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Replication of cytomegalovirus in human epitheloid diploid cell line.

Human diploid BAMB cells with epitheloid morphology, which had been derived from amniotic fluid cells, were capable of supporting the replication of human cytomegalovirus (CMV), without prior treatment of the cells with halogenated pyrimidines. The growth of this virus in BAMB cells and in human diploid fibroblastoid (LEP) cells was compared in parallel tests. Virus replication was slower and less efficient in the former than in the latter system. The most characteristic morphological feature of the CMV-infected BAMB cells was the formation of multinucleated giant cells which frequently contained more than a hundred nuclei; such cells were not seen in LEP cultures. The development of ultrastructural changes was slower in BAMB cells than in LEP cells. The additional most marked differences concerned the place of viral envelopment and the production of cytoplasmic dense bodies. While in LEP cells most nucleocapsids were enveloped from the inner leaflet of the nuclear membrane, in the other system a great majority of the particles acquired their envelopes by budding into vacuoles. Cytoplasmic dense bodies were rare in infected LEP cells but very frequent in BAMB cells. Budding of these structures into vacuoles was also observed.

Cell Line↗

Chromosomes in tumors derived from mouse tumor x diploid cell hybrids obtained in vitro.

Hybrids formed in vivo between Cl.1D tumor cells and host cells have been shown to carry a copy of each chromosome pair contributed by the host cell parent (1). However, in these hybrids, the tissue type of the host cell parents remained unknown. In the present study, hybrids between the malignant Cl.1D fibroblasts and either normal diploid fibroblasts (CF hybrids) or normal thymocytes (CT hybrids) were examined. These hybrids produced tumors when injected into host mice. Metaphases of growing hybrid cell tumors were analyzed. Neither CF nor CT malignant hybrids showed loss of any specific chromosome pair contributed by the normal cell parent. Since elimination of any chromosome pair contributed by the diploid fibroblast parent is not a prerequisite for CF hybrid tumoral growth, it seems unlikely that malignancy of hybrids results from nonexpression of normal alleles of those genes putatively implied in malignancy of Cl.1D cells.

Aneuploidy↗

Evidence for gene silencing by DNA methylation in normal human diploid fibroblasts.

Human diploid fibroblasts, strain MRC-5, were permeabilized by electroporation and treated with 5-methyl deoxycytidine triphosphate (5-methyl dCTP) in the S phase of the cell cycle. The frequency of TGR HPRT- cells was increased up to 20-fold in comparison to control untreated cultures. Representative TGR clones were unable to grow in HAT, and these were treated with 5-azacytidine (5-aza-CR). In many cases subsequent growth in HAT medium was observed, but in others it is likely that the cells had run out of growth potential. The results provide the first evidence of the silencing and reactivation of a gene in normal diploid mammalian cells.

Azacitidine↗

Apparent bisexual behavior of yeast strains obtained from hybridization of industrial yeasts of the genus Saccharomyces with auxotrophic diploids.

During a genetic study of some hybrids of brewer's and distiller's yeast strains with impaired sporulation characteristics and genetically marked auxotrophic aa and alpha alpha diploids, strains which showed positive mating reactions with both a and alpha haploid tester strains were observed. These strains proved to be homothallic and sporulated freely. The original hybrids, which appeared to be tetraploid, usually yielded sporulating single-spore clones on dissection of asci formed from them, with few or no mating strains among them. Dissection of asci from these clones yielded some single-spore clones which showed mating reactions with one or the other or both haploid tester strains, and further selection produced strains which on sporulation and dissection yielded single-spore clones which were apparently bisexual and sporulated freely. These strains proved to be homothallic, yielding single-spore clones which were all of the a mating type, and in which the mating reaction and, possibly, the action of the genes for homothallism were impaired, so that sporulating, non-mating diploids and haploids of both mating types were present in cultures originally obtained as single-spore clones.

Hybridization, Genetic↗

Structural analysis of the rDNA intergenic spacer of Brassica nigra: evolutionary divergence of the spacers of the three diploid Brassica species.

EcoRI restriction of the B. nigra rDNA recombinants, isolated from a lambda genomic library, showed that the 3.9-kb fragment corresponded to the Intergenic Spacer (IGS), which was sequenced and found to be 3,928 bp in size. Sequence and dot-matrix analyses showed that the organization of the B. nigra rDNA IGS was typical of most rDNA spacers, consisting of a central repetitive region and flanking unique sequences on either side. The repetitive region was composed of two repeat families-RF 'A' and RF 'B.' The B. nigra RF 'A' consisted of a tandem array of three full-length copies of a 106-bp sequence element. RF 'B' was composed of 66 tandemly repeated elements. Each 'B' element was only 21-bp in size and this is the smallest repeat unit identified in plant rDNA to date. The putative transcription initiation site (TIS) was identified as nucleotide position 3,110. Based on the sequence analysis it was suggested that the present organization of the repeat families was generated by successive cycles of deletions and amplifications and was being maintained by homogenization processes such as gene conversion and crossing-over.A detailed comparison of the rDNA IGS sequences of the three diploid Brassica species-namely, B. nigra, B. campestris, and B. oleracea-was carried out. First, comparisons revealed that B. campestris and B. oleracea were close to each other as the repeat families in both showed high sequence homology between each other. Second, the repeat elements in both the species were organized in an interspersed manner. Third, a 52-bp sequence, present just downstream of the repeats in B. campestris, was found to be identical to the B. oleracea repeats, thereby suggesting a common progenitor. On the other hand, in B. nigra no interspersion pattern of organization of repeats was observed. Further, the B. nigra RF 'A' was identified as distinct from the repeat families of B. campestris and B. oleracea. Based on this analysis, it was suggested that during speciation B. campestris and B. oleracea evolved in one lineage whereas B. nigra diverged into a separate lineage. The comparative analysis of the IGS helped in identifying not only conserved ancestral sequence motifs of possible functional significance such as promoters and enhancers, but also sequences which showed variation between the three diploid species and were therefore identified as species-specific sequences.

Base Sequence↗

Polymorphism and concerted evolution in a tandemly repeated gene family: 5S ribosomal DNA in diploid and allopolyploid cottons.

5S RNA genes and their nontranscribed spacers are tandemly repeated in plant genomes at one or more chromosomal loci. To facilitate an understanding of the forces that govern 5S rDNA evolution, copy-number estimation and DNA sequencing were conducted for a phylogenetically well-characterized set of 16 diploid species of cotton (Gossypium) and 4 species representing allopolyploid derivatives of the diploids. Copy number varies over twentyfold in the genus, from approximately 1,000 to 20,000 copies/2C genome. When superimposed on the organismal phylogeny, these data reveal examples of both array expansion and contraction. Across species, a mean of 12% of nucleotide positions are polymorphic within individual arrays, for both gene and spacer sequences. This shows, in conjunction with phylogenetic evidence for ancestral polymorphisms that survive speciation events, that intralocus concerted evolutionary forces are relatively weak and that the rate of interrepeat homogenization is approximately equal to the rate of speciation. Evidence presented also shows that duplicated 5S rDNA arrays in allopolyploids have retained their subgenomic identity since polyploid formation, thereby indicating that interlocus concerted evolution has not been an important factor in the evolution of these arrays. A descriptive model, one which incorporates the opposing forces of mutation and homogenization within a selective framework, is outlined to account for the empirical data presented. Weak homogenizing forces allow equivalent levels of sequence polymorphism to accumulate in the 5S gene and spacer sequences, but fixation of mutations is nearly prohibited in the 5S gene. As a consequence, fixed interspecific differences are statistically underrepresented for 5S genes. This result explains the apparent paradox that despite similar levels of gene and spacer diversity, phylogenetic analysis of spacer sequences yields highly resolved trees, whereas analyses based on 5S gene sequences do not.

Base Sequence↗

Copia-like retrotransposable element evolution in diploid and polyploid cotton (Gossypium L.).

Copia-like retrotransposable elements were identified in allotetraploid cotton, Gossypium hirsutum, and two species representing its diploid progenitors, G. herbaceum and G. raimondii. These elements are present in high copy number in all three species. Because the two diploid genomic groups have been isolated on opposite sides of the world for 6-11 million years, horizontal transfer of elements between these species is highly unlikely. Elements were intensively sampled to generate a model of copia-like retrotransposable element evolution in systems where vertical transmission is the sole probable means of descent. Copia-like retrotransposon diversity is equally great in all three Gossypium species. Despite this high heterogeneity, analysis of 89 partial reverse transcriptase sequences resulted in the recognition of nine sharply differentiated retrotransposon lineages, each containing elements that share high sequence similarity. No evidence of horizontal transfer from other taxa was obtained. Phylogenetic analyses demonstrate that element topologies are incongruent with Gossypium phylogeny. Consideration of processes that obscure phylogenetic reconstruction of multigene families (including sampling error, variable degrees of orthology and paralogy, differential lineage age and lineage loss and/or proliferation) demonstrates that incongruence between organismal and retrotransposon trees is expected under conditions in which vertical processes are the sole means of transmission. Identification of closely related elements between species allowed rates of copia-like retrotransposon sequence evolution to be estimated as approximately 10(-9) nucleotide substitutions/site/year. These rates are consistent with the interpretation that these retrotransposons have been evolving under functional constraints for most of the time frame bracketed by the species studied. Extrapolation of these results to previous studies that sampled from more highly divergent taxa indicates that horizontal transfer need not be invoked to explain observed phylogenetic patterns.

Amino Acid Sequence↗

Age-related accumulation of phosphatidylcholine hydroperoxide in cultured human diploid cells and its prevention by alpha-tocopherol.

The levels of phosphatidylcholine hydroperoxide in serially cultured human fetal diploid fibroblasts at various population doubling levels were determined by high-performance liquid chromatography combined with chemiluminescence detection. This methodology utilizes a mixture of cytochrome c and luminol as post-column hydroperoxide group specific luminescent reagents. The cellular hydroperoxide content increased with age from 0.34 to 27.72 pmol/10(6) cells. At the end of the cells' in vitro lifespan (51st population doubling level), the hydroperoxide content per 10(6) cells reached about 80 times the level found in cells of the 20th population doubling level. Supplementation of exogenous alpha-tocopherol to the culture medium prevented hydroperoxide accumulation, but did not extend the lifespan in vitro. The results indicate that substantial intracellular phospholipid hydroperoxide accumulation occurred in the course of aging of human fetal diploid fibroblasts.

Cell Line↗

Carcinogenesis in vitro. II. Chemical transformation of diploid human cell cultures: A rare event.

Seventy-five diploid human cell s-rains were subjected to a number of chemical carcinogens, including urethane and polycyclic hydrocarbons. In most cases, no visible morphological alterations were induced by any treatment. Development of morphologically altered foci was noticed in urethane-treated cultures derived from a patient with von Recklinghausen's disease. This disease is transmitted by an autosomal dominant gene, and has a high rate of spontaneous transformation of neurofibromas to neurofibrosarcomas. Attempts to isolate continuous cell lines from altered foci were successful in only two of several attempts. These continuous cell lines demonstrate altered morphology, loss of contact inhibition, accelerated growth rate, and have attained over 240 generations in a period of 140 weeks. Untreated control cultures became terminal by the 20th generation. Giemsa banding procedures showed that the chromosomal complement consisted of heteroploid human chromosomes. A second diploid cell strain derived from the above patient's sibling, also suffering from von Recklinghausen's disease, likewise was morphologically altered by urethane. Chemical transformation of human cells is difficult to induce; however, selection of genetically predisposed cells and prolonged, intermittent, and repeated chemical treatment may be important factors in achieving transformation.

Adult↗

Characterization of a new human diploid cell line--IMR-91.

A human diploid fibroblast cell line has been established from the lung tissue of a male fetus. This has been characterized and frozen away in large quantity. A smaller quantity of fibroblastlike cells from skin has also been established, partially characterized, and placed in frozen storage from the same fetus. This project is in support of the National Institute on Aging research in general cell biology. The present lines designated IMR-91 lung and IMR-91 skin complement the previous human diploid fibroblast culture (IMR-90) established from a female fetus. The lack of random inactivation of one of the two X chromosomes in the present male line reduces the genetic heterogeneity inherent in the female line.

Cell Division↗

Development of improved media and culture conditions for clonal growth of normal diploid cells.

Multiplication of normal diploid cells in culture is controlled by a complex set of interacting extracellular variables. The amount of serum protein needed for colony formation by such cells is affected directly by many of the other variables, including the nature of the culture surface, the type of trypsinization procedure used, and the qualitative and quantitative composition of the culture medium. By a sequential process of adjusting all of these variables to optimum values for cellular multiplication with minimal amounts of serum protein, we have been able to obtain clonal growth of normal human and chicken cells with less than 500 microgram per ml dialyzed serum protein. Precise quantitative adjustment of nutrient concentrations is particularly important. The multiplication-promoting functions of serum can be classified operationally as "replaceable" (those that can be replaced by modifying the medium or the culture conditions) and "nonreplaceable" (those that we have not yet been able to replace). Elimination of the requirement for replaceable functions of serum has improved greatly the specificity and sensitivity of the bioassay for the nonreplaceable functions. The nonreplaceable multiplication-promoting activity from fetal bovine serum for human diploid fibroblasts has been separated from fetuin and serum albumin and purified approximately 15-fold.

Blood Proteins↗

Fructose-induced enhanced mitogenicity of diploid human cells: possible relationship with cell differentiation.

Fructose strongly stimulates the growth of normal diploid human skin fibroblasts (SFs) and induces marked changes in their morphology and lipid accumulation. This mitogenic effect occurs despite very low fructose consumption and depends on the presence of glutamine. The cell kinetics of cultured fructose-fed human skin fibroblasts were different from those fed on glucose: in the presence of fructose a high proliferative index persisted at Day 14 of culture and the duration of the total cell cycle and of the G1 + 1/2 M and S phases was slightly shorter. The mitogenic effect of fructose on SF was largest in the presence of human serum: it was small or undetectable when fibroblasts were cultured in media supplemented with dialyzed human serum, fetal bovine serum, or serum substitutes. This suggests that serum growth factor(s) mediate the mitogenic effect of fructose. Only normal diploid human cells seem to be sensitive to this mitogenic effect of fructose: the long-term growth of normal human liver cells on fructose was slightly better or similar to that on glucose. In contrast, fructose could only support limited growth of hamster fibroblastic Nil cells and of a transformed human fibroblastic line, which grew better with glucose.

Animals↗

Recombinagenic effect of cryptolepine in uvsH+//uvsH+ and uvsH//uvsH diploid strains of Aspergillus nidulans.

Recombinagenic potential of the alkaloid cryptolepine was evaluated in two diploid strains of Aspergillus nidulans--a wild type strain (uvsH+//uvsH+) and a DNA-repair-deficient one (uvsH//uvsH). Treatment of both strains with cryptolepine failed to alter colony growth in culture; its recombinagenic potential was determined by the homozygotization index (in which events of mitotic exchange may cause expression of genes previously masked by the dominant allele). Mitotic crossing-overs were induced by 7 and 14 mg/L doses of cryptolepine in both diploid A. nidulans strains.

Alkaloids↗

BAC libraries of Triticum urartu, Aegilops speltoides and Ae. tauschii, the diploid ancestors of polyploid wheat.

Triticum urartu, Aegilops speltoides and Ae. tauschii are respectively the immediate diploid sources, or their closest relatives, of the A, B and D genomes of polyploid wheats. Here we report the construction and characterization of arrayed large-insert libraries in a bacterial artificial chromosome (BAC) vector, one for each of these diploid species. The libraries are equivalent to 3.7, 5.4 and 4.1 of the T. urartu, Ae. speltoides, Ae. tauschii genomes, respectively. The predicted levels of genome coverage were confirmed by library hybridization with single-copy genes. The libraries were used to estimate the proportion of known repeated nucleotide sequences and gene content in each genome by BAC-end sequencing. Repeated sequence families previously detected in Triticeae accounted for 57, 61 and 57% of the T. urartu, Ae. speltoides and Ae. tauschii genomes, and coding regions accounted for 5.8, 4.5 and 4.8%, respectively.

Base Sequence↗

Inheritance and genetic mapping of tuber eye depth in cultivated diploid potatoes.

Tuber eye depth of the potato (Solanum tuberosum L.) is an important trait for the processing quality and appearance of potatoes. In the present study, we used a cultivated diploid potato family (12601) of 107 plants to dissect the mode of inheritance and to map the gene(s) controlling the trait. The family segregated for both eye depth (deep vs shallow) and tuber shape (round vs long) traits. The deep eye (Eyd) phenotype was found to be associated with round tubers (Ro) in most progeny clones. Further evaluation of this population with molecular markers including simple sequence repeats, amplified fragment length polymorphism, and sequence-characterized amplified regions revealed that the primary locus for eye depth is located on chromosome 10. This map location was confirmed by evaluating a second diploid family (12586). The results of this study led to the following conclusions: (1) there is a major locus controlling the eye depth trait; (2) deep eye (Eyd) is dominant to shallow (eyd); (3) the Eyd/eyd locus is located on chromosome 10; and (4) the Eyd/eyd locus is closely linked with the major locus for tuber shape (Ro/ro), at a distance of about 4 cM.

Agriculture↗

Quantitative trait loci and candidate gene mapping of aluminum tolerance in diploid alfalfa.

Aluminum (Al) toxicity in acid soils is a major limitation to the production of alfalfa (Medicago sativa subsp. sativa L.) in the USA. Developing Al-tolerant alfalfa cultivars is one approach to overcome this constraint. Accessions of wild diploid alfalfa (M. sativa subsp. coerulea) have been found to be a source of useful genes for Al tolerance. Previously, two genomic regions associated with Al tolerance were identified in this diploid species using restriction fragment length polymorphism (RFLP) markers and single marker analysis. This study was conducted to identify additional Al-tolerance quantitative trait loci (QTLs); to identify simple sequence repeat (SSR) markers that flank the previously identified QTLs; to map candidate genes associated with Al tolerance from other plant species; and to test for co-localization with mapped QTLs. A genetic linkage map was constructed using EST-SSR markers in a population of 130 BC(1)F(1) plants derived from the cross between Al-sensitive and Al-tolerant genotypes. Three putative QTLs on linkage groups LG I, LG II and LG III, explaining 38, 16 and 27% of the phenotypic variation, respectively, were identified. Six candidate gene markers designed from Medicago truncatula ESTs that showed homology to known Al-tolerance genes identified in other plant species were placed on the QTL map. A marker designed from a candidate gene involved in malic acid release mapped near a marginally significant QTL (LOD 2.83) on LG I. The SSR markers flanking these QTLs will be useful for transferring them to cultivated alfalfa via marker-assisted selection and for pyramiding Al tolerance QTLs.

Aluminum↗

Coalescent patterns in diploid exchangeable population models.

A class of two-sex population models is considered with N females and equal number N of males constituting each generation. Reproduction is assumed to undergo three stages: 1) random mating, 2) exchangeable reproduction, 3) random sex assignment. Treating individuals as pairs of genes at a certain locus we introduce the diploid ancestral process (the past genealogical tree) for n such genes sampled in the current generation. Neither mutation nor selection are assumed. A convergence criterium for the diploid ancestral process is proved as N goes to infinity while n remains unchanged. Conditions are specified when the limiting process (coalescent) is the Kingman coalescent and situations are discussed when the coalescent allows for multiple mergers of ancestral lines.

Algorithms↗