Genomic instability in interspecific cell hybrids III. Repression of Colcemid resistance in hybrids suggests preferential β-tubulin expression

A Colcemid-resistant Chinese hamster line with an altered form of β-tubulin was used in studies of the expression of spindle proteins in interspecific cell hybrids. Eight hybrids between this line, and a Colcemid-sensitive mouse cell line, were studied. The altered hamster β-tubulin was not expressed as an increased resistance to Colcemid in any hybrid. Since the complete hamster chromosome complement was represented among the hybrids, the absence of altered β-tubulin is not due to segregation of the mutant hamster β-tubulin gene. We suggest either that the hamster β-tubulin gene is repressed in hybrids, or that hamster β-tubulin is excluded from the spindle in hybrid cells. We compare these findings with previous reports of the repression of other highly active, moderately repeated constitutive genes in interspecific hybrids.     

Is transformation associated with an increased error frequency in mammalian cells?

Acute starvation of mammalian cells for amino acids results in translational errors that may be detected by two-dimensional polyacrylamide gel electrophoresis. Using this as an assay for error frequency in mammalian cells, we investigated the hypothesis that neoplastic transformation was associated with an increased error frequency which in turn leads to an increased mutation rate and a decreased efficiency of regulatory controls (phenomena of tumor progression). Although we found that transformation was not always associated with an increased level of mistranslation we showed that SV40 transformation increased the level of translational errors in all cell types tested.     

Does 8-Azaguanine induce or select for chromosome number instability in human cell hybrids?

Segregant clones resistant to 8-Azaguanine (8AG) obtained from hybrids between clonal derivatives of the EUE line were analysed for chromosome number distribution. In almost all cases the 8AG^R clones show chromosome losses and a greater variability in chromosome numbers than that of the parental hybrids and their non-selected subclones. A study of the karyotypic evolution of 8AG^R segregant clones maintained for a prolonged period either in 8AG or in drug-free medium did not reveal any correlation between chromosome variability and the presence of 8AG. The preadaptive nature of the segregation to 8AG resistance is also indicated by the results of a fluctuation test.     

Is glucose transport enhanced in virus-transformed mammalian cells? A dissenting view

Much of the literature on the uptake of glucose by untransformed and transformed animal cells is based on experiments carried out with 2-deoxy-D-glucose (2-DOG). Results obtained with this analog can be ambiguous, since 2-DOG can be phosphorylated by hexokinases of animal cells. An intracellular trapping mechanism is thus provided. Therefore, the total flux of 2-DOG into the cell is a resultant of both transport and hexokinase action, and the measurement of total 2-DOG incorporation is a valid measurement of transport only if 2-DOG is phosphorylated as rapidly as it enters the cell. Evidence is presented here that this is not necessarily the case, significant levels of free intracellular 2-DOG approaching external concentrations were found in untransformed and transformed mouse 3T3 cells even at early times during uptake. Differences in total intracellular 2-DOG between untransformed and transformed cells were accounted for entirely by 2-deoxyglucose phosphate. Thus, it appears the apparent increase of 2-DOG uptake accompanying transformation in these cell lines is not due to an effect on the transport process, but on enhanced phosphorylation, which is a reflection of an alteration in the regulation of glycolysis. The ambiguity introduced by phosphorylation can be oviated by the use of an analog that cannot be phosphorylated, such as 3-O-methyl-D-glucose. The rate of transport and efflux of this sugar was not found to be different in untransformed versus transformed 3T3 cells. Moreover, deficiencies of this analog as a substrate for the glucose transport system are pointed out.     

Gene expression of differentiated parent in teratocarcinoma cell hybrids. Repression or reprogramming?

The hybrid cell line H422 was constructed by fusing embryonal carcinoma (EC) cells of the PCC4AzaRCapR cell line with lymphocytes from a 129-tw32 mouse inbred strain female. An apparently complete extinction of stage-specific gene products of the lymphocyte parent was inferred from comparison of protein maps in two-dimensional PAGE of parental and hybrid cells, and from the reactivity patterns of monoclonal antibodies in radioimmunobinding assays. Furthermore, the hybrid cells display a true EC phenotype and EC functions, tumorigenicity and pluripotency. Taken together, the available evidence suggests either that the lymphocyte genome is a 'silent passenger' as far as the differentiated functions of hybrid cells are concerned, or that the developmentally restricted lymphocyte genome is reprogrammed in the hybrid cells back to a non-determined stage and both genomes thus act in a concerted manner.     

Genomic models with genotype × environment interaction for predicting hybrid performance: an application in maize hybrids

Key message

A new genomic model that incorporates genotype?×?environment interaction gave increased prediction accuracy of untested hybrid response for traits such as percent starch content, percent dry matter content and silage yield of maize hybrids.

Abstract

The prediction of hybrid performance (HP) is very important in agricultural breeding programs. In plant breeding, multi-environment trials play an important role in the selection of important traits, such as stability across environments, grain yield and pest resistance. Environmental conditions modulate gene expression causing genotype?×?environment interaction (G?×?E), such that the estimated genetic correlations of the performance of individual lines across environments summarize the joint action of genes and environmental conditions. This article proposes a genomic statistical model that incorporates G?×?E for general and specific combining ability for predicting the performance of hybrids in environments. The proposed model can also be applied to any other hybrid species with distinct parental pools. In this study, we evaluated the predictive ability of two HP prediction models using a cross-validation approach applied in extensive maize hybrid data, comprising 2724 hybrids derived from 507 dent lines and 24 flint lines, which were evaluated for three traits in 58 environments over 12 years; analyses were performed for each year. On average, genomic models that include the interaction of general and specific combining ability with environments have greater predictive ability than genomic models without interaction with environments (ranging from 12 to 22%, depending on the trait). We concluded that including G?×?E in the prediction of untested maize hybrids increases the accuracy of genomic models.

     

Is the mammalian cell plasma membrane a barrier to oxygen transport?

Oxygen transport in the Chinese hamster ovary (CHO) plasma membrane has been studied by observing the collision of molecular oxygen with nitroxide radical spin labels placed in the lipid bilayer portion of the membrane at various distances from the membrane surface using the long-pulse saturation-recovery electron spin resonance (ESR) technique. The collision rate was estimated for 5-, 12-, and 16-doxylstearic acids from spin-lattice relaxation times (T1) measured in the presence and absence of molecular oxygen. Profiles of the local oxygen transport parameters across the membrane were obtained showing that the oxygen diffusion-concentration product is lower than in water for all locations at 37 degrees C. From oxygen transport parameter profiles, the membrane oxygen permeability coefficients were estimated according to the procedure developed earlier by Subczynski et al. (Subczynski, W. K., J. S. Hyde, and A. Kusumi. 1989. Proceedings of the National Academy of Sciences, USA. 86:4474-4478). At 37 degrees C, the oxygen permeability coefficient for the plasma membrane was found to be 42 cm/s, about two times lower than for a water layer of the same thickness as the membrane. The oxygen concentration difference across the CHO plasma membrane at physiological conditions is in the nanomolar range. It is concluded that oxygen permeation across the cell plasma membrane cannot be a rate-limiting step for cellular respiration. Correlations of the form PM = cKs between membrane permeabilities PM of small nonelectrolyte solutes of mol wt less than 50, including oxygen, and their partition coefficients K into hexadecane and olive oil are reported. Hexadecane: c = 26 cm/s, s = 0.95; olive oil: c = 23 cm/s, s = 1.56. These values of c and s differ from those reported in the literature for solutes of 50 less than mol wt less than 300 (Walter, A., and J. Gutknecht. 1986. Journal of Membrane Biology. 90:207-217). It is concluded that oxygen permeability through membranes can be reliably predicted from measurement of partition coefficients.     

Is cytoskeletal tension a major determinant of cell deformability in adherent endothelial cells?

We tested the hypothesis that mechanical tension in thecytoskeleton (CSK) is a major determinant of cell deformability. To confirm that tension was present in adherent endothelial cells, weeither cut or detached them from their basal surface by a microneedle. After cutting or detachment, the cells rapidly retracted. This retraction was prevented, however, if the CSK actin lattice was disrupted by cytochalasin D (Cyto D). These results confirmed thatthere was preexisting CSK tension in these cells and that the actinlattice was a primary stress-bearing component of the CSK. Second, todetermine the extent to which that preexisting CSK tension could altercell deformability, we developed a stretchable cell culture membranesystem to impose a rapid mechanical distension (and presumably a rapidincrease in CSK tension) on adherent endothelial cells. Altered celldeformability was quantitated as the shear stiffness measured bymagnetic twisting cytometry. When membrane strain increased 2.5 or 5%,the cell stiffness increased 15 and 30%, respectively. Disruption ofactin lattice with Cyto D abolished this stretch-induced increase instiffness, demonstrating that the increased stiffness depended on theintegrity of the actin CSK. Permeabilizing the cells with saponin andwashing away ATP and Ca²⁺ did notinhibit the stretch-induced stiffening of the cell. These resultssuggest that the stretch-induced stiffening was primarily due to thedirect mechanical changes in the forces distending the CSK but not toATP- or Ca²⁺-dependent processes.Taken together, these results suggest preexisting CSK tension is amajor determinant of cell deformability in adherent endothelial cells.

     

Do clusters of replication units in the mammalian cells exist?

Evidence against the existence of clusters of synchronously operating units in human and Chinese hamster cells has been presented by means of the DNA fiber autoradiography. The analysis of DNA molecule autoradiographs obtained by different labelling protocols shows that the appearance of tandemly arrayed units may be an artifact associated with fibre autoradiography. The data obtained indicate that the method of measuring centre-to-centre distances previously used to estimate replication unit size is not reliable. An attempt to define the heterogeneity of replication unit size shows that the distances between the centres of adjacent replication units vary from 5 to 500 μm, average value 120 μm.     

RNA interference: towards a functional genomics in mammalian cells?

The discovery of the induction of RNA degradation by double stranded RNA in C. elegans, "RNA interference", makes it possible to envision systematic studies of gene function in mammalian cells. Indeed, in spite of the existence in mammals of the interferon response to double stranded RNA, the introduction of small interfering RNA can induce a sequence specific inhibition of gene expression either through RNA degradation or by blocking translation. Although the inhibition is transient and usually not complete, strategies have been developed to achieve long term gene silencing. The issue of target specificity is still not completely clear and will probably constitute a limitation of this approach. However, because of the unprecedented ease with which large scale screens can be performed, RNA interference has already established itself as the tool of choice to initiate functional genomics in mammalian cells.     

Is astrocyte laminin involved in axon guidance in the mammalian CNS?

This paper provides evidence for the expression of laminin on glia in correlation with axon elongation and nerve pathway formation during embryonic development of the mouse optic nerve and other parts of the central nervous system (CNS). We show that punctate deposits of laminin on immature glial cells precede the entrance of the first optic axons into the nerve, and remain in close association with growing axons. Furthermore, we show that in one particular region of the optic pathway that the retinal ganglion cell axons avoid in normal animals (i.e., the pigmented area of the distal nerve) the punctate laminin matrix is missing. As the optic nerve matures punctate laminin deposits disappear, and laminin is reduced in the astroglial cytoplasm. The close correlation of the punctate form of laminin with early axonal growth is true not only in the optic nerve but also in some other parts of the CNS. We demonstrate such punctate laminin deposits in a model of astrocyte-induced regeneration of the corpus callosum in acallosal mice (G. Smith, R. Miller, and J. Silver, 1986, J. Comp. Neurol. 251, 23-43), and in glia associated with several normal developing axon trajectories, such as the corpus callosum, fornix, and pathways in the embryonic hindbrain. In all of these regions punctate laminin deposits are found on astroglia that are associated with early growing axons. Our results indicate that the punctate form of laminin, produced by astrocytes, may be an important factor involved in axon elongation and nerve pathway formation in the mammalian CNS.