dynamics, the second considers the fixation index in each class, and the third block provides the change in gene frequencies. The first two blocks settle to equilibrium at a rate determined by the population turnover time while the last block after a while is dominated by a slowly changing variable, the average gene frequency. The dynamics of the gene frequency take the usual form for a continuous time slow selection model, and this provides explicit, epidemiologically justified expressions for the genotypic fitnesses. We apply the method to other disease transmission patterns (SEI and SIS) and discuss how suitable time averages extend our results to diseases with temporally varying incidence.

Alleles↗

The cytonuclear effects of facultative apomixis. I. Disequilibrium dynamics in diploid populations.

We comprehensively analyze the cytonuclear effects of generalized mixed mating, including all combinations of selfing, outcrossing, and apomixis, the asexual production of seeds. After first deriving the time-dependent solutions for nonrandom associations (disequilibria) between a diallelic cytoplasmic marker and the alleles and genotypes at a diploid nuclear locus, we delimit all possible dynamical behaviors and the conditions under which each occurs. As in standard mixed mating systems, all disequilibria ultimately decay to zero except when outcrossing is absent, in which case permanent disequilibria result if the allelic association is initially nonzero. When at least some outcrossing is present, any initial allelic association decays at a constant geometric rate, whereas genotypic disequilibria may first increase in magnitude or change sign. Although selfing and apomixis tend to retard the decay of disequilibria (or approach to equilibrium) and often to the same extent, apomixis can have a stronger effect under some conditions. We also determine the dynamics of cytonuclear disequilibria in specific examples that may be of particular interest for empirical studies of hybrid zones. The results suggest several practical guidelines for experimental design and data analysis and show how the cytonuclear disequilibrium dynamics under mating system alone furnish a quantitative baseline for null hypotheses against which to test for the presence of other evolutionary forces.

Data Interpretation, Statistical↗

Repair of DNA strand breaks in progeric fibroblasts and aging human diploid cells.

The rate of rejoining of DNA strand breaks induced by 10 krad of gamma-irradiation has been studied in normal human diploid skin fibroblasts and in skin fibroblasts from six patients with symptoms of progeria. Although slightly more rapid in very early passage, the repair rate in normal cells was similar throughout most of their life span in vitro. The appearance of cells with reduced repair capacity was evident as the cultures became senescent. The progeric fibroblasts varied greatly in their response to irradiation. The rate of repair was greatly reduced in two strains, whereas in two others extensive DNA degradation was consistently observed in unirradiated cells. Degradation was apparently related to the radiation received from the incorporated radiolabel. Normal repair was seen in progeric fibroblasts transformed by SV40 virus.

Aging↗

Time-lapse cinemicrophotographic studies of cell division patterns of human diploid fibroblasts (WI-38) during their in vitro lifespan.

Genealogies of human diploid embryonic lung fibroblasts, WI-38 were prepare from analysis of filmed sequences of clones at passages 20, 28 and 53. The results indicate heterogeneity in cell division patterns, interdivision time and migration activity. The relationship of the cell division patterns to age of culture is difficult to assess at this time because of the heterogeneity of the clones, however, the late passage culture appeared to be more variable in terms of sister-sister, and mother-daughter relationships. The passage 28 culture was representative of a highly proliferating clone, exhibiting short interdivision times and a synchronous division pattern. The passage 20 and 53 clones exhibited longer interdivision times and less synchronous division pattern than the passage 28 clone. A gradual lengthening of average interdivision time with successive generations has been observed in all genealogies regardless of passage level of the donor culture. A portion of daughter cells in the fifth and sixth generation exhibited lower interdivision time than the mother cell. The effects of nutrients, space, and mitotic inhibitors or stimulators on interdivision time of the cells within the clones is discussed.

Cell Aggregation↗

Low-serum medium development for human diploid fibroblast microcarrier cultures.

A new medium supplement mixture, PPRF92, has been developed to enable the serial subculture of human diploid fibroblasts (MRC-5 cells) on microcarriers. Furthermore, the PPRF92 supplements enable cell growth at serum levels as low as 1%. Through an optimization programme, the PPRF92 supplements have evolved into a simple mixture with the concentrations of key components at a level that makes the overall cost very competitive with medium containing 10% foetal bovine serum (FBS). Furthermore, the PPRF92 supplement mixture is most efficacious when FBS is replaced with the cheaper, and more widely available, adult bovine serum (ABS). Although medium exchange with serum is necessary in order to achieve confluence on microcarriers, the PPRF92 mixture is only necessary at the initiation of each passage. Using the medium replenishment protocol that has been developed in our laboratory, MRC-5 cells were successfully serially passaged through 13 bead-to-bead transfers on microcarriers in DMEM/F12 medium enriched with the PPRF92 supplement mixture reported here, and 1% ABS.

Blood↗

Determinants of a genotoxic effect of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in human diploid fibroblasts.

The induction of micronuclei was studied in human diploid fibroblasts incubated in the presence of the tobacco-specific nitrosamine NNK. We used four fibroblast strains having a high capacity of O6-alkylguanine DNA alkyltransferase (13.0-23.3 pmol O6-methylguanine repaired per 8 x 10(6) cells) and four strains that showed no detectable repair capacity. Incubation with NNK doubled the frequency of micronuclei in repair-deficient cells but failed to evoke any effect in the proficient cell strains. Control experiments were performed with the direct methylating agent MNNG and in the presence of inhibitors of either metabolic activation or alkyltransferase. The results showed that the genotoxicity of NNK is dependent on the relationship between its metabolic activation and the constitutive DNA repair. This supports earlier findings that low constitutive levels of O6-alkylguanine DNA alkyltransferase may increase susceptibility to lung cancer after exposure to DNA methylating agents.

Adult↗

Adaptation of hepatitis A virus to high titre growth in diploid and permanent cell cultures.

A hepatitis A virus isolate originally obtained from the feces of a clinically ill patient and passaged in diploid human embryonic kidney and lung cells was adapted to grow in MRC-5, Cercopithecus aethiops muscle and in Vero cells. Three different adaptation methods were applied. Either method proved to be suitable to finally give high virus titres of cell-bound as well as cell-free virus in the supernatant of infected cultures during 10 to 15 passages. An easily performable immunoperoxidase staining method was used for the titration of hepatitis A virus in microtitre plates. Cytopathogenic changes in MRC-5 cell cultures infected with fully adapted virus are described.

Cells, Cultured↗

Histone gene expression in human diploid fibroblasts: analysis of histone mRNA levels using cloned human histone genes.

The cellular abundance of H2A, H2B, H3 and H4 histone mRNA sequences was determined prior to and at various times after stimulation of non-dividing human diploid fibroblasts to proliferate. The representation of histone mRNAs was quantitated by electrophoretic fractionation of total cellular RNAs, diffusion transfer to nitrocellulose and hybridization with a series of cloned genomic human histone sequences. The levels of mRNAs for the four core histones were observed to be temporally and quantitatively coupled with both DNA replication and histone protein synthesis. Therefore, a contribution to the regulation of histone gene expression at a transcriptional level is suggested.

Cells, Cultured↗

Ultrastructure of the compound eye of the diploid female beetle, Xyleborus ferrugineus.

The compound eye of female (diploid) Xyleborus ferrugineus beetles was examined with scanning and transmission electron microscopy. The eye is emarginate, and externally consists of roughly 70-100 facets. Each ommatidium is composed of a thickly biconvex lenslet with about 50 electron dense and rare layers. The lens facet overlies a crystalline cone of the acone type which is roughly hourglass-shaped. Pigment cells envelop the entire ommatidium, and pigment granules also are abundant throughout the cytoplasm of the 8 retinular cells. The rhabdomeres of 2 centrally situated photoreceptor cells effectively fuse into a rhabdom that extends from the base of the crystalline cone deeply into the ommatidium. Six distal peripheral retinular cells encircle the 2 central cells, and their rhabdomeres join laterally to form a rhabdomeric ring around the central rhabdom. The rhabdom and rhabdomeric ring are effectively separated by the cytoplasm of the two central retinular cells which contains the usual organelles and an abundance of shielding pigment granules. Eight axons per ommatidium gather in a tracheae-less fascicle before exiting the eye through the fenestrate basement membrane. No tracheation was observed among the retinular cells. Each Semper cell of each observed crystalline cone contained an abundance of virus-like particles near the cell nucleus. The insect is laboratory reared, and the visual system seems very amenable to photoreceptor investigations.

Animals↗

Haploid and diploid expression of a Brassica campestris anther-specific gene promoter in Arabidopsis and tobacco.

The anther-specific cDNA clone Bcp1 from Brassica campestris is expressed in both the haploid pollen and diploid tapetum, as shown by in situ hybridization. We have isolated Bgp1, a genomic clone homologous to Bcp1. The coding region and extensive 5' flanking sequences of Bgp1 have been sequenced, and the coding region shows 88% identity with Bcp1. RNA gel blot analysis confirmed the expression of Bgp1-specific transcripts in B. campestris pollen. A 767 bp 5' DNA fragment was fused to the reporter gene beta-glucuronidase (gus) and introduced into both Arabidopsis thaliana and Nicotiana tabacum by transformation. This 5' fragment directed high-level expression in the pollen and tapetum of transgenic Arabidopsis. In transgenic tobacco however, the same construct was expressed only in pollen. A series of 5' deletion constructs has been created and used to transform A. thaliana to analyse the 5' region of Bgp1. The results indicate that Bgp1 expression in the tapetum and pollen of Arabidopsis requires the presence of different 5' DNA sequences.

Amino Acid Sequence↗

Chromosomal replication of Drosophila virilis. II. Organization of active origins in diploid brain cells.

DNA fiber autoradiography was used to determine parameters underlying the DNA replication of the eukaryotic chromosome in Drosophila diploid brain cells organ culture. The average rate of fork movement, estimated from 4 different labelling intervals, is 0.35 micron/min at 25 degrees C. Of the tandem arrays 93% show patterns which are compatible with bidirectional replication, 7% show unidirectional replication. The unidirectional mode of replication is interpreted as being a consequence of the experimental schedule (using hot-cold pulse labelling) combined with the occurrence of termination signals. - Some autoradiograms showed the expected two grain tracks of different densities; others showed only a high density track. The latter were most prominent in arrays of short replicons (less than 10 micron) which correlate with replicating satellite sequences. - The majority of replicons fall into size classes less than 100 micron. The frequency distribution is skewed towards larger replicon sizes; it spans 2-238 micron, has a mean of x = 25.6 micron and a median of approximately 21.0 micron. If the distribution is corrected for supposed satellite replicons, the median increases of approximately 31.0 micron. - In experiments using warm-hot pulse labelling, arrays were scored which must have been a consequence of fixed termination signals. Furthermore, grain tracks diverging from weak labelled centers often have different lengths, indicating that these replicons contain two diverging replicating sections of unequal length.

Autoradiography↗

Chromosome constitution of in vitro segregated haploid and diploid cells of the mouse.

The composition in segregated haploid sets of paternal and maternal chromosomes has been studied in order to verify whether their composition is uniparental of mixed, fixed or variable. Primary cultures where prepared using kidneys from hybrids of strains of Mus musculus in which the parental chromosomes are distinguishable; the maternal set consists of 20 teleocentric chromosomes, the paternal set of 9 metacentric chromosomes, derived by Robertsonian fusion and 2 telocentrics. Applying Seabright's banding technique, an analysis of segregated haploid and diploid cells, which have originated spontaneously through polyploidisation-segregation processes was carried out. It was concluded that the haploid sets have a variable composition of paternal and maternal chromosomes.

Animals↗

Human DNA replication: fiber autoradiographic analysis of diploid cells from normal adults and from Fanconi's anemia and ataxia telangiectasia.

Fiber autoradiograms prepared from radioactive DNA of diploid skin fibroblasts and lymphocytes from normal adult humans show patterns of replication of chromosomal DNA that are closely similar to those from other mammalian cells. The rate of replication fork movement is 0.64 micrometer/min and 0.42 micrometer/min for fibroblasts and lymphocytes respectively. In both types of cells, replication units show a median length of about 70 micrometer. Replication proceeds bidirectionally from a central origin in most units and adjacents units initiate synthesis synchronously. In skin fibroblasts from individuals with Fanconi's anemia or ataxia telangiectasia, the autoradiographic patterns of DNA replication are the same as in controls. This suggests that S phase DNA synthesis is normal in these disorders.

Anemia, Aplastic↗

The central localization of the small and early replicating chromosomes in human diploid metaphase figures.

Centromere-center distances are analyzed in 700 metaphase plates, which belong to four different samples. The descriptive analysis of the chromosome distribution shows that smaller, earlier replicating, genet-dense chromosomes are preferentially found near the metaphase plate center, surrounded by longer chromosomes which finish their replication rather late during S phase. This general pattern is highly constant in diploid metaphase samples and independent of sex, culture time, and number of individuals used in the sample. There is accumulating evidence that this overall distribution is not the result of technical artifacts. The metaphase plate data are complementary to the concept of an interphase nucleus structure in which late-replicating, genetically less active chromatin is accumulated at the periphery of the nucleus, while other, earlier replicating chromatin is connected with the intranuclear matrix. Although the currently available data should not be overinterpreted, an extension of the "bodyguard" hypothesis, which was suggested for C heterochromatin, provides a functional interpretation for these data: The peripherally localized, late-replicating genetic material protects the centrally localized euchromatin against mutagens, clastogens, and maybe also against viruses.

Adult↗

The organization of ribosomal genes in diploid and tetraploid species of the genus Odontophrynus (Amphibia, Anura).

The organization of the repeat unit of the ribosomal genes was determined in populations of Odontophrynus americanus 2n, 4n, O. occidentalis 2n and O. barrioi 2n (Amphibia, Anura) from South America. HindIII, EcoRI and BamHI restriction patterns of rDNA from single individuals were probed with two rDNA clones from Xenopus laevis. Variations in length and sequence were found among different species, populations and specimens. A general pattern could be established from the most frequent type of repeat observed in each case, so that diploid populations could be distinguished from tetraploid ones. The molecular evolution of variant repeats is discussed. It is suggested that the reduced size of the non-transcribed spacers in the tetraploids from São Paulo could play a role in the control of rDNA transcription. Distinct restriction maps were tentatively correlated to the varying localization of active rDNA clusters on metaphase chromosomes of corresponding populations.

Animals↗

Comparative study of immature erythroid cells of the diploid Bufo ictericus and the tetraploid Odontophrynus americanus (Amphibia, Anura): ultrastructural cytochemical detection of nucleic acids and polysaccharides, and mapping of the element phosphorus.

In the present study, we used ultrastructural cytochemistry to analyze the distribution of nuclear and cytoplasmic nucleic acids and polysaccharides, and electron spectroscopic imaging to map the element phosphorus in immature erythroid cells taken from two amphibians, the diploid Bufo ictericus and the tetraploid Odontophrynus americanus. In the cytoplasm of cells from the tetraploid species, we detected numerous inclusions containing a material that was similar to the dispersed chromatin seen in the nucleus of these cells. The RNase-gold complex labeled both the dispersed nuclear chromatin and the cytoplasmic inclusions. The Thiéry technique showed that glycoconjugates were present in all the membranous complexes of the erythroid cells of both types of amphibians under study, although they were absent within or around the cytoplasmic RNA inclusions. Electron spectroscopic imaging revealed the presence of phosphorus in these inclusions. These data suggest that an increase in RNA synthesis occurs in tetraploid amphibian cells, probably as a result of an alteration in the mechanisms of gene regulation.

Animals↗

Weigle reactivation of diploid M13 phage.

The limited ability of ultraviolet (UV)-irradiated E. coli cells to W-reactivate UV-irradiated, single-stranded DNA phages fd and M13 was investigated. The kinetics of induction for W-reactivation of UV-irradiated fd phage are different from that for other SOS functions. W-reactivation of UV-irradiated M13 phage was studied using phage particles that contain at least two single-stranded DNA genomes. No effect on the extent of W-reactivation of diploid phage was observed, compared to that of normal haploid phage, indicating that the mechanism of W-reactivation of single-stranded DNA phages does not involve recombination between partially replicated genomes.

Coliphages↗

Absence of functional RNA encoded by a silent chromosome in non-complementing diploids obtained from protoplast fusion in Bacillus subtilis.

The molecular basis for lack of phenotypic expression of one chromosome in Bacillus subtilis non-complementing diploid clones (Ncd cells) was investigated. Correlations between chromosomal inactivation and absence of functional transcripts were determined with wild-type prophage phi 105 or a thermoinducible mutant phi 105 cts23, on either the expressed or the silent chromosome. It appears that no significant amount of phage mRNA is detectable in Ncd cells carrying the prophage in the inactive chromosome. However, phi 105 mRNA represents 0.23% of total cellular mRNA in an Ncd strain with the prophage in the expressed chromosome and 0.28% in the parental lysogenic strain. The lack of an mRNA repressor of phi 105 prophage from the silent chromosome was confirmed by the absence of repressor activity in Ncd clones with a temperature sensitive mutant phi 105 located in the silent chromosome. After heat induction, no phage production was observed. As expected these clones do not exhibit phi 105 immunity when superinfected with the same phage. The combined data of the present and previous work suggest that control of phenotypic suppression of Ncd strains should, at the transcription level, involve a different DNA tertiary organisation in one of the two chromosomes.

Bacillus subtilis↗