Sister chromatid differentiation and isolabeling of chromosomes

Summary Isolabeling observed during sister chromatid differentiation (SCD) was studied from human skin fibroblasts by the fluorescence-plus-Giemsa (FPG) technique. Bromodeoxyuridine (BrdU) was fed to exponentially dividing cells for 52 h to enable completion of two consecutive cycles of DNA replication. During this period, the late-replicating regions of some chromosomes were able to go through three replication cycles. These chromosome regions had evidently incorporated BrdU bifiliarly in both chromatids and hence, on staining with FPG, appeared isostained (isolabeled). Thus, incubation of exponentially dividing cells with BrdU for a period longer than that required for two cell cycles appears to be a suitable method for revealing the late-replicating regions of the genome, such as the X chromosome in a human female, as isolated.In another experiment with Indian muntjac chromosomes, isolabeled segments were darkly stained, which suggested unifilar incorporation of BrdU. In this case, unequal crossing-over or an unequal distribution of thymine residues probably is responsible for the isolabel.     

Molecular cloning of unintegrated and a portion of integrated moloney murine leukemia viral DNA in bacteriophage lambda.

A covalently closed circular form of unintegrated viral DNA obtained from NIH 3T3 cells freshly infected with Moloney murine leukemia virus (M-MLV) and a port of the endogenous M-MLV from the BALB/Mo mouse strain have been cloned in bacteriophage lambda. The unintegrated viral DNA was cleaved with restriction endonuclease HindIII and inserted into the single HindIII site of lambda phage Charon 21A. Similarly high-molecular-weight DNA from BALB/Mo mice ws cleaved sequentially with restriction endonucleases EcoRI and HindIII and separated on the basis of size, and one of the two fractions which reacted with an M-MLV-specific complementary DNA was inserted into the HindIII site of Charon 21A. Recombinant clones containing M-MLV-reacting DNA were analyzed by restriction endonuclease mapping, heteroduplexing, and infectivity assays. The restriction endonuclease map of the insert derived from unintegrated viral DNA, lambda x MLV-1, was comparable to published maps. Electron microscope analysis of the hybrid formed between lambda x MLV-1 DNA and 35S genomic M-MLV RNA showed a duplex structure. The molecularly cloned lambda x MLV-1 DNA contained only one copy of the long terminal repeat and was not infectious even after end-to-end ligation of the insert DNA. The insert DNA derived from endogenous M-MLV, lambda x MLVint-1, contained a DNA stretch measuring 5.4 kilobase pairs in length, corresponding to the 5' part of the genomic viral RNA, and cellular mouse DNA sequences measuring 3.5 kilobase pairs in length. The viral part of the insert showed the typical restriction pattern of M-MLV DNA except that a single restriction site, PvuII, in the 5' long terminal repeat was missing. Reconstructed genomes containing the 5' half derived from the integrated viral DNA and the 3' half derived from the unintegrated viral DNA were able to induce XC plaques after transfection in uninfected mouse fibroblasts.     

Antiviral activity of substituted 5-chlorodiphenylamine-2-carboxylic acid hydrazides and their N-benzylidene derivatives

The substituted 5-chlorodiphenylamine-2-carboxylic acid hydrazides and their N-benzylidene derivatives were tested for their antiviral activity against gomphrena mosaic virus in a hypersensitive hostin vitro as well asin vivo. Most of the compounds exhibited potential antiviral activity. These compounds may be useful in controlling viral infections in garden as well as in field crops.     

Functional antagonism between c-Jun and MyoD proteins: a direct physical association.

The product of the proto-oncogene Jun inhibits myogenesis. Constitutive expression of Jun in myoblasts interferes with the expression and the function of MyoD protein. In transient transfection assays Jun inhibits transactivation of the MyoD promoter, the muscle creatine kinase enhancer, and a reporter gene linked to MyoD DNA-binding sites. Conversely, MyoD suppresses the transactivation by Jun of genes linked to an AP-1 site. We demonstrate that both in vivo and in vitro MyoD and Jun proteins physically interact. Mutational analysis suggests that this interaction occurs via the leucine zipper domain of Jun and the helix-loop-helix region of MyoD.     

Forcing expression of a soybean root glutamine synthetase gene in tobacco leaves induces a native gene encoding cytosolic enzyme

Glutamine synthetase (GS; EC 6.3.1.2) is present in different subcellular compartments in plants. It is located in the cytoplasm in root and root nodules while generally present in the chloroplasts in leaves. The expression of GS gene(s) is enhanced in root nodules and in soybean roots treated with ammonia. We have isolated four genes encoding subunits of cytosolic GS from soybean (Glycine max L. cv. Prize). Promoter analysis of one of these genes (GS15) showed that it is expressed in a root-specific manner in transgenic tobacco and Lotus corniculatus, but is induced by ammonia only in the legume background. Making the GS15 gene expression constitutive by fusion with the CaMV-35S promoter led to the expression of GS in the leaves of transgenic tobacco plants. The soybean GS was functional and was located in the cytoplasm in tobacco leaves where this enzyme is not normally present. Forcing this change in the location of GS caused concomitant induction of the mRNA for a native cytosolic GS in the leaves of transgenic tobacco. Shifting the subcellular location of GS in transgenic plants apparently altered the nitrogen metabolism and forced the induction in leaves of a native GS gene encoding a cytosolic enzyme. The latter is normally expressed only in the root tissue of tobacco. This phenomenon may suggest a hitherto uncharacterized metabolic control on the expression of certain genes in plants.     

Soil surface CO2 flux in a Minnesota peatland

Soil surface CO₂ flux was measured in hollow and hummock microhabitats in a peatland in north central Minnesota from June to October in 1991. We used a closed infrared gas exchange system to measure soil CO₂ flux. The rates of CO₂ evolution from hummocks (9.8 ± 3.5 g m⁻² d⁻¹, [mean ± SE]) were consistently higher than those from hollows (5.4 ± 2.9 g m⁻² d⁻¹) (the hummock values included the contribution of moss dark respiration, which may account for 10–20% of the total measured flux). The soil CO₂ flux was strongly temperature-dependent (Q₁₀ ≈ 3.7) and appeared to be linearly related to changes in water table depth. An empirical multiplicative model, using peat temperature and water table depth as independent variables, explained about 81% of the variance in the CO₂ flux data. Using the empirical model with measurements of peat temperature and estimates of hollow/hummock microtopographic distribution (relative to water table elevation), daily rates of “site-averaged” CO₂ evolution were calculated. For the six-month period (May–October), the total soil CO₂ released from this ecosystem was estimated to be about 1340 g CO₂ m⁻². Published as Paper No. 9950, Journal Series, Nebraska Agricultural Research Division, University of Nebraska, Lincoln, NE, USA.     

The effects of herbicides and mycoparasites at different moisture levels on carpogenic germination in Sclerotinia sclerotiorum

Colonization of plant roots by fluorescent pseudomonads has been correlated with disease suppression. One mechanism may involve altered defense responses in the plant upon colonization. Altered defense responses were observed in bean (Phaseolus vulgaris) inoculated with fluorescent pseudomonads. Systemic effects of root inoculation by Pseudomonas putida isolate Corvallis, P. tolaasii (P9A) and P. aureofaciens REW1-I-1 were observed in bean leaves from 14-day-old plants. SDS- polyacrylamide gel electrophoresis demonstrated that levels of certain acid-soluble proteins increased in the leaf extracts of inoculated plants. Plants inoculated with REW1-I-1 produced more of a 57 M_r protein, and plants inoculated with isolates P9A and REW1-I-1 produced more of a 38 M_r protein. Northern hybridization revealed enhanced accumulation of mRNAs, that encode the pathogenesis-related protein PR1a, in leaves of plants inoculated with P. putida and REW1-I-1. Only REW1-I-1, but not P9A or P. putida induced symptoms of an hypersensitive response on tobacco leaves, bean cotyledons, and in bean suspension cultures. Phenolics and phytoalexins accumulated in bean cotyledons exposed to REW1-I-1 for 24 h but little change in levels of these compounds occurred in cotyledons inoculated with P9A and P. putida. Both suspension culture cells and roots treated with REW1-I-1 rapidly evolved more hydrogen peroxide than those exposed to P9A and P. putida. However, roots from 14-day-old plants colonized by P9A, P. putida or REW1-I-1 did not have higher levels of phenolics, phytoalexins or mRNAs for two enzymes involved in phenolic biosynthesis, phenylalanine-ammonia lyase and chalcone synthase. A selective induction of plant defense strategies upon root colonization by certain pseudomonads is apparent.