The developmental origin of primordial germ cells and the transmission of the donor-derived gametes in mixed-sex germline chimeras to the offspring in the chicken

A novel system has been developed to determine the origin and development of primordial germ cells (PGCs) in avian embryos directly. Approximately 700 cells were removed from the center of the area pellucida, the outer of the area pellucida, and the area opaca of the stage X blastoderm (Eyal-Giladi and Kochav, 1976; Dev Biol 49:321–337). When the cells were removed from the center of the area pellucida, the mean number of circulating PGCs per 1 μl of blood was significantly decreased to 13 (P < 0.05) in the embryo at stage 15 (Hamburger and Hamilton, 1951: J Morphol 88:49–92) as compared to intact embryos of 51. When the removed recipient cells from the center of the area pellucida were replenished with 500 donor cells, no reduction in the PGC number was observed. The removal of cells from the outer of area pellucida or from the area opaca had no effect on the number of PGCs. When another set of the manipulated embryos were cultured ex vivo to hatching and reared to sexual maturity, the absence of germ cells and the degeneration of seminiferous tubules were observed in resulting chickens derived from the blastoderm from which the cells were removed from the center of the area pellucida. Chimeric embryos produced by the male donor cells and the female recipient contained the female-derived cells at 97.2% in the whole embryo and 94.3% in the erythrocytes at 5 days of incubation. At 5–7 days of incubation, masculinization was observed in about one half of the mixed-sex embryos. The proportions of the female-derived cells in the whole embryo and in the erythrocytes were 76.5% and 80.2% at 7 days to 55.7% and 62.5% at 10 days of incubation, respectively. When the chimeras reached their sexual maturity, they were test mated to assess donor contribution to their germline. Five of six male chimeras (83%) and three of five female chimeras (60%) from male donor cells and a female recipient embryo from which 700 cells at the center of area pellucida were removed were germline chimeras. Three of the five male germline chimeras (60%) and one of the three female germline chimeras (33%) transmitted exclusively (100%) donor-derived gametes into the offspring. When embryonic cells were removed from the outer of area pellucida or area opaca, regardless of the sex combination of the donor and the recipient, the transmission of the donor-derived gametes was essentially null. The findings in the present studies demonstrated, both in vivo and in vitro, that the PGCs originate in the central part of the area pellucida and that the developmental fate to germ cell (PGCs) had been destined at stage X blastoderm in chickens. Mol. Reprod. Dev. 48:501–510, 1997. © 1997 Wiley-Liss, Inc.     

Stimulation of human B lymphocytes by Nocardia-delipidated cell mitogen and derived fractions from N. opaca. Structure-activity relationship

Nocardia-delipidated cell mitogen (NDCM), a particulate fraction prepared from Nocardia opaca, is able to stimulate the proliferation of small resting human B lymphocytes and their differentiation into Ig-secreting cells. This fraction contains two active structures: the cell wall peptidoglycan (PG) and a fraction (Cy I) derived from the cytoplasmic compartment. Treatment of insoluble PG with various bacteriolytic enzymes showed that the minimal structure required for mitogenic activity is more complex than that required for the differentiation of human lymphocytes. The mitogenic activity of cell wall fractions varies in different bacterial species; that prepared from N. opaca is the more potent. Both mitogenic structures of N. opaca induce higher responses in infant and adult PBL as compared to cord lymphocytes. The differentiation of B lymphocytes into Ig-secreting cells induced by PG fractions is T-dependent.     

Effects of L-tryptophan on morphogenesis and growth in the early chick blastoderm

Summary Chick blastoderms, suppliedin vitro andin ovo with L-tryptophan at the primitive streak stage, showed in their continued development typical retardation of brain formation and somitogenesis in the embryo, whereas heart formation remained unaffected. In contrast to an overall reduction in size observed at the higher L-tryptophan concentrations, a moderate enlargement of the area opaca, compared with the controls, was found at the lower concentrations. This enlargement was combined with an increased flattening of the ectodermal area opaca cells and a reduction of the number of microvilli covering these cells. As a simultaneous supply of glucose could reduce, to some extent, the morphogenetic disturbances, these might partly be ascribed to a blocking of gluconeogenesis from L-tryptophan, but the overall reduction in size mentioned, together with the observation of a reduced decomposition of intracellular yolk granules in the L-tryptophan-treated blastoderms, indicates that impairment of intracellular yolk granule decomposition was the principal disturbance. The possible role of serotonin—probably formed from the L-tryptophan supplied—is suggested as a regulating factor in this connexion.     

Colocalization of laminin and fibronectin in bovinelens epithelial cells in vitro

Summary The distribution and organization of the extracellular matrix (ECM) proteins laminin, fibronectin, entactin, and type IV collagen were investigated in primary colonies and secondary cultures of bovine lens epithelial cells using species-specific antisera and indirect immunofluorescence microscopy. Primary cell colonies fixed in formaldehyde and permeabilized with Triton X-100 displayed diffuse clonies. In contrast, thick bundles of laminin and fibronectin were located on the basal cellsurfaces and in between cells in the densely packed center of the colonies, and as “adhesive plaques” and fine extracellular matrix cords in the sparsely populated (migratory) outer edge of the colonies. The distribution of ECM proteins observed in secondary lens epithelial cell cultures was similar to that observed at the periphery of the primary colony. Extraction of the secondary cell cultures with sodium deoxycholate confirmed that laminin and fibronectin were deposited on the basal cell surface. Indeed, the patterns of laminin and fibronectin deposition suggested that these proteins codistribute. These results establish that lens epithelial cells in culture can be used as a model system to study the synthesis and extracellular deposition of the basement membrane proteins, laminin and fibronectin. Supported by Public Health Service grant EY05570 from the National Eye Institute Bethesda, MD.     

Differential lectin-mediated agglutinabilities of the embryonic and the first extraembryonic cell line of the early chick embryo

Summary The lectin-mediated agglutinability of cells dissociated from different areas of the gastrulating chick embryo was investigated. Differences in agglutinability were quantified by using a Coulter counter. Cells from the area pellucida (AP) and those from the endoderm of the area opaca (AOEn) are agglutinated by Concanavalin A (Con A), wheat germ agglutinin (WGA) andRicinus communis agglutinin (RCA). In cells from both areas the greatest agglutination response is obtained with RCA. Trypsinization of AOEn cells enhances their agglutinability with Con A, WGA and RCA. The lectin-induced agglutinability of cells from the area pellucida is similar in EDTA-dissociated and trypsinized cells.Cells from the AP are significantly more agglutinable with Con A than those of the AOEn regardless whether the former are obtained by trypsinization or dissociation with EDTA. The higher agglutinability of cells of the area pellucida with Con A, as well as the differential enhancement by trypsin of the agglutinability of AOEn cells with Con A, WGA, and RCA may reflect a difference in the cell surface glycoreceptors between the cells of the are pellucida (predominantly embryonic) and the first extraembryonic (AOEn) cell line. These cells have been shown to sort out from each other at the earliest stages of development.     

Post-nodal mesoblast caudalizes the host axis and inhibits cell population growth, and induces new primitive streaks in chick embryos

One to eight post-nodal fragments (PN) or Hensen's nodes (HN) from full primitive streak stage chick embryos were transplanted onto the area pellucida or area opaca of stage 4 embryos and cultured for 20 h. Thirteen morphological and numerical parameters were affected in the host embryos and analyzed by multiple logistic regression for parametric hierarchy. In the area pellucida, both PN and HN transplants inhibited cell population growth while only PN caudalized the host axis and induced supernumerary primitive streaks expressing the mesoderm-specific gene Brachyury. In the area opaca, neither grafts influenced host axis morphogenesis, but PN inhibited the cell population growth significantly. Tracking [(3)H]TdR labeled grafts showed that PN cells migrated towards the host axis and participated in the formation of supernumerary somites and hearts. When placed near the host axis, PN caudalized it and inhibited cell population growth.     

Expression of fibronectin receptor (integrin) in the uterus of rats in relation to the estrous cycle

Summary The expression and localization of fibronectin receptor (integrin), fibronectin, laminin and collagen type IV in the endometrium of the rat uterus during each period of the estrous cycle were investigated by immunofluorescent microscopy. Fibronectin receptor was observed at the epithelial cells of the endometrium and at vascular endothelial cells. At proestrus, when epithelial cells actively migrate, fibronectin receptor was observed at the basal and lateral epithelial cell surfaces. During estrus, fibronectin receptor had begun to disappear, little fibronectin receptor was observed at metestrus or diestrus. No prominent changes in the localization of fibronectin (seen at the vascular endothelial cells and in the stroma) or of laminin and collagen type IV (seen at the muscles and at the basement membranes of the epithelial and vascular endothelial cells) were observed in relation to the estrous cycle. Thus, uterine epithelial cells, like epithelial cells of the healing cornea, increase their expression of fibronectin receptor during active migration, probably facilitating their attachment to stromal fibronectin. This fibronectin-fibronectin receptor mechanism may underlie epithelial repair, whether the defect results from physiological processes or from an insult.     

Fibronectin in cultured rat keratinocytes: distribution, synthesis, and relationship to cytoskeletal proteins

The aim of this study was to investigate whether epidermal cells can synthesise fibronectin and whether the distribution of this glycoprotein is related to the adhesion and cytoskeletal organisation of these cells. The production of fibronectin by newborn rat epidermal cells was shown by indirect immunofluorescence staining of cultures grown in the absence of a feeder layer using an antiserum which had been cross-adsorbed with foetal calf serum proteins to remove antibodies which recognised serum fibronectin. The distribution of fibronectin in areas of cell-cell and cell-substratum contact, characteristically in the form of short radial stitches, was examined in more detail using immunoelectron microscopy with colloidal gold as marker. This showed the close proximity of fibronectin to the cell membrane, with the ventral surface and fine cellular processes showing the heaviest labelling, and also revealed evidence of a relationship between external fibronectin and internal structure in epidermal cells. Immunofluorescence showed that tonofilaments (keratin) and microtubules were present as fibrillar arrays but were not related to fibronectin distribution. Vimentin and desmin were absent. Actin was distributed as a circumferential bundle of filaments, with finer stands running radially to the edge. The latter were reminiscent of the radial fibronectin stitches and a spatial correspondence between fibronectin and actin was confirmed by double-label immunofluorescence which revealed many instances of overlap and colinearity of actin and fibronectin filaments. The ability of keratinocytes to produce fibronectin suggests that these cells can contribute to the formation of the basement membrane in skin. The localisation of fibronectin and its close association with actin also suggests that it is involved in keratinocyte adhesion and is related to the internal organisation of these cells.     

Expression of fibronectin receptor (integrin) in the uterus of rats in relation to the estrous cycle.

The expression and localization of fibronectin receptor (integrin), fibronectin, laminin and collagen type IV in the endometrium of the rat uterus during each period of the estrous cycle were investigated by immunofluorescent microscopy. Fibronectin receptor was observed at the epithelial cells of the endometrium and at vascular endothelial cells. At proestrus, when epithelial cells actively migrate, fibronectin receptor was observed at the basal and lateral epithelial cell surfaces. During estrus, fibronectin receptor had begun to disappear, little fibronectin receptor was observed at metestrus or diestrus. No prominent changes in the localization of fibronectin (seen at the vascular endothelial cells and in the stroma) or of laminin and collagen type IV (seen at the muscles and at the basement membranes of the epithelial and vascular endothelial cells) were observed in relation to the estrous cycle. Thus, uterine epithelial cells, like epithelial cells of the healing cornea, increase their expression of fibronectin receptor during active migration, probably facilitating their attachment to stromal fibronectin. This fibronectin-fibronectin receptor mechanism may underlie epithelial repair, whether the defect results from physiological processes or from an insult.     

Transient stimulation of glucose metabolism by insulin in the 1-day chick embryo

Effects of insulin upon glucose metabolism were investigated in chick embryos explanted in vitro during the first 30 h of incubation. Insulin stimulated the glucose consumption of the chick gastrula (18 h) and neurula (24 h), but had no effect on the late blastula (0 h:laying) and on the stage of six to eight somites (30 h). The increase in glucose consumption concerned both the embryonic area pellucida (AP) and extraembryonic area opaca (AO). AP responded to a greater extent (50%) and at a lower range of concentrations (0.1-1.0 ng/ml) than AO (30%; 1-100 ng/ml). Insulin had no effect on the oxygen consumption of blastoderms, whereas it stimulated the aerobic lactate production (approximately 70% of the additional glucose consumption was converted to lactate). The nanomolar range of stimulating concentrations suggests that insulin has a specific effect in the chick embryo, and that it could modulate glucose metabolism in ovo as well. The transient sensitivity of the embryo to insulin is discussed in relation to behavior of mesodermal cells.     

The pluripotency of the extraembryonic mesodermal cells of the early chick blastoderm: effects of the AP and AOV environments

This study investigates the developmental potential of the extraembryonic mesodermal cells of the early chick blastoderm. [3H]Thymidine-labeled mesodermal fragments from the extraembryonic area pellucida (AP) and area opaca vasculosa (AOV) were transplanted into the AP or AOV of nonlabeled host blastoderms in culture, and their fate followed autoradiographically. All the homotopically transplanted mesodermal cells differentiated in accordance with their normal fates. However, not all the heterotopically transplanted mesodermal cells did so, for some of the stage 8 AP extraembryonic mesodermal cells (normally nonerythropoietic) gave rise to blood cells when transplanted into the AOV. We also observed that the stage 4-5 AOV mesoderm continues to migrate peripherally when heterotopically transplanted into the AP, at a time when the AP mesodermal cells are nonmigratory. In support of our premise that the stage 8-9 AP extraembryonic mesoderm has the potential to form blood, we observed a clear-cut production of hemoglobin when the latter mesoderm was co-cultured on coverslips with stage 4 AOV endoderm.     

Primordial germ cells of the young chick blastoderm originate from the central zone of the area pellucida irrespective of the embryo-forming process

Early chick blastoderms (stages X-XII) were divided by a circular cut into two fragments. In one experimental group, the area opaca was separated from the marginal zone and the central disc of the area pellucida, while in another group the area opaca plus marginal zone were separated from the central disc. Other blastoderms of equivalent stages were each cut into three strips of equal size (either perpendicular or parallel to the axis of symmetry). The fragments were isolated and incubated for 43-48 h after which they were PAS-stained, whole-mounted and checked for the presence of primordial germ cells (PGCs). The results showed that most of the PGCs originated from the central disc and not from the periphery of the area pellucida and that they segregated from this zone even if no embryonic axis developed in the explant. In such cases, the PGCs were found to be dispersed throughout the entire explant, usually in association with forming blood islands. When an axis did develop in the explant, the PGCs were found to be concentrated around its anterior end, in a pattern resembling the germinal crescent. No indication of a quantitative regulation of PGCs was found in the explants and the sum of PGCs, calculated for the complementary fragments of a blastoderm, matched the range of numbers in control blastoderms. Our results suggest that PGCs may already be determined as early as stage X and that their further differentiation is independent of the embryo-forming process.     

Migration of chick blastoderm under the vitelline membrane: the role of fibronectin

In the earliest stages of its development the chick blastoderm is a flattened disc at the surface of the yolk. It gradually increases in diameter, partially because the cells are rapidly proliferating, but also because the cells at the periphery (the margin of overgrowth) are migrating in a centrifugal direction. These cells utilize the inner surface of the vitelline membrane as their substratum. In the normal blastoderm, these cells at the edge of the spreading blastoderm are the only cells which are attached to the vitelline membrane. This investigation is concerned with the possible role played by fibronectin in the interaction between these migrating cells and the vitelline membrane. Chick blastoderms, explanted by the New (1955) technique have been treated with synthetic peptides that mimic the adhesive recognition signal of the fibronectin molecule. The pentapeptide GRGDS (containing the specific RGD cell adhesion sequence) caused the edge cells of the blastoderm to detach within minutes, and the expansion of the blastoderm was inhibited for about 4 hr. After this period there was gradual recovery and the cells reattached and spreading resumed. Examination of the margin of the blastoderm by scanning electron microscopy showed that cell processes were lost soon after treatment with GRGDS but concomitant with reattachment and the resumption of spreading, the cell processes reformed. The pentapeptide GRDGS (with the amino acids G and D inverted) produced a brief inhibition of spreading, but after an hour these blastoderms spread at the same rate as controls. Immunocytochemical staining with anti-fibronectin demonstrated that fibronectin was not only present at the interface of the edge cells and the vitelline membrane, but also between the epiblast and the hypoblast. These results indicate that tissue movement during blastoderm spreading is dependent upon fibronectin and that the specific RGD amino acid sequence, and presumably the VLA/integrin family of receptors, is involved in this embryonic morphogenetic movement.     

Changes in the distribution of a major fibroblast protein, fibronectin, during mitosis and interphase

The distribution of a major fibroblast protein, fibronectin, was studied by immunofluorescence and immunoscanning electron microscopy in cultures of human and chicken fibroblasts during different phases of the cell cycle. The main findings were: (a) In interphase cells, the intensity of surface-associated fibronectin fluorescence correlated with that of intracellular fibronectin fluorescence. (b) The intensity of the fluorescence of both surface-associated and intracellular fibronectins was not changed in cells that were synthesizing DNA. (c) Mitotic cells had reduced amounts of surface-associated but not of intracellular fibronectin. The surface fibronectin that remained on meta-, ana-, or telophase cells had a distinct punctate distribution and was also localized to strands attaching the cells to the substratum. Fibronectin strands first reappeared on the surface of flattening cytoplasmic parts of telophase cells. (d) Fibronectin was also detected in extracellular fibrillar material on the growth substratum, particularly around dividing cells. Thus, surface-associated fibrillar fibronectin was present during G(1), S, and G(2) but in cells undergoing mitosis the distribution was altered and the amount appeared to be reduced. The observations on the distribution of surface-associated fibronectin suggest that rather than being involved in growth control this fibronectin plays a structural role in interactions of cells with the environment.     

Vasculogenesis in the early quail blastodisc as studied with a monoclonal antibody recognizing endothelial cells

QH1, a monoclonal antibody that recognizes quail endothelial and haemopoietic cells, was applied to quail blastodiscs in toto, in order to analyse by immunofluorescence the emergence of the vascular tree. The first endothelial cells were detected in the area opaca at the headfold stage and in the area pellucida at the 1-somite stage. Single cells then interconnected progressively, especially in the anterior intestinal portal and along the somites building up the linings of the heart and dorsal aortas. This study demonstrates that endothelial cells differentiate as single entities 4 h earlier in development than hitherto detected and that the vascular network forms secondarily. The horseshoe shape of the extraembryonic area vasculosa is also a secondary acquisition. A nonvascularized area persists until later (at least the 14-somite stage) in the region of the regressing primitive streak.     

Localization of embryonal acetylcholinesterase during the early development of the chick embryo

The presence of "embryonic" acetylcholinesterase activity, as described by Drews (1975) was investigated during early chick embryonic development, mainly in the following systems: a) primitive streak and Hensen's node during gastrulation movements; b) area opaca during blood islets and vessels differentiation; c) mesoderma of lateral laminae, during delamination movements. The demonstration of enzymic activity was performed with slightly modified histochemical methods. The enzyme was thus localized around the nuclei, in the cytoplasm and associated to plasma membrane of cells engaged in morphogenetic movements. The enzyme activity localized at the plasma membrane was supposed to be involved in the regulation of membrane functions concerning intercellular communications, such as inductive message, perhaps mediated by ion fluxes.     

Rigidity sensing at the leading edge through alphavbeta3 integrins and RPTPalpha

Cells require optimal substrate stiffness for normal function and differentiation. The mechanisms for sensing matrix rigidity and durotaxis, however, are not clear. Here we showed that control, Shp2-/-, integrin beta1-/-, and talin1-/- cell lines all spread to a threefold greater area on fibronectin (FN)-coated rigid polyacrylamide surfaces than soft. In contrast, RPTPalpha-/- cells spread to the same area irrespective of rigidity on FN surfaces but spread 3x greater on rigid collagen IV-coated surfaces than soft. RPTPalpha and alphavbeta3 integrins were shown previously to be colocalized at leading edges and antibodies to alphavbeta3 blocked FN rigidity sensing. When FN beads were held with a rigid laser trap at the leading edge, stronger bonds to the cytoskeleton formed than when held with a soft trap; whereas back from the leading edge and in RPTPalpha-/- cells, weaker bonds were formed with both rigid and soft laser traps. From the rigidity of the trap, we calculate that a force of 10 pN generated in 1 s is sufficient to activate the rigidity response. We suggest that RPTPalpha and alphavbeta3 at the leading edge are critical elements for sensing FN matrix rigidity possibly through SFK activation at the edge and downstream signaling.     

The Blood Island is a Site of Formation of the Primary Embryo Thrombocyte in the Chick Blastoderm

Using the immunohistological technique we inquired at what developmental stage and in which site of chick blastoderm does the embryo thrombocyte (ET) begin to differentiate. An anti-ET antibody was raised against rabbits by injecting ETs isolated from blood of 10 day chick embryos. By applying the indirect staining method to smear preparations of blood collected from developing embryos it was confirmed that cytoplasm of the ET showed more intense staining than that of the erythroid cell and that the ET population could be distinguished from the erythrocyte population by this antibody. Cells showing the intense staining could be detected first in blood islands of the area opaca vasculosa of stage 9+ blastoderms. These embryo thromboblasts were found singly or in groups of a small number at dorsal periphery of cell clusters in the blood island. The electron microscopy revealed that embryo thromboblasts appeared in the same position in the stage 9+ blastoderm. At stage 10+ or later embryo thromboblasts were also present adhering to the vascular endothelium or free in the vessel lumen. We conclude that ETs start differentiating from primitive mesenchymal cells localized in the blood island of the area opaca vasculosa at stage 9 or earlier, migrate thereafter to vessel lumen, and enter the blood stream.     

Fibronectin and cell shape in vivo: studies on the endometrium during pregnancy

The rat endometrium during pregnancy was used as a model system to study fibronectin in vivo. Fibronectin distribution on stromal fibroblasts, as determined by indirect immunofluorescence staining, was studied in relationship to cell shape during decidual transformation. Fibroblasts of the estrus endometrial stroma were elongated cells with a fibrillar pattern of fibronectin on their surfaces. During days 1-6 of pregnancy, as these elongated cells acquired a round morphology, fibronectin changed first to a patched distribution on the cells'a surfaces and then disappeared. The change in fibronectin was specific for the fibroblasts since over the same time period there was no decrease in fibronectin found associated with blood vessels or in the epithelial-stromal basement membrane. These results support the proposed relationship between cell surface fibronectin and cell shape that has been inferred from in vitro experiments. After implantation, fibronectin distribution was studied in relationship to the position of the conceptus. In the stroma proximal to the implanting conceptus, fibronectin was absent except around blood vessels, which may help explain how decidual tissue could act as a barrier to trophoblast invasion. Finally, fibronectin distribution was studied in the uterus after parturition. Debris in the uterine lumen was coated with fibronectin, which may be important in the rapid removal of this material by phagocytic cells. Also, fibronectin associated with the epithelial-stromal basement membrane was reorganized after reepithelialization had occurred.     

No correlation exists between the conjugative transfer of the autotrophic character and that of plasmids in Nocardia opaca strains

A new isolate of Nocardia opaca was obtained by enrichment culture for aerobic lithoautotrophic growth on CO2 and H2. This strain, MR22, is very similar to N. opaca MR11 (formerly 1b) in functioning as a donor for genetic information determining the ability to grow lithoautotrophically (Aut character) in matings with Aut- strains of N. opaca or closely related heterotrophic species. The strain contains a plasmid, pHG33 of about 110 kb. A mutant was isolated from strain MR22 which was plasmid-free, and had lost the Aut character, resistance to 50 microM-thallium salt and susceptibility to the nocardia-specific bacteriophage phi B1. As a recipient of the Aut character, this plasmid-free mutant was as well suited as plasmid-bearing Aut- strains of N. opaca. In matings with the mutant as recipient the frequency of Aut+ transconjugants per donor was 3 X 10(-4) with N. opaca MR11 (pHG31-a, Aut+, Tlr, Strs, phi B1s) and 2 X 10(-3) with N. opaca MR22 (pHG33, Aut+, Tlr, Strs, phi B1r) as donor. Phenotypic characterization of the transconjugants, which had been selected for the Aut marker, revealed that in many cases the Aut marker had been transferred without plasmid transfer. Furthermore, plasmid-free, Aut+ transconjugants functioned as donors for the Aut marker. Both plasmid-free and plasmid-bearing transconjugants transferred the Aut marker to the Aut- strains of N. opaca with a frequency which was one or two orders of magnitude higher than that of the wild-type strains. The plasmids pHG31-a and pHG33 code for thallium resistance (50 microM-thallium acetate). The frequency of thallium-resistant transconjugants was 10(-1) to 10(-2) per donor; all thallium-resistant transconjugants contained the donor plasmid. We conclude that the plasmids pHG31-a of strain MR11 and pHG33 of strain MR22 of N. opaca carry the genetic information for thallium resistance but not the Aut character. As plasmid-free Aut+ strains can function as donors the Aut character is assumed to reside on the chromosome and to function as an independent self-transmissible genetic element.