The influence of pretreatment on cell stage progression and the time of DNA synthesis in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) uninucleate microspores

The objective of this study was to correlate the time that DNA synthesis first occurs in haploid microspores of barley with cell cycle and plant morphological stages and to subsequently assess the influence of pretreatments on DNA synthesis at different stages of microspore development. Spikes with microspores in early, mid, and late uninucleate stages of the two-rowed barley cultivars Manley and Igri were subjected to two commonly used pretreatments. First, during cold pretreatment for 28 days there was a slow increase in relative DNA values as well as asymmetric nuclear divisions in some microspores. Second, during a 4-day cold plus 0.3 M mannitol pretreatment, there was very little change in the microspore stage or DNA values indicating that for the duration of this pretreatment the progression of the cell cycle was essentially suspended at all stages, both in Igri and Manley. The results are discussed relative to the potential for genetic transformation of microspores.     

Analysis of a high frequency transformation system for Ophiostoma ulmi, the causal agent of Dutch elm disease

Summary A transformation system for Ophiostoma ulmi (Buism.) Nannf. was developed and analyzed. Protoplasts were generated from actively budding yeastlike cells by digestion with NovoZym 234 in MgSO₄ after pretreatment with 2-mercaptoethanol. Protoplast regeneration was most efficient when 0.6 M sucrose was used as the osmoticum. Several plasmids containing fusions between fungal promoters and a bacterial gene for hygromycin phosphotransferase successfully transformed O. ulmi to hygromycin resistance. One of these vectors, pPS57, which contains a promoter for isopenicillin N synthetase from Penicillium chrysogenum, consistently conferred the greatest resistance to hygromycin. Linearization of the vector and inclusion of 2-mercaptoethanol in the transformation reaction resulted in enhanced transformation efficiency. Approximately 4 × 10³ transformants/g DNA per 10⁷ protoplasts were obtained using the optimized procedure. Southern hybridization after alternating field and standard electrophoresis suggested random insertion of tandem repeats (some greater than 250 kb) into the fungal chromosomes. Antibiotic resistance was stable through mitosis. However, expression of the transforming DNA after meiosis was highly variable.     

Rapid-mixing studies of radiosensitivity with thiol-depleted mammalian cells

A moderate reduction in the non-protein thiol content of V79 379A Chinese hamster cells, obtained by pretreatment with buthionine sulphoximine (BSO), diethyl maleate (DEM) or N-ethyl maleimide (NEM), increase both the absolute radiosensitivity of the cells in hypoxia and the radiosensitizing effect of adding oxygen 7 ms after irradiation. Combined pretreatment of cells with BSO and NEM removes most of the non-protein thiol and some of the protein thiol; such treatment further increases the radiosensitivity of hypoxic cells but there is no further effect of adding oxygen 7 ms after irradiation. Addition of 2-mercaptoethanol to cells 7 ms after irradiation gives protection factors that increase with increasing severity of thiol depletion. Substantial radioprotection can still be observed when 2-mercaptoethanol is added 70 ms after irradiation of cells pretreated with BSO and NEM; there is no effect of adding 2-mercaptoethanol to such cells 50s after irradiation. These observations support the repair-fixation model of radiation damage and suggest that, in addition to the established role of non-protein thiol in chemical repair of radiation damage, other endogenous reducing agents such as protein thiol may be important in determining cellular radiosensitivity. A relatively long-lived thiol-modifiable component of radiation damage has been observed within hypoxic thiol-depleted cells.     

Use of 2-mercaptoethanol in cell culture]

Survival and growth in in vitro cultivation of lymphocytes, lymphoma cells and some other cells including human carcinomas are profoundly improved by 2-mercaptoethanol. These cells hardly take up cystine, an essential nutrient in the culture medium, but in the presence of 2-mercaptoethanol they can utilize cystine. Recently it has been found that 2-mercaptoethanol is effective in the in vitro cultivation of pathogenic trypanosomes and in the in vitro development of bovine embryos. The mechanisms by which 2-mercaptoethanol improves the survival and growth of these cells are described.     

DNA extraction during giemsa differentiation of chromatids singly and doubly substituted with BrdU

Human lymphocytes were cultured in ³H-labelled BrdU. Cells were pretreated to induce differentiation, autoradiographed and Giemsastained. DNA extraction was deduced if grain counts were lower in differentiated mitoses compared with untreated controls. — The differentiation method involved sequential pretreatments with short wave UV and 2 × SSC at 60 ° C. This removed 34% of label from first division cells (with TB.TB chromosomes) but relatively more (53%) from second division (TB.BB chromosomes). In second division cells, about two thirds of label was lost from pale (BB) chromatids but only one third from dark (TB) chromatids. The UV and SSC pretreatments acted in collaboration, since neither alone reduced grain counts significantly. — On testing other methods, similar preferential DNA extraction was obtained with Perry and Wolff's FPG method, and with the hot salt pretreatment of Korenberg and Freedlender. However, good Giemsa differentiation could also be obtained using Hoechst 33258 and light pretreatments without any DNA loss. Reverse differentiation patterns (TB pale, BB dark) induced by warm acids resulted in extraction of nearly two thirds of ³H-BrdU label, but relative loss was the same from pale and dark chromatin. Direct reverse staining using alkaline Giemsa did not result in any loss of label. — Thus preferential DNA loss from pale stained chromatin underlies differentiation methods using light plus hot salt pretreatments, but it is not obligatory for good differentiation using other techniques.     

Effect of 2-mercaptoethanol on the electrophoretic behavior of rat and dogfish metallothionein and chromatographic evidence of a naturally occurring metallothionein polymerization

1. Metallothionein behavior in SDS-PAGE has been characterized. 2. It has been found that metallothionein behavior in this electrophoretic system depends upon the reducing environment. Migration as a well-defined protein band is only achieved in the presence of 2-100 mM 2-mercaptoethanol. 3. Within those 2-mercaptoethanol levels, both rat and dogfish metallothionein migrate as a protein with a molecular weight several times higher than that expected by amino acid analyses. This is not due to molecule oxidations, since this effect is promoted by the presence of 2-mercaptoethanol. 4. No effect of 2-mercaptoethanol on metallothionein behavior is found in conventional PAGE. 5. The present results suggest that to study the effect of 2-mercaptoethanol in SDS-PAGE is a simple and accurate way to identify a protein as metallothionein. 6. It has also been found that metallothionein aggregates naturally in the absence of ionic strength.     

Novikoff hepatoma deoxyribonucleic acid polymerase. Sensitivity of the beta-polymerase to sulfhydryl blocking agents.

Unlike other beta-class eukaryotic DNA polymerases, the enzyme purified from the Novikoff hepatoma is inhibited by both sulfhydryl blocking agents N-ethylmaleimide (NEM) and p-hydroxymercuribenzoate (pHMB). The degree of sensitivity varies depending on the enzyme purity, pH of the reaction, and the presence of sulfhydryl reducing agents. Novikoff beta-polymerase activity is unaffected by the presence of 2-mercaptoethanol (2-Me) or dithiothreitol (DTT); however, the combination of 2-mercaptoethanol and NEM or pHMB acts to reverse the inhibition of the sulfhydryl blocking agent. The reversal of inhibition involves more than just a titration of NEM with 2-mercaptoethanol since a) the combination of these two reagents actually stimulates the DNA polymerase, and b) dithiothreitol did not reverse the inhibition. Binding of the polymerase to DNA did not affect the enzyme sensitivity to NEM.     

Mouse lymphoma L1210 cells can be arrested in the G1 phase by adjusting cellular cysteine and glutathione

Mouse lymphoma L1210 cells (NCI line) that have low ability to take up cystine became deficient in cellular cysteine and glutathione in normal culture media. The cells entered the resting state during culture when they were seeded at high cell densities. They remained viable and were mostly present in the G1 or G0 phase. In the growth-arrested state, the cellular glutathione content was one order of magnitude lower than in the exponentially growing phase in the presence of 2-mercaptoethanol. In the arrested state, DNA synthesis was almost inhibited, and RNA and protein synthesis decreased markedly. Transfer of the cells to medium containing 2-mercaptoethanol, which improves the utilization of cystine by these cells, produced the rapid recovery of RNA and protein synthesis. DNA synthesis slowly increased, reaching a maximum after a lag period.     

Electroporation of cells

By measuring uptake of the membrane impermeable dye. phenosafranine, it can be shown that the plasma membrane of intact cells within cell aggregates can be reversibly permeabilized by electroporation. However, the plant cell wall is a barrier to DNA uptake by intact cells, although under certain circumstances expression of DNA, electroporated into intact cells, can be demonstrated. The level of expression is about 20–50 times lower than that obtained by electroporation of protoplasts, and depends on cell wall properties and pretreatments of cell aggregates. In contrast, efficient transformation of whole cells of bacteria and yeasts can be achieved by electroporation. Factors which influence DNA transfer into whole plant cells and the possibility of stable transformation are discussed.     

Enhancement by alkylating agents of chemical carcinogen transformation of hamster cells in culture

When Syrian hamster embryo cells were pretreated with a weak chemical carcinogen, methyl methanesulfonate (MMS) or ethyl methanesulfonate (EMS), or with a physical agent such as X-irradiation prior to being exposed to a potent cancer-producing chemical, transformation (crisscrossing of cells not seen in control) occurred up to nine times more often than when the cells were not pretreated. The degree of enhancement appears independent of carcinogen dose. The transformation frequency associated with the carcinogens benzo(a)pyrene (BP), dimethylbenz(a)anthracene (DMBA), 3-methylcholanthrene (MCA), N-acetoxy-2-acetylaminofluorene (AcAAF), and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was increased. There are similarities in the enhancement produced by pretreatment of hamster cells with X-irradiation and with alkylating agents: with both, maximum enhancement occurred approx. 48 h after treatment and lethality attributable to the pretreatment was 10–20% relative to control. However, enhancement produced by X-irradiation pretreatment was slightly greater than that obtained with MMS. The exact cause of the enhancement in transformation resulting from the interaction of these agents is not yet known, but the enhancement associated with MMS pretreatment cannot be related to partial cell synchronization or disruption in the cell cycle. Hamster cells pretreated with 250 μM of MMS demonstrated no alteration in normal cel DNA synthesis through 48-h post-treatment. Analysis of unscheduled DNA synthesis by autoradiography or by alkaline sucrose gradients indicated that the damaged DNA was rapidly repaired after treatment. Therefore, repair of DNA damage as it is now understood is probably not involved.     

In Vitro Construction of Pseudovirions of Human Papillomavirus Type 16: Incorporation of Plasmid DNA into Reassembled L1/L2 Capsids

Lack of permissive and productive cell cultures for the human papillomaviruses (HPVs) has hindered the study of virus-neutralizing antibodies and infection. We developed a cell-free system generating infectious HPV16 pseudovirions. HPV16 L1/L2 capsids, which had been self-assembled in insect cells (Sf9) expressing virion proteins L1 and L2, were disassembled with 2-mercaptoethanol (2-ME), a reducing agent, and reassembled by removal of 2-ME in the presence of a β-galactosidase expression plasmid. Plasmid DNA purified together with the reassembled capsids was resistant to DNase I digestion. The reassembled pseudovirions mediated DNA transfer to COS-1 cells, as monitored by induced β-galactosidase activity. Transfer was inhibited by anti-HPV16 L1 antiserum but not by antisera against L1s of HPV6 and HPV18. Construction in vitro of HPV pseudovirions containing marker plasmids would be potentially useful in developing methods to assay virus-neutralizing antibodies and to transfer exogenous genes to HPV-susceptible cells.     

Full replacement of 2-mercaptoethanol by cysteine plus selenium compounds in augmenting DNA synthesis of mitogen-stimulated mouse spleen lymphocytes

Mouse spleen lymphocytes require 2-mercaptoethanol for maximal mitogenic activation in vitro. Previous studies indicate that the lymphocytes are defective in the cystine transport activity and that they require 2-mercaptoethanol to utilize cystine. 2-Mercaptoethanol catalytically carries cysteine moiety into the cells in a mixed disulfide form. Because cysteine is easily oxidized to cysteine in the culture medium, it has been not easy to precisely examine the effect of near-physiological concentrations of cysteine on the activation of lymphocytes. By controlling the cysteine content in the medium, we have reviewed the effect of cysteine to see if cysteine replaces 2-mercaptoethanol in enhancing the DNA synthesis of lipopolysaccharide-stimulated lymphocytes. It was found that cysteine was less effective than 2-mercaptoethanol, and that cysteine fully replaced 2-mercaptoethanol when a selenium compound was supplemented. The effects of cysteine and selenium compounds were apparently independent and additive. Among the selenium compounds examined, sodium selenite and L-selenocystine were much more effective in stimulating DNA synthesis than sodium selenate and L-selenomethionine.     

Effects of the N-Substitution and Physical Form of Chitosan and the Modification of its Nonreducing End Groups on the Rates of Hydrolysis by Chitinase from Streptomyces griseus

Zymolyase was purified from Zymolyase-60,000 by ion-exchange chromatography with sugar and gel filtration. Zymolyase was separated into the two protein fractions A and B. Neither alone could lyse yeast cells, but together showed high lytic activity. Zymolyase A and B were β-1,3-glucanase and alkaline protease, respectively. On stepwise treatment of yeast cells with the enzymes, yeast cells were lysed only by treatment with Zymolyase A after pretreatment with Zymolyase B. Zymolyase A lysed yeast cells in the presence of 2-mercaptoethanol, but B could not even at high concentration. Zymolyase B decreased the turbidity of a yeast cell suspension by about 13%.     

Contrasts in the actions of protein antibiotics on deoxyribonucleic acid structure and function.

The protein antibiotics neocarzinostain (NCS), macromomycin (MCR), and auromomycin (AUR), which is closely related to MCR, have been compared for their in vitro and in vivo actions on deoxyribonucleic acid (DNA). NCS, markedly stimulated by 2-mercaptoethanol, is much more active in inducing strand scissions in superhelical pMB9 and linear duplex lambda DNA than AUR, which is slightly inhibited by 2-mercaptoethanol. Purified MCR, even at very high levels, does not give any significant amount of cutting with either DNA substrate. 2-Propanol stimulates the activity of NCS but inhibits that of AUR. On the other hand, the antioxidant alpha-tocopherol strongly inhibits DNA breakage by both drugs. The intercalating drugs ethidium bromide, daunorubicin, proflavin, and actinomycin D at low concentrations inhibit DNA scission by AUR. The levels of intercalators required to inhibit NCS activity to comparable levels are about 10 times higher than those for AUR. Although MCR has virtually no in vitro DNA cutting activity, it is, like AUR and NCS, cytotoxic, as measured by the inhibition of DNA synthesis and induction of DNA strand breakage in HeLa cells.     

Induction of lymphokine-activated killer activity in rat splenocyte cultures: The importance of 2-mercaptoethanol and indomethacin

Summary The role of 2-mercaptoethanol and indomethacin in the induction of lymphokine-activated killer (LAK) activity by interleukin-2 (IL-2) in rat splenocyte cultures was investigated. Spleens from 4-month-old male rats of five different strains were tested. Splenocytes were cultured for 3–5 days in the presence of IL-2 (1000 U/ml) and LAK activity was assessed by 4-h⁵¹Cr release assays with P815 and YAC-1 cells as targets. LAK activity could be induced by IL-2 in splenocytes from all rat strains, but only when 2-mercaptoethanol was present in the culture medium. Optimal LAK activity was induced when the 2-mercaptoethanol concentration in splenocyte cultures was at least 5 µM. Different rat strains showed differences in levels of in vitro induction of LAK activity. In the presence of 2-mercaptoethanol the level of LAK activity induced by IL-2 was high in BN and Lewis rats, intermediate in Wistar and Wag rats, and low in DZB rats. In the absence of 2-mercaptoethanol no or minimal LAK activity was induced. Furthermore we observed that addition of 50 µm indomethacin to the culture medium in the presence of 2-mercaptoethanol augmented the induction of LAK activity to some extent. In the absence of 2-mercaptoethanol, addition of indomethacin resulted only in low levels or no induction of LAK activity. We conclude that for optimal induction of LAK activity by IL-2 in rat splenocyte cultures 2-mercaptoethanol is essential, while indomethacin can only marginally further improve this induction.     

The control of DNA replication in hybrids between neutrophils and fibroblasts

Abstract. DNA synthesis regulation in heterokaryons between mouse neutrophils and cultured cells of various proliferative potentials has been studied. The following features have been found. Both immortalized and non-immortalized cells can reactivate DNA synthesis in neutrophil nuclei. The reactivation ability of cultured cells increases after immortalization and is not changed by further transformation. Neutrophils inhibit the entry of cultured cell nuclei into S phase and have no effect on ongoing DNA synthesis. Malignant cells are much less sensitive to the inhibitory action of neutrophils than non-malignant ones. Non-malignant immortalized cells are as sensitive to this effect as non-immortalized cells. Neutrophil karyoplasts do not influence DNA synthesis in partner cultured cell nuclei. Cycloheximide pretreatment of neutrophils drastically diminishes their inhibitory effect.     

An effective method for isolation of DNA from pig faeces and comparison of five different methods

Polymerase chain reaction (PCR) detection of microorganism in faecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this paper, we describe a new modified method for extracting PCR-quality microbial community DNA from pig faecal samples, which combines the pretreatment with polyformaldehyde, and subsequent DNA lysis in the presence of CTAB, salt, PVP, and β-mercaptoethanol, followed by isolation of nucleic acids using chloroform (no phenol) based protocol. The method resulted in a 1.3- to 11-fold increase in DNA yield when compared to four other widely used methods. Genomic DNA extracted from all five methods was assessed by both agarose gel electrophoresis and polymerase chain reaction for amplification of 16S rDNA specific fragments. The results showed that the improved method represented a reproducible, simple, and rapid technique for routine DNA extraction from faecal specimens and was notably better than using the QIAamp^® DNA Stool Mini Kit.     

Relationship Between Prophage Induction and Transformation in Haemophilus influenzae

The interaction between transformation and prophages of HP1c1, S2, and a defective phage of Haemophilus influenzae has been investigated by measurement of (i) the effect of prophage on transformation frequency and (ii) the effect of transformation on phage induction. The presence of any of the prophages does not appreciably alter transformation frequencies in various Rec(+) and Rec(-) strains. However, exposure of competent lysogens to transforming deoxyribonucleic acid (DNA) may induce phage but only in Rec(+) strains, which are able to integrate transforming DNA into their genome. Transformation of Rec(+) lysogens with DNA irradiated with ultraviolet (UV) light causes the production of even more phage than results from unirradiated DNA, but this indirect UV induction is not as effective as direct induction by UV irradiation of lysogens. Both types of UV induction are influenced by the repair capacity of the host. Wild-type cells contain a prophage and can be induced by transformation to produce a defective phage, which kills a small fraction of the cells. Defective phage in wild-type cells are also induced by H. parainfluenzae DNA, and a much larger fraction of the cells is killed. Strain BC200, which is highly transformable but is not inducible for defective phage, is not killed by H. parainfluenzae DNA, suggesting that wild-type cells are killed by killed by this DNA because of phage induction. A minicell-producing mutant, LB11, has been isolated. Some phage induction occurs in this strain when the cells are made competent, unlike the wild type. A large majority of LB11 cells surviving the competence regime are killed by exposure to transforming DNA.     

Preparation of mycelial and yeast-like spheroplasts fromMucor rouxii by a lytic enzyme preparation fromPenicillium islandicum

Spheroplasts ofMucor rouxii were prepared from mycelial and yeast-like cells by use of aPenicillium islandicum enzymatic complex. This enzyme preparation, which presents high chitosanase and chitinase activities, was produced by growingP. islandicum either on mycelial or yeast-like walls ofM. rouxii. The presence of magnesium sulfate as an osmotic stabilizer was critical to obtain high yields of spheroplasts from mycelial forms. In the case of yeast-like cells, pretreatment with β-mercaptoethanol followed by magnesium sulfate was essential for extensive spheroplast production.     

The use of microwave irradiation as a pretreatment toin situ hybridization for the detection of measles virus and chicken anaemia virus in formalin-fixed paraffin-embedded tissue

Summary Microwave irradiation was investigated as a pretreatment toin situ hybridization on formalin-fixed, paraffin-embedded tissue. Two probe/tissue systems were used: a single-stranded RNA probe for the detection of measles virus nucleocapsid genome in subacute sclerosing panencephalitis brain tissue, and a double stranded DNA probe for chicken anaemia virus in thymus of chicken infected with the virus. Microwaving, when used as sole pretreatment, was not as effective as the more traditional enzyme pretreatments forin situ hybridization. However, when used in combination with existing pretreatments, a significant increase was found in hybridization signal in both brain and thymus tissue. This was emphasized when combination enzyme/microwave pretreatments were used prior to detection of measles virus byin situ hybridization in a series of five archival subacute sclerosing panencephalitis cases. The use of microwave irradiation would be recommended as a means of supplementingin situ hybridization methods, especially when using long-term formalin fixed paraffin-embedded tissue.