Evolution of karyotypic abnormalities and C-MYC oncogene amplification in human colonic carcinoma cell lines

Cell lines (COLO 320 DM and COLO 320 HSR), established from a human neuroendocrine tumor, contain an amplified cellular oncogene (c-myc). We have previously shown that the homogeneously staining regions (HSRs) of a marker chromosome in the COLO 320 HSR cells that evolved in culture from COLO 320 DM cells contain amplified c-myc. Molecular hybridization in situ has now been used to demonstrate that the HSRs are on both arms of what was once an X chromosome. We also show that amplified c-myc copies are present in the isolated double minute chromosomes (DMs) of the COLO 320 DM cells that were characteristic of the tumor cells initially established from the patient. The results suggest that the amplified c-myc appeared first as DMs and was subsequently transposed to engender HSRs on an X chromosome. The initial COLO 320 tumor cell may have acquired two early replicating (i.e., active) X chromosomes and lost the late replicating (i.e., inactive) X.     

Stereoscopic back-scattered electron imaging of silver-stained proteins in nucleoli

By using simultaneously the AgNOR silver staining method, back-scattered electron imaging mode and stereo-tilt in scanning electron microscopy (SEM), it is possible to observe the nucleus through the cell surface, the nucleolus, and the tri-dimensional distribution of the AgNOR-associated acidic proteins. In C3H10T1:2 cells and their 7-12-dimethylbenz--anthracene-treated transformants, the staining demonstrates several intranucleolar silver-staining granules (SSG), surrounded by a weakly staining region. The SSG may represent the fibrillar center (FC) and the weakly staining region, the fibrillar dense component (FD). This component can link several SSG together to form a rope-like structure. In cells with no visible nucleolus and inactive nucleolar organizer regions (NORs) the silver-staining granules are less numerous, close together and the presumed fibrillar dense components are not visible. The SSG are located more peripheraly, and the weakly staining region and the rope-like structure are less prominent in control cell nucleoli than in transformed cells with a comparatively high rate of RNA synthesis.     

Effect of serine hydroxamate and methyl alpha-D-glucopyranoside treatment on nucleoside polyphosphate pools, RNA and protein accumulation in Streptomyces hygroscopicus

The accumulation of RNA and protein and the kinetics of nucleoside triphosphate and guanosine polyphosphate pools during amino acid starvation and carbon source downshift were investigated in Streptomyces hygroscopicus. RNA accumulation was controlled stringently during both amino acid starvation and carbon source downshift. The pool size of ppGpp increased dramatically under these conditions. However, the intracellular concentrations of nucleoside triphosphates were low and the concentration of guanosine polyphosphates was much lower than in Escherichia coli. The possible significance of this phenomenon in the regulation is discussed.     

Use of plasmid pULB113 (RP4::Mini-Mu) to construct a genomic map ofAeromonas hydrophila

Plasmid pULB113 (RP4::Mini-Mu) promoted homologous gene transfer inAeromonas hydrophila; transfer of chromosomal markers occurred at frequencies of between 10^–3 and 10^–4 per donor cell regardless of the marker selected; this indicated chromosome transfer from multiple origins. With a variety of amino acid biosynthetic markers, a single circular map of this bacterium was constructed.     

The effect of triacontanol on growth, photosynthesis and photorespiration in Chlamydomonas reinhardtii and Anacystis nidulans

Chlamydomonas reinhardtii Dangerad 11–32(90) (−), which exhibits C3 properties, and Anacystis nidulans (Strain no. UTEX 625), which exhibits C4 properties, were used to study the effects of triacontanol on growth, photosynthesis and photorespiration. Photosynthetic rate was measured as CO2 uptake and the O2 inhibition of photosynthesis was used as a measure of photorespiration. Triacontanol dissolved in chloroform and dispersed in Tween-20 and triacontanol colloidally dispersed in an aqueous solution of sodium tallow alkyl sulfate were tested. Chlamydomonas cultures increased significantly in cell number after 4 days, and in chlorophyll content after 3 days of treatment with 2.3 × 10⁻⁸ M TRIA in chloroform/Tween-20. In cultures of Anacystis the chlorophyll content became significantly higher 3 days after treatment with 2.3 × 10⁻⁹ M TRIA and the cell number was noticeably higher than the controls.
CO₂ uptake by triacontanol-treated Chlamydomonas cultures was about the same in both 2 and 21% O2, and the O2 inhibition was significantly reduced as compared with the controls. Photosynthesis in Anacystis was O2-insensitive under the experimental condition used. When Anacystis was treated with triacontanol there was no change in the rate of CO2 uptake and no change in the O2 sensitivity of its CO2 uptake. It appears that triacontanol affects some process which regulated the balance between photosynthesis and photorespiration, but other processes which result in increased growth are probably also affected.     

Cytological evidence for somatic diploidization in dikaryotic cells ofArmillariella mellea

Dikaryotic hyphae isolated from basidiocarps ofArmillariella mellea are unstable in aseptic culture and change into monokaryotic hypae. During monokaryotization the nuclei of a dikaryon fuse and fusion nucleus immediately divides resulting in two uninucleate cells, from which the monokaryotic mycelium originates. Similar fusion of two nuclei takes place in matings of compatible singlespore isolates. It is concluded that the resulting monokaryotic mycelium is diploid.     

New data on the toxic and radioprotective effect of cysteine onEscherichia coli 15 T- cells

Summary Exponential phase cultures ofE. coli 15 T^- cells growing on glucosemineral medium were supplemented with 2 mM l-cysteine-HCl. The optical density, acid soluble SH (AS-SH) as well as the DNA, RNA, and protein synthesis of the cultures were examined during this treatment and after return to normal growth conditions. Similarly, the survival of cells irradiated by a standard X-ray dose in cysteine-free buffer was measured.The development of radioresistance during the cysteine treatment as well as the loss of this acquired radioresistance after return to normal growth conditions could be divided into two phases: a) an instantaneous and b) a slow change of radioresistance. Phase a seems to be related to the changes occurring in the AS-SH content of the bacteria, while phase b is apparently dependent on the alterations in the synthesis of macromolecules.This work was partly presented at the 6th Annual Meeting of the European Society for Radiation Biology, Interlaken 1968.     

Effects on microbial activity by extraction of indigenous cells from soil slurries

Abstract: Possible effects on the physiological activity and culturability of soil microorganisms by different soil dispersion procedures, and effects on activity caused by extracting bacteria from soil, were investigated. There was no apparent difference in cfu's with dispersion of a silty loam soil and a loamy sand soil with pyrophosphate as compared to dispersion in NaCl. Substrate-induced respiration was reduced in the silty loam soil, and methanol oxidation was reduced in the loamy sand soil with dispersion in pyrophosphate, and the soil pH was irreversibly increased by the treatment. Extracted bacterial fractions had lower numbers of culturable cells as percentage of the total number of bacteria in each fraction, lower respiration rates and no methanol oxidation activity as compared to the soil slurry both before and after extraction. The physiological activity was apparently not affected by the number of cells extracted. This indicates that the increased extraction rate of indigenous soil bacteria obtained by effective disruption of aggregates and detachment of cells from surfaces, only results in increased extraction of cells that have been physiologically changed as a result of the extraction process.     

Elements within the beta-lactoglobulin gene inhibit expression of human serum albumin cDNA and minigenes in transfected cells but rescue their expression in the mammary gland of transgenic mice.

Two new beta-lactoglobulin (BLG)/human serum albumin (HSA) hybrid gene vectors were constructed and tested for expression in COS-7 cells and in transgenic mice. The HSA sequences were inserted between the second and sixth BLG exons. Transient transfection experiments with these vectors as well as a series of additional vectors with either the BLG 5'- or 3'- intragenic sequences revealed that sequences within BLG exon 1/intron 1/exon 2 abrogated BLG- directed HSA expression in vitro, regardless of the presence of HSA introns or the origin of the 3' polyadenylation signal. In contrast, the same BLG expression cassette enabled the efficient expression of HSA cDNA or minigene in the mammary gland of transgenic mice with subsequent secretion of the corresponding protein into the milk of 56 and 82%, respectively of the mouse strains at levels up to 0.3 mg/ml. Previous attempts to express HSA cDNA inserted into exon 1 of the BLG gene had failed [Shani,M., Barash,I., Nathan,M., Ricca,G., Searfoss,G.H., Dekel,I., Faerman,A., Givol,D. and Hurwitz,D.R. (1992) Transgenic Res. 1, 195- 208]. The new BLG expression cassette conferred more stringent tissue specific expression than previously described BLG/HSA constructs [Barash,I, Faerman,A., Ratovitsky,T, Puzis,R., Nathan,M., Hurwitz,D.R. and Shani, M. (1994) Transgenic Res. 3, 141-151]. However, it was not able to insulate the transgenes from the surrounding host DNA sequences and did not result in copy number dependent expression in transgenics. Together, the in vitro and in vivo results suggest both positive and negative regulatory elements within the BLG intragenic sequences evaluated. The new BLG construct represents an extremely valuable vector for the efficient expression of cDNAs in the mammary gland of transgenic animals.