Characterization of WiDr: A human colon carcinoma cell line

Summary We describe the establishment and characterization of WiDr, a cell line derived from a human colon carcinoma. It produces carcinoembryonic antigen in culture, and has a doubling time of 15 hr with plating efficiency of 51%. The HLA antigenic profile and the allozyme genetic signature (composed of eight gene-enzyme systems) of WiDr cells are different from those of HeLa cells. Furthermore, WiDr cells possess three marker chromosomes, again distinct from the HeLa marker chromosomes. Finally, it is highly tumorigenic in four different xenogeneic animal models. Based on these studies, WiDr represents a useful model cell line for tumor cell biology investigations.     

Characterization of the WIDR: a human colon carcinoma cell line.

We describe the establishment and characterization of WiDr, a cell line derived from a human colon carcinoma. It produces carcinoembryonic antigen in culture, and has a doubling time of 15 hr with plating efficiency of 51%. The HLA antigenic profile and the allozyme genetic signature (composed of eight gene-enzyme systems) of WiDr cells are different from those of HeLa cells. Furthermore, WiDr cells possess three marker chromosomes, again distinct from the HeLa marker chromosomes. Finally, it is highly tumorigenic in four different xenogeneic animal models. Based on these studies, WiDr represents a useful model cell line for tumor cell biology investigations.     

Application of DNA fingerprints for cell-line individualization.

DNA fingerprints of 46 human cell lines were derived using minisatellite probes for hypervariable genetic loci. The incidence of 121 HaeIII DNA fragments among 33 cell lines derived from unrelated individuals was used to estimate allelic and genotypic frequencies for each fragment and for composite individual DNA fingerprints. We present a quantitative estimate of the extent of genetic difference between individuals, an estimate based on the percentage of restriction fragments at which they differ. The average percent difference (APD) among pairwise combinations from the population of 33 unrelated cell lines was 76.9%, compared with the APD in band sharing among cell lines derived from the same individual (less than or equal to 1.2%). Included in this survey were nine additional cell lines previously implicated as HeLa cell derivatives, and these lines were clearly confirmed as such by DNA fingerprints (APD less than or equal to 0.6%). On the basis of fragment frequencies in the tested cell line population, a simple genetic model was developed to estimate the frequencies of each DNA fingerprint in the population. The median incidence was 2.9 X 10(-17), and the range was 2.4 X 10(-21) to 6.6 X 10(-15). This value approximates the probability that a second cell line selected at random from unrelated individuals will match a given DNA fingerprint. Related calculations address the chance that any two DNA fingerprints would be identical among a large group of cell lines. This estimate is still very slight; for example, the chance of two or more common DNA fingerprints among 1 million distinct individuals is less than .001. The procedure provides a straightforward, easily interpreted, and statistically robust method for identification and individualization of human cells.     

Genetic structure of gilthead seabream, Sparus aurata, in the Central Mediterranean Sea

The gilthead seabream, Sparus aurata, represents an important economic resource for Mediterranean aquaculture. In spite of its wide geographic distribution and economic importance, only recently studies have been carried out on the genetic composition of natural populations, which have revealed a picture of a heterogeneous degree of genetic differentiation among S. aurata populations. In this study an allozyme analysis of samples from six different collecting sites along the Italian and Croatian coasts was carried out, covering an area in the Central Mediterranean sea that has yet to be investigated through gene-enzyme systems. Data on 26 gene loci, 10 of which are polymorphic, indicate a slight but significant genetic structure (F_ST = 0.0167) of the species. A hierarchical analysis of population subdivision made it possible to identify three different assemblages found in the Adriatic Sea, Tyrrhenian Sea and Sardinian Channel, though an isolation by distance model can be rejected. The results are discussed in the light of previous literature and taking conservation into consideration.     

A negative genetic interaction map in isogenic cancer cell lines reveals cancer cell vulnerabilities

Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large‐scale sequencing efforts. Using genome‐scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co‐culture competition assays to generate a high‐confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non‐isogenic cancer cell lines. For example, the PTEN^?/? DiE genes reveal a signature that can preferentially classify PTEN‐dependent genotypes across a series of non‐isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model.     

REALIZED GENE FLOW VIA POLLEN IN ARTIFICIAL POPULATIONS OF MUSK THISTLE,CARDUUS NUTANS L.

Realized gene flow via pollen was measured in four adjacent, artificial populations of musk thistle, Carduus nutans L. by observing the distribution of electrophoretic markers at two allozyme loci. Realized gene movement declined exponentially with distance. Dispersal distances of the marker alleles averaged 5.0 m. Estimates of effective neighborhood sizes based on the movement of these genetic markers ranged from 126 to 378 individuals. When measures of seed movement were included, estimates of effective population sizes range from 1,281 to 3,844 individuals. It is, therefore, unlikely that chance effects play a major role in shaping the genetic structure of well-established musk thistle populations.     

SPERM-MEDIATED GENE FLOW AND THE GENETIC STRUCTURE OF A POPULATION OF THE COLONIAL ASCIDIAN BOTRYLLUS SCHLOSSERI

The genetic structure of populations of sessile and sedentary organisms is often characterized by microgeographic differentiation in gene frequencies and deviations from panmixia. In many terrestrial botanical systems, restricted gene flow via seed and pollen dispersal may have an important role in promoting such genetic patterns. Until recently, however, limited dispersal of the sexual propagules of benthic invertebrates has not been considered to play a comparable role in aquatic systems. Based on paternity analyses in the field using rare allozyme markers, it appears that concentrations of sibling sperm of the sessile, colonial ascidian Botryllus schlosseri decline rapidly within 50 cm of a source colony. In combination with spatially restricted dispersal of brooded larvae, limited dispersal of sperm should enhance the potential for genetic diversification and inbreeding. However, analysis of allelic and genotypic frequencies at three independent, polymorphic allozyme loci using F-statistics provides little evidence for microgeographic variation in gene frequencies. This lack of differentiation can be explained in terms of the absolute number (rather than concentration) of gametes and larvae dispersing from a point source, which—depending on diffusion and geometric assumptions—may actually increase with distance. In contrast to the absence of differentiation, levels of inbreeding are high, even within the confines of 25 times 25–cm quadrats. The absence of genetic diversification and presence of inbreeding caution against inferring levels and causes of gene flow from indirect analysis of genetic structure and, conversely, making predictions about genetic and breeding structure based solely on direct analysis of gene flow.     

Molecular and morphological divergence in the butterfly genus Lycaeides (Lepidoptera: Lycaenidae) in North America: evidence of recent speciation

Male genital morphology, allozyme allele frequencies and mtDNA sequence variation were surveyed in the butterfly species Lycaeides idas and L. melissa from across much of their range in North America. Despite clear differences in male genital morphology, wing colour patterns and habitat characteristics, genetic variation was not taxonomically or geographically structured and the species were not identifiable by either genetic data set. Genetic distances (Nei's D=0.002–0.078, calculated from allozyme data) between all populations of both species were within the range commonly observed for conspecific populations of other butterflies. The most frequent mtDNA haplotype was present in individuals of both species in populations from southern California to Wisconsin. We conclude that speciation has probably happened recently and the lack of genetic differentiation between the species is the product of either (1) recent or ongoing gene flow at neutral loci, and/or (2) an insufficiency of time for lineage sorting. The evolution of male genital morphology, wing colour patterns and ecological characteristics has proceeded more rapidly than allozyme or mtDNA evolution.     

DEVELOPMENT OF THE HYBRID SWARM BETWEEN PECOS PUPFISH (CYPRINODONTIDAE: CYPRINODON PECOSENSIS) AND SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS): A PERSPECTIVE FROM ALLOZYMES AND mtDNA

A comparison of allozyme and mtDNA frequencies was used for insight into a situation in the Pecos River, Texas where contact between the endemic pupfish (Cyprinodon pecosensis) and an introduced congener (C. variegatus) has resulted in rapid, geographically extensive genetic introgression. Temporal changes in mean frequencies of diagnostic allozyme markers indicate that the clinal pattern of introduced genetic material (Echelle and Connor 1989) is slowly decreasing in amplitude. Significant rank concordance in diagnostic allele frequencies among sites and across sampling years indicates directional influences upon temporal allele frequency change. These observations are consistent with the theory of gene flow in neutral clines. Levels of introgression indicated by each of four allozyme loci and mtDNA were roughly equivalent. The early history of the hybrid swarm is explained by genetic swamping, possibly mediated by selection for C. variegatus or C. variegatus × C. pecosensis, at a time when the normally abundant endemic species had been catastrophically depleted. High frequencies of an introduced GPI-A allele in all samples of intergrades suggests that the introduced genome originated with a single founding event.     

Molecular evidence for historical and recent population size reductions of tiger salamanders (Ambystoma tigrinum) in Yellowstone National Park

Population declines caused by natural and anthropogenic factors can quickly erode genetic diversity in natural populations. In this study, we examined genetic variation within 10 tiger salamander populations across northern Yellowstone National Park in Wyoming and Montana, USA using eight microsatellite loci. We tested for the genetic signature of population decline using heterozygosity excess, shifts in allele frequencies, and low ratios of allelic number to allelic size range (M-ratios). We found different results among the three tests. All 10 populations had low M-ratios, five had shifts in allele frequencies and only two had significant heterozygosity excesses. These results support theoretical expectations of different temporal signatures among bottleneck tests and suggest that both historical fish stocking, recent, sustained drought, and possibly an emerging amphibian disease have contributed to declines in effective population size.     

On reliable discovery of molecular signatures

Background  

Molecular signatures are sets of genes, proteins, genetic variants or other variables that can be used as markers for a particular phenotype. Reliable signature discovery methods could yield valuable insight into cell biology and mechanisms of human disease. However, it is currently not clear how to control error rates such as the false discovery rate (FDR) in signature discovery. Moreover, signatures for cancer gene expression have been shown to be unstable, that is, difficult to replicate in independent studies, casting doubts on their reliability.     

Capturing Genetic Variation during Ecological Restorations: An Example from Kankakee Sands in Indiana

Genetic variation in populations, both natural and restored, is usually considered crucial for response to short‐term environmental stresses and for long‐term evolutionary change. To have the best chance of successful long‐term survival, restored populations should reflect the extant variation found in remnants, but restored sites may suffer from genetic bottlenecks as a result of founder effects. Kankakee Sands is a large‐scale restoration being conducted by The Nature Conservancy (TNC) in northwestern Indiana. Our goal was to test for loss of genetic variation in restored plant populations by comparing them with TNC’s seed source nursery and with local remnant populations that were the source of nursery seed and of the first few restored sites. Allozyme analysis of Baptisia leucantha, Asclepias incarnata, Coreopsis tripteris, and Zizia aurea showed low levels of allozyme diversity within all species and reductions in polymorphism, alleles per locus, and expected heterozygosity between remnants and restorations for all species except A. incarnata. Almost all lost alleles were rare; restored populations contained almost 90% of alleles at polymorphic loci that occurred in remnants at frequencies greater than 1%. Allele frequencies for most loci did not differ between remnants and restored sites. Most species showed significant allele frequency differentiation among remnant populations and among restored sites. Our results indicate that seed collection techniques used at Kankakee Sands captured the great majority of allozyme variation present in seed source remnant populations.     

Latitudinal variability of Drosophila melanogaster: Allozyme frequencies divergence between European and Afrotropical populations

Allelic frequencies at five polymorphic loci were determined in seven European and six Afrotropical populations of Drosophila melanogaster. African populations, which may be considered as ancestral for the species, showed a greater genetic diversity as measured by the number of alleles found. Within each geographic group (Europe or tropical Africa) genetic distances between local populations were very small (D=0.027). By contrast, the average distance between European and African populations (D=0.389) was more than 12 times bigger. It was previously known that various morphological or physiological differences, which probably reflect genetic adaptations to different environments, exist between these temperate and tropical populations. Data presented here suggest that the divergence in allozyme frequencies also reflects some selective mechanisms.     

Experimental studies on some genetic effects of marine pollution

Following the results of a series of investigations carried out to estimate the degree of marine pollution by utilizing certain marine filter feeders, such as the blue musselMytilus galloprovincialis, research has been planned to detect possible genetic effects of pollutants, with special attention to those acting at the population level. The possible selective role of pollutants has been studied both in natural (Mytilus) and in experimental (Tisbe holothuriae) populations by utilizing some electrophoretically-detected gene-enzyme systems as genetic markers. For some of the seven polymorphic loci studied inMytilus (AP, LAP, 6-PGD, IDH_s, IDH_m, PGI, PGM) significant changes in gene frequencies have been detected which can be related to the degree of pollution in the sampling areas. In the more polluted areas these changes were accompanied by a decrease in the frequency of heterozygotes. Similar changes in gene frequencies also occurred in laboratory populations of the copepodTisbe, reared under various experimental conditions. In particular, certain alleles of two loci, PGI-1 and AP-1, exhibited an increase in frequency, especially in populations cultured at various levels of oil pollution. This trend appeared more significant for the locus PGI. The fact that equilibria are reached and that the less favoured alleles are nevertheless maintained in the populations, even at extremely low frequencies, suggests the balanced nature of these enzyme polymorphisms. The significance of the above findings is briefly discussed.     

MAJOR GENETIC DIFFERENCES BETWEEN CROWN-OF-THORNS STARFISH (ACANTHASTER PLANCI) POPULATIONS IN THE INDIAN AND PACIFIC OCEANS

Spatial variation in allelic frequencies at nine allozyme loci were assayed in 20 populations of the crown-of-thorns starfish, Acanthaster planci, collected throughout the Pacific and Indian Oceans. These data were analyzed together with published data, for the same loci, from an additional 19 populations, giving a total sample size of approximately 1800 individuals. There was a marked discontinuity between the Indian and Pacific Ocean populations, but those off Western Australia and from the Southeast Asian region had a strong Pacific affinity. The genetic groups were congruent with the distributions of two color morph groups: gray-green to red-brown forms in the Pacific and a blue to pale red form in the Indian Ocean. These patterns of genetic structure are similar to those described for the starfish Linckia laevigata, which has similar life-history characteristics. Vicariant events may have influenced some populations within the Pacific, but the allozyme data cannot resolve the effects of these events clearly. Patterns of variation within regions were consistent with isolation by distance, but, at larger scales, were obscured by regional vicariance and some outliers, particularly by apparently high levels of gene flow between Japan and the Great Barrier Reef, Australia. Apparent gene flow between population pairs was not closely related to present-day ocean currents. The results demonstrate a strong influence of allopatric separation on genetic divergence at large geographic scales, but also show evidence of slow rates of change in gene frequencies consistent with the large population sizes of this species. Low levels of divergence between groups demonstrate the genetic structure is recent (Pleistocene) and are likely responses to changes in climate and sea level.     

Patterns of morphometric and allozyme variation in Aedes albopictus

A survey of microgeographic variation using morphometric and allozyme analyses was conducted on 19 US populations of Aedes albopictus (Skuse) (Diptera: Culicidae), a mosquito that was recently introduced into the US. There was considerable variation within and among populations both in morphometric traits and allele frequencies. A multivariate discriminant analysis enabled the separation of populations into distinct groups; separation among the populations in the morphometric analysis was incomplete with an average of 70% of the individuals being correctly classified. In the allozyme analysis, the discrimination was complete. The populations from Texas were placed close together in the morphometric analysis, whereas in the allozyme analysis a geographic clustering of populations could not be detected. A test of association between the distance matrices derived from the morphometric and allozyme analyses was statistically nonsignificant. The results are discussed in the context of the colonization of the US by A. albopictus. The possible factors underlying the differences in the patterns of variation derived from morphometric and allozyme analyses are also discussed.     

Genetic resources of wild barley in the Near East: structure, evolution and application in breeding

Genetic diversity and structure of populations of the wild progenitor of barley, Hordeum spontaneum, from three countries, Israel, Turkey and Iran, in the Near East Fertile Crescent, are compared and contrasted. The analysis is based on electrophoretically discernible allozymic variation in proteins encoded by 27 shared loci in 2125 individuals representing 52 populations of wild barley. The results indicate that: (a) H. spontaneum in the Near East Fertile Crescent is very variable genetically; (b) genetic differentiation of populations includes some clinal but primarily regional and local patterns often displaying sharp geographic differentiation over short distances; (c) the average relative genetic differentiation (G_st) was 54% within populations, 39% among populations, and 8% between the three countries; (d) allele distribution is characterized by a high proportion of unique alleles (51%), and a high proportion of common alleles that are distributed either locally or sporadically; (e) discriminant analysis by allele frequencies successfully clustered wild barley of each of the three countries (96% correct classification); (f) a substantial portion of the patterns of allozyme variation in the wild gene pool is significantly correlated with the environment and is predictable ecologically, chiefly by a combination of humidity and temperature variables; (g) natural populations of wild barley are, on the average, more variable than two composite crosses and land races of cultivated barley. The spatial patterns and environmental correlates and predictors of genetic variation of H, spontaneum in the Fertile Crescent indicate that genetic variation in wild barley populations is not only rich but at least partly adaptive and predictable by ecology and allozyme markers. Consequently, conservation and utilization programmes should optimize sampling strategies by following the ecological-genetic factors and allozyme markers as effectively predictive guidelines.     

Lack of allozyme variability amongVarroa mite populations

A gene-enzyme analysis was carried out on the honeybee miteVarroa jacobsoni. By means of gel electrophoresis, 17 presumptive gene loci, belonging to 14 enzyme systems, were scored. Adult female mites were collected in 12 apiaries from European countries and in one apiary from Beijing (China). The populations analysed were monomorphic for all the loci considered, almost all the individuals being homozygous for the same alleles. The breeding system and stochastic mechanisms during the spread ofV. jacobsoni over the world may account for the uncommonly low level of genetic variability observed. The genetic identity found among the analysed samples suggests that taxonomic differentiation is absent even among mite populations living in the different honeybee races considered.This research was supported by the Institut Technique de l'Apiculture (ITAPI), Bures-Sur-Yvette, France.     

Growth rate correlates to individual heterozygosity in the European eel, Anguilla anguilla L

Heterozygosity-fitness correlations (HFCs) have been reported in populations of many species. We provide evidence for a positive correlation between genetic variability and growth rate at 12 allozyme loci in a catadromous marine fish species, the European eel (Anguilla anguilla L.). More heterozygous individuals show a significantly higher length and weight increase and an above average condition index in comparison with more homozygous individuals. To a lesser extent, six microsatellite loci show a similar pattern, with positive but not significant correlations between heterozygosity and growth rate. The HFCs observed could be explained by an effect of either direct allozyme over-dominance or associative overdominance. Selection affecting some of the allozyme loci would explain the greater strength of the HFCs found at allozymes in comparison with microsatellites and the lack of correlation between MLH at allozymes and MLH at microsatellites. Associative overdominance (where allozyme loci are merely acting as neutral markers of closely linked fitness loci) might provide an explanation for the HFCs if we consider that allozyme loci have a higher chance than microsatellites to be in linkage disequilibrium with fitness loci.     

Spatiotemporal allozyme divergence caused by aridity stress in a natural population of wild wheat, Triticum dicoccoides, at the Ammiad microsite, Israel

Spatiotemporal diversity at 35 allozyme loci was assayed over 6 years in 1,207 individuals of wild emmer wheat (Triticum dicoccoides)from a microgeographic microsite, Ammiad, north Israel. This analysis used new methods and two additional sample sets (1988 and 1993) and previous allozymic data (1984–1987). This microsite includes four major habitats (North-facing slope, Valley, Ridge, and Karst) that show topographic and ecological heterogeneity. Significant temporal and spatial variations in allele frequencies and levels of genetic diversity were detected in the four subpopulations. Significant associations were observed among allele frequencies and gene diversities at different loci, indicating that many allele frequencies change over time in the same or opposite directions. Multiple regression analysis showed that variation in soil-water content and rainfall distribution in the growing season significantly affected 10 allele frequencies, numbers of alleles at 8 loci, and gene diversity at 4 loci. Random genetic drift and hitchhiking models may not explain such locus-specific spatiotemporal divergence and strong allelic correlation or locus correlation as well as the functional importance of allozymes. Natural ecological selection, presumably through water stress, might be an important force adaptively directing spatiotemporal allozyme diversity and divergence in wild emmer wheat at the Ammiad microsite. Received: 3 July 2000 / Accepted: 1August 2000