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Two non-small cell carcinoma cell lines from the major histopathologic groups of human lung cancers have been karyotyped: HS-24 was established from a squamous cell carcinoma, and SB-3 was obtained from a metastasis of a poorly differentiated adenocarcinoma. Endoreduplication is characteristic for both cell lines. Subsequent loss of chromosomes led finally to hypotetraploid karyotypes with modal chromosome numbers of 66-68 and 70-72 for HS-24 and SB-3, respectively. The structural analysis was performed by G- and C-banding. Stable overrepresentation of chromosomes 7, 8, 12, and 16 was found. Both cell lines developed a characteristic set of disomic and stable markers. Chromosomes involved in markers were 1, 2, 5, 6, 10, 11, 16, and 17. Consistent numerical and structural normality for chromosomes 4, 18, and 21 was observed.

Human non-small lung cancer cell lines HS-24 (established from a primary squamous cell carcinoma) and SB-3 (established from a metastasis of a primary adenocarcinoma of the lung into the adrenal gland) were analysed for the proteinases tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator inhibitor (PAI-1). The proteinases were characterized by activity measurements, inhibition studies, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis. Cell-associated proteinases were determined in cell lysates, secreted proteinases in cell conditioned culture media. Both cell lines were found to secrete uPA and PAI-1, whereas tPA could be detected only in HS-24 conditioned media. No cathepsin B activity could be detected in media of both cell lines. However, activation experiments and western blot analysis showed, that at least HS-24 secrete an inactive precursor. Cell lysates of HS-24 and SB-3 show PA activity, but on a low level. Cathepsin B activity was also found to be low in HS-24 lysates. However, SB-3 lysates show high cathepsin B activity. Further characterization of the proteinases by their sensitivity against several inhibitors suggests that they are similar to the corresponding proteinases of normal, nonmalignant cells.

In a natural population of Drosophila persimilis (McDonald Ranch, Napa Valley, California), KL and MD chromosomal arrangement frequencies undergo a seasonal cycle, with MD common in spring and KL common in summer. Samples collected from spring and summer provided isofemale strains established as homozygous KL and MD pairs (kinlines) with each pair derived from a single heterokaryotype wild progenitor. Haploid doses of chromosomes 2 and 4 were controlled by marker-cross derivations of kinlines. Percentage onset of female receptivity was measured from cultures at 25 degrees and at 15 degrees , using ten kinlines from spring and nine from summer collections, with fast-mating hybrid males as standard testers. Mating tests consisted of 20 tester males x 20 females of specific age, karyotype, and kinline observed for 30 min. At 25 degrees females became receptive at 48 hr after eclosion: parental line (KL(i)/KL(i) and MD(i)/MD(i)) females were approximately equal at 55 to 60% receptive, while among hybrids, MD(i)/MD(j) homokaryotype females were significantly less receptive (68%) than all other outbred combinations (73 to 77%). At 15 degrees , females became receptive at four days of age, with increases on the fifth and sixth days: both parental line and outbred MD/MD females were significantly more receptive (28% at four days and 62% at six days) than all heterokaryotype females (20 to 26% at four days and 55 to 59% at six days), which in turn were more receptive than KL/KL parental and outbred females (10% at four days and 40% at six days). Heterosis was expressed at 25 degrees , but not at 15 degrees . Thus, dominance for female receptivity was temperature dependent. Females polymorphic for these third chromosomal karyotypes possess differential temperature sensitivity for onset of receptivity and are likely to contribute in a significant way to the observed seasonal frequency cycle in the natural population from which they have been derived.

The highly metastasizing ASML cells and the non-metastasizing AS cells, arisen as spontaneous tumors of the rat, were confronted with rat lung tissue in vitro. Small cubes of the lung were allowed to heal their cut edges, then tumor cells were added. Both tumor cell types adapted their shape to the environment, penetrated the superficial layer of lung cells, either of epithelial or of fibroblastoid character and settled on the basal lamina, which, however, was not pierced. In a second set of experiments the tumor cells were inoculated intravenously into the living animal. The lung loaded with tumor cells was excised and cut into cubes which were then incubated in vitro. Here also both tumor cell types exhibited an invasive behavior but the basal lamina of the vessels in which the tumor cells have been arrested was not penetrated. These data indicate that tumor cell behavior is strongly dependent on the environment and the complete invasion or extravasation must be considered as an inducible process.

The morphological characteristics of two rat tumor cell lines were studied by means of scanning electron microscopy. While the ASML-cells (Ascites, Solid, Metastases, Lung) when spread via the lymphatic vessels form metastases in the lung, the AS-cells (Ascites, Solid) do not form secondary tumors. The ASML-cells do not attach on glass or plastic but grow in suspension; AS-cells, can be cultivated as monolayers. The ASML-cells form loosely packed clusters when they are shaken in a gyratory shaker, AS-cells produce well organized aggregates in which the intercellular contacts are very intensive. When a mixture of cells from both lines is aggregated the two types sort out and the line-specific aggregate or cluster is formed without interaction between the two types. ASML-cells remain spherical when confronted with pieces of the aortic wall and there are no signs of reaction with the underlying endothelial cells. The endothelium is drastically altered when AS-cells are seeded on top of the aortic wall: the tumor cells flatten, penetrate the endothelial layer and induce the endothelial cells to retract. These phenomena can also be seen in confrontations with the mesothelium of the diaphragm. As also cells from aggregates or clusters behave as the single cells we conclude that the newly formed heterotypic contacts are favored over homotypic ones. In addition they have proved to be more intensive.

In order to study the process of invasion in more detail we developed an in vitro model of the vessel wall. Rat tumour cells derived from an adenocarcinoma of the pancreas, BSp73 AS--of high invasive but low metastatic capacity--and BSp73 ASML--not invasive but highly metastatic--were compared for their mode of invasion into confluent monolayers of endothelial cells. Corneal as well as vascular endothelial cells were plated alternatively onto the basal lamina-like bovine lens capsule that was mounted in a combi-ring dish or reconstituted extracellular matrix (Basement Membrane Matrigel) as substrata. The endothelial monolayers were confronted with AS- and ASML-tumour cells. The interaction of the various cell types was followed by scanning and transmission electron microscopy. The invasive cell type AS was able to force the endothelial cells to retract and subsequently undermined the endothelial cell layer. In the noninvasive cell population ASML most cells remained in the typical roundish morphology and did not interact with the endothelial cell layers. Only a very minor fraction of ASML populations was able to attach to and also invade into the endothelial cell monolayer. It could be shown that AS-cells individually and as small groups penetrated the endothelial cell layer. The results of transmission and scanning electron microscopy suggest that endothelial cell retraction and underlapping of adjacent endothelial cells by tumour cells play an important role in invasion and extravasation through blood vessels. Against all expectations, the nonmetastasizing tumour cell variant (AS-cells) exhibited a dramatic invasive behaviour whereas the highly metastatic ASML-variant mostly retained its spherical shape and showed invasive activity only in exceptional cases.

Previous discovery that Drosophila melanogaster females tend to discriminate in mating against phenotypes of earliest courting males prompted a study of the Hawaiian species D. silvestris. Tibial bristle variation in males from opposite coasts of the island of Hawaii functions in courtship, and the possibility that females can distinguish males differing in the tibial trait is explored. Mating tests, designed to give each female and male an alternative choice between two individuals of opposite sex every 30 min, consisted of intrapopulation tests with a strain derived from an eastern (Kilauea) population and interpopulation tests between that strain and one derived from a western (Kahuku) population. Males were given initial combat tests, with "winners" then used in mating (except one test with "loser" males). Matings (52-55%) were classified into categories according to the readiness of the female to mate and sequence of courtship. Low-threshold females (accepting the first male after less than four courtship bouts) occurred at 30-35%. Among intrapopulational tests, females (with higher threshold) accepted first- and second-courting males about equally (25:36, respectively), but for male success in mating, the winning of initial intermale combats and the uniformity of courtship effort tended to be important criteria. Among interpopulation tests, homogamic matings were nearly equal (25% each), but heterogamic matings contrasted in that Kilauea females were reluctant to mate with Kahuku males (14%), while reciprocal matings occurred most frequently (34%). Females favored males second to court, particularly when a Kilauea male (with extra tibial bristles) was the second male. Thus a morphological feature likely to be influential in mating is demonstrated to be so; and sexual selection is operating via male-male combat plus discrimination in favor of particular opposite-sex individuals in this species.

The highly metastatic and nonmetastatic variants of the rat tumor BSp73 have been tested for motility in vitro by a phagokinetic assay. Surprisingly, the nonmetastatic variant is locomotional, whereas the highly metastatic variant exhibited stationary motility only. Soluble factors of normal rat serum and medium conditioned by regenerating normal rat lung, or solid constituents of the extracellular matrix material which was exudated by normal rat lung fibroblasts or bovine cornea endothelial cells as well as collagen type III could not stimulate locomotory ability in these tumor cells. However, by contact to rat lung cells they acquired passive mobility. The significance of these cellular properties for their metastatic behavior is discussed.

Pieces of the endothelium of the aorta of BDX rats were confronted with two syngeneic-tumor-cell variants. While the AS variant is non-metastasizing, the ASML cells metastasize spontaneously via the lymphatic vessels. By means of scanning-electron microscopy the adhesion phenomena and the various stages of invasive activity of the non-metastasizing variant (AS) as well as the retraction of the endothelial cells depending on the time of confrontation were studied. Though the metastasizing variant (ASML) adhered firmly to the endothelium, we found neither signs of invasive activity of these tumor cells nor retraction phenomena of the endothelial cells. Aggregates of AS- and clusters of ASML-cells, respectively, behaved in exactly the same way as single cells: while the ASML-clusters remained inactive, the AS-aggregates exhibited invasive activities. Those cells which were in intimate contact with the endothelium started to leave the aggregate, thereby forming a foot-like layer beneath the rest of the aggregate; these cells began to invade.

The rat tumor cell system BSp73AS/BSp73ASML was investigated for its structural aspects. The non-metastasizing line BSp73AS has less nuclear atypism than the metastasizing line BSp73ASML. Microvilli are scanty and variable in length and structure; in BSp73ASML they cover the cells densely, are short and homogeneous in size. In vitro adhesion and spreading tests show structurally flexible BSp73AS cells which flatten completely onto substrata. BSp73ASML cells remain spherical and develop only small attachment areas. In vitro aggregation of the BSp73AS cells leads to round, tightly packed aggregates, their outer cell layer is epitheloid BSp73ASML cells form clusters of mostly ball-shaped cells. Surprisingly, as judged from EM images, the intercellular junctions in BSp73ASML cell clusters are qualitatively the same as in BSp73AS cell aggregates. However, the extent of apposition between cells in BSp73AS aggregates is much higher than between cells in the BSp73ASML clusters. BSp73ASML cells lack the ability to vary their shape. This is the prominent difference revealed by these tests between the two cell lines. Hence, this fact could play an important role in their different metastatic behavior in vivo. We speculate that the ability to vary the cell shape is necessary for expansive growth in the host environment but not for the metastatic spread.

In continuation of a previous paper (1), we have prolonged the time of confrontation of two rat tumor cell variants (BSp73 AS and ASML) with normal epithelial cells and broadened the spectrum of confrontation partners. In addition to the aorta we have also used small veins, the ductus thoracicus as a lymphatic vessel, the diaphragm, and lung fragments. The organ sections were preserved for a longer period of time by incubating them in a gyratory shaker. Under these conditions the vessel material and diaphragm remained morphologically intact for up to 12 hrs; lung cubes concealed the cut surfaces by immediate wound healing, and were preserved intact for up to 40 days. All endothelia and the mesothelium of the diaphragm were destroyed by the nonmetastasizing AS cells, but the basal lamina remained intact by morphological criteria even after 18 hrs of exposure to the AS cells. The metastasizing ASML cells attached by filopodia mostly to the basal lamina of the vessels, but were unable to destroy neither the endothelia nor the basal lamina. The superficial cell layers of the lung cubes, however, were penetrated by the cells of both tumor lines, but to a different degree.

Previous tests for mating activity of Drosophila persimilis homokaryotype KL (Klamath) and MD (Mendocino) chromosomal arrangements (northern California population: Redwoods) had shown KL to mate faster on the average than MD in homogamic tests. A strain (double-cross hybrid of four KL lines from the same population) with reliable high mating activity was developed for testing the sexes separately. Five pairs of KL-MD homokaryotype strains were chosen to be tested by the criterion that each pair had been derived from a separate wild KL/MD progenitor. Strains were crossed within arrangements in a diallelic design (20 inter- and five intrastrain crosses tested in 16 replicates per cross) to provide mating activity indices of four sets: KL females, KL males, MD females, MD males. Mating tests employed ten virgin experimental flies with ten tester (double-cross hybrid) flies of the opposite sex in 30-min observation periods. All flies were matured for 5 days at 25 degrees C before testing. Among parental strains, females were consistently higher in mating activity than males for both KL and MD arrangements. Most interstrain hybrids were heterotic, with KL and MD females not significantly different. However, hybrid MD males displayed greatest variation and had lowest mating activity, while KL males were the least variable and highest in mating activity. With heterosis in the hybrids, there was no predictability (additivity) from performance of parental strains to hybrid offspring. Mating activities of the two sexes were uncorrelated, indicating either that the sexes have independent genetic systems controlling mating activity or that the expression of the same genetic system is influenced by sex. Since the hybrid males of the two karyotypes displayed different courtship activity while the females were at about an equal level of receptivity, intrasexual selection among males is likely to be important in nature.

Chromatin depleted nuclei ('nuclear matrix') of Ehrlich ascites cells were characterized and fragmented by glycerol shot technique (particle fragmentation). The preparations reveal that 'nuclear matrix' is entirely composed of granules and fibres. Prominent size classes of granules are 10 to 20 nm and 25 to 40 nm, respectively. Most of the granules remain attached to fibres during the fragmentation process. The diameter of the fibres corresponds with double-stranded DNA visualized under identical conditions. The RNP-like nature of the particles is shown by their proteinase K/RNase sensitivity. Since the 'nuclear matrix' architecture becomes instable in high salt buffer after pretreatment with RNase which changes the RNP-particle-like material it must be inferred that the RNP/DNA interaction is a prerequisite for the high salt stability of the 'nuclear matrix' complex.

The colonization of the lung by the rat tumor cells BSp73 ASML which have the ability to metastasize via the lymphatic system was studied at the ultrastructural level. Tumor cells arriving in the lung after i.v. injection become transiently embolized; within hours, however, they begin to extravasate from the blood capillaries. Swelling cellular protrusions open a limited area between endothelium and basal lamina through which tumor cells erupt. Tumor cells then form metastases in the interstitial tissue and, in an apparently lymphotropic action, intravasate the lymphatic vessels in a similar manner to a reverse diapedesis-like process. Within the lympatic system they settle, spread, and build up extensive tumor foci particularly in the subpleural region.

The structure of 50 S E. coli ribosomal subunits was studied by electron microscopy as these particles were gradually depleted of proteins by incubation with 0.5 to 6.0 m LiCl. Changes observed in the structure of the depleted subunits were correlated with the location of the deleted ribosomal proteins on the control 50 S particle. These changes were particularly striking in the "crown" region, the site of a considerable number of the proteins necessary for the biological activity of the 50 S subunit. Protein L 16, the first to be removed by the LiCl treatment, was found to be essential for the structural integrity of the large subunit through interactions with ribosomal proteins residing in the left-hand side crest and the interface. Based on electron microscopic evidence, a scheme was proposed for the structural changes accompanying the stepwise unfolding of the 50 S E. coli subunit by LiCl.

Chromosomal DNA is associated with polypeptides covalently bound to internal DNA ends. Since these polypeptides can only be released from chromosomal DNA by enzymes or other agents hydrolysing phosphodiester bonds they were termed 'the most tightly bound' (MTB) polypeptides in DNA. Antibodies developed against the MTB polypeptides are shown to form immunocomplexes with major 'nuclear matrix' polypeptides as well as with polypeptides which are still associated with 'nuclear matrix' DNA isolated by means of SDS/proteinase K and phenol. Immuno-complex formation is revealed by immunoblotting and by indirect immunofluorescence. Thus, since MTB polypeptides, major 'nuclear matrix' polypeptides and 'nuclear matrix' DNA-associated polypeptides share common antigenic sites they can be considered to be identical or at least closely related. This suggests that a fraction of distinct 'nuclear matrix' polypeptides is either transiently or permanently linked to DNA by covalent bonds. Consistently, isolated eukaryotic 'bulk' DNA is inevitably associated with residual 'nuclear matrix' polypeptides.

Ribulosebisphosphate carboxylase from the hydrogen bacterium Alcaligenes eutrophus having a molecular weight of 534000 and consisting of eight large and eight small subunits has been crystallized by microdialysis using inorganic as well as organic precipitating agents. Crystals have tetragonal space group P42212, a = b = 11.27 nm, c = 20.14 nm, and contain one quarter molecule per asymmetric unit. X-rays are diffracted to 0.35-nm resolution on still photographs. Light optical diffractions of electron micrographs of thin sectioned crystals displayed patterns which could be interpreted on the basis of the unit cell determined by X-rays. Packing considerations are in accord with our earlier proposal regarding the subunit arrangement of this enzyme which differs from that reported for tobacco ribulosebisphosphate carboxylase.

DNA (cytosine-5-)-methyltransferase was purified as a single polypeptide (190 kDa by SDS-PAGE) from mouse P815 mastocytoma cells. This enzyme transfers methyl groups to unmethylated as well as to hemimethylated DNA sites with a strong preference for the hemimethylated substrate. A structural analysis of the isolated enzyme by electron microscopical techniques was undertaken. On the basis of the results obtained, we propose a model for the enzyme structure. This model describes the enzyme as a hemi-elliptical globular structure with dimensions of 5.4-6.7 nm for the height h and 10.3-10.8 nm for the diameter d, respectively; this globular structure bears a small appendix at the flat side. A molecular mass of 235-250 kDa is calculated from the measured dimensions. Limited trypsin digestion of the enzyme led to a 160-kDa fragment which preserved the gross morphology of the original material. The possible structure function relationships are discussed.

The applicability of conventional radiochromatographic procedures to the detection and quantification of specific, carcinogen-induced structural modifications in the DNA of mammalian cells is limited by the necessity of using radioactively labelled agents and by the relatively large amounts of DNA required for analysis of low levels of DNA modification. Recently developed immunoanalytical methods have improved this situation considerably. High-affinity monoclonal antibodies (MAB), in combination with radio- and enzyme-immunoassays, now permit the sensitive detection of alkyldeoxynucleosides in small samples of hydrolysed DNA from tissues and cultured cells exposed previously to non-radioactive (e.g., environmental) alkylating N-nitroso carcinogens. Furthermore, MAB can be used to quantify by direct immunofluorescence (and with the aid of computer-based image analysis of electronically intensified fluorescence signals) specific alkylation products in the DNA of individual cells. With this method, the present detection limit for, e.g. O6-ethyl-2'-deoxyguanosine (O6-EtdGuo) is of the order of 7 X 10(2) O6-EtdGuo molecules per diploid genome. Therefore, cells (e.g. from biopsy material) can now be monitored directly for the presence of specific carcinogen-DNA adducts, or with respect to their capacity to remove enzymatically such modified structures from DNA. In combination with transmission electron microscopy, MAB also permit the direct visualization of specific carcinogen-modified sites in DNA. Thus, O6-EtdGuo can be localized in double-stranded DNA molecules by the binding of a MAB specifically directed against this ethylation product.

Metastatic (ASML 14-1, ASmv) and non metastatic (AS17-4) cell lines of the rat BSp73 pancreatic adenocarcinoma were investigated for amplification and expression of oncogene DNA. The c-myc gene was amplified, but only in one metastatic variant, ASML. The degree of amplification (3.5-fold) increased after prolongued in vitro cultivation (17.5-fold). All three tumor cell lines expressed c-myc and ras mRNA. Ras expression was at the same level as in rat liver. C-myc expression was considerably above the level in rat liver, but also differed considerably between the metastatic variants. In the metastatic ASML cells the c-myc gene was localized by in situ hybridization on a marker chromosome derived from chromosome 7. The karyotypes of the metastatic variants are different and have no common marker chromosomes. Our results obtained with the BSp73 tumor model do not support a role of the c-myc gene in the metastatic process.

The events during the settlement of BSp73AS (AS) tumor cells in the syngeneic rat lung are described. Although AS cells show highly invasive behavior in vitro, subcutaneous primary tumors grow solidly without detectable metastatic spreading. However, AS cells when applied to the syngeneic rats via the tail vein give rise to lung colonies which grow rapidly at the site of the cells' primary arrest in the capillaries. The colonization comprises formation of microemboli, penetration of the endothelium including the basal lamina, and invasion of the lung tissue. Within two weeks, large colonies develop, thereby compressing, invading, and destroying their surroundings without detectable preference in direction. This establishment of AS tumors in the lung reflects the high invasive potential of AS cells and displays many of the morphological features observed during the formation of colonies of metastatic cell lines. Thus, we conclude that a nonmetastatic tumor cell line, such as AS, may possess almost the whole set of properties necessary for successful metastasis.

Application of a monoclonal antibody (Mab ER-6; Rajewsky et al., 1980) specific for O6-ethyl-2'-deoxyguanosine (O6-EtdGuo), in conjunction with a protein-free spreading procedure for double-stranded DNA molecules and transmission electron microscopy, permits the visualization of antibody molecules complexed to O6-EtdGuo residues formed in DNA upon reaction with the carcinogen N-ethyl-N-nitrosourea (EtNU) (Nehls et al., 1984). To obtain information on the distribution of O6-EtdGuo in native DNA exposed to EtNU in vitro, samples of purified rat brain DNA were briefly incubated with EtNU at concentrations differing by a factor of 8 (0.5 and 4 mg of EtNU/ml, respectively). As determined in DNA hydrolysates by competitive radioimmunoassay, the resulting DNA preparations contained O6-EtdGuo at O6-EtdGuo/2'-deoxyguanosine molar ratios of 15.1 X 10(-5) and 116 X 10(-5), respectively. Interspace distances between Mab-binding sites in both sets of ethylated DNA were determined by electron microscopy both in individual DNA fragments of different size, and in computer-generated, long-thread DNA constructs. Comparative statistical analyses by a newly developed MOLRANDO computer program show a non-random distribution pattern of Mab-binding O6-EtdGuo residues.

Structures of 50S ribosomal subunits, CsCl and ethidium bromide core particles from these subunits have been investigated by electron microscopy and image processing by FAIRS. This method revealed structural details which are obscured in individual images, and enabled to distinguish six crown forms, different in their side protuberances, and two kidney forms. Crown forms were imaged as symmetrical or asymmetrical forms. The latter type was far more frequent in untreated populations than the first. The depletion of proteins by both agents caused stepwise degradation of the side protuberances in the crown forms thereby transforming asymmetrical to symmetrical forms. It is concluded from these findings that asymmetrical and symmetrical forms in untreated populations represent also structurally different particles. From the higher complexity in terms of component composition and structure it is concluded that the asymmetrical crown forms are more likely to represent the native structure of isolated 50S subunits than the symmetrical forms. Existing models for this subunit are discussed in terms of this finding.

The rat tumor cell lines BSp73AS (AS, non-metastasizing with pronounced adherent capacities) and BSp73ASML (ASML, highly metastasizing with reduced adherent capacities) were cytogenetically investigated. The ASML cell line is reportedly derived from the AS cell line. Both lines exhibited abnormal numerical and structural chromosomal characteristics. The metastasizing ASML cells showed a higher chromosome number (modal number: 62-63) than the nonmetastasizing AS cells (modal number:48). The AS karyotype was characterized by the presence of a large metacentric marker chromosome resulting from a Robertsonian translocation (Rb 6.7). This chromosome is as such absent in ASML cells but perhaps it may be present in these cells with a major part of chromosome 7 being deleted. The most interesting feature of the ASML karyotype was the presence of double minutes (DMs) and a homogeneously staining region (HSR) at the telomeric end of chromosome 6. These were peculiar to the ASML cells, being absent in the AS cells. DMs and HSR are reported to be correlated with the resistance to various drugs and with the acquired virulence of tumor cells through gene amplification. Therefore, we assume that in the metastasizing ASML cell line the DMs and HSR were established through genetic selection and that they are probably related to the acquired metastasizing capacity of these cells.

A monoclonal antibody (AK 30-10) is described which specifically reacts with DNA both in double and single-stranded forms but not with other molecules and structures, including deoxyribonucleotides and RNAs. When used in immunocytochemical experiments on tissue sections and permeabilized cultured cells, this antibody detects DNA-containing structures, even when the DNA is present in very small amounts. Examples of high resolution detection include the DNA present in amplified extrachromosomal nucleoli, chromomeres of lampbrush chromosomes, mitochondria, chloroplasts and mycoplasmal particles. In immunoelectron microscopy using the immunogold technique, the DNA was localized in distinct substructures such as the "fibrillar centers" of nucleoli and certain stromal centers in chloroplasts. The antibody also reacts with DNA of chromatin of living cells, as shown by microinjection into cultured mitotic cells and into nuclei of amphibian oocytes. The potential value and the limitations of immunocytochemical DNA detection are discussed.

Lines containing two third-chromosome inversions (Arrowhead and Pikes Peak) of Drosophila pseudoobscura selected for fast and slow mating speed were crossed to a multichromosomal, dominant marker inversion stock, and strains with various combinations of chromosomal derivation were tested for their mating activity. When lines were selected isodirectionally with the known third-chromosome control, the third chromosome was found to possess major epistatic control over mating speed, followed by the fourth, with no appreciable effect from the first and second. However, polygenic modification mating activity was found throughout the genome when selection was applied opposite to the original third-chromosome control. The work reported here strengthens the evidence that the adaptive role of chromosomal polymorphism has a behavioral basis in mating activity.

The unicellular green alga Chlorella pyrenoidosa shows normal heterotrophic growth and pigment synthesis in the presence of inhibitors of chloroplast transcription and translation.Cells treated with 100 μg/ml of rifampin, 25 μg/ml of lincomycin or 25 μg/ml of spectinomycin for 48 h do not contain any chloroplast ribosomes, 16 S or 23 S RNA as shown with cell fractionation and gel electrophoresis. Labeling experiments with [5(-3) H]uridine indicate that rifampin blocks completely the formation of 16 S and 23 S RNA whilst cytoplasmic rRNA synthesis is unaffected.Production of dry matter and of cell nitrogen are unaffected by treatment of the cells with antibiotics. Chlorophyll production is almost normal as compared with the untreated control culture. The release of daughter cells is partially inhibited by rifampin treatment. In such cultures, giant cells can be observed among cells of normal size.The O2-production of rifampin-treated cells remains unaffected up to 24 h after application of the drug. Later on, the oxygen evolution declines and, after 72 h, oxygen is consumed even during illumination. The O2-consumption in the dark is markedly enhanced after rifampin treatment.Electron microscopy of rifampin-treated Chlorella shows that apparently normal chloroplast membranes are formed. The thylakoids are arranged in grana-like structures whereas in untreated cells they form only stacks of two or three thylakoids. In the chloroplasts of rifampin-treated Chlorella cells, no more ribosomes are found.The cellular location of synthesis of some chloroplast constituents is discussed.