The dynamical structure of the RNA in alfalfa mosaic virus studied by 31P-nuclear magnetic resonance

The structure of the viral RNA in alfalfa mosaic virus (AlMV) was investigated by means of 31P-nuclear magnetic resonance (NMR). It was found that the 31P-NMR line width of AlMV Top a particles is significantly smaller than that of the larger Bottom particles. At low temperatures, the totational correlation time of the 31P nuclei essentially equals the tumbling rate of the virus particle, indicating that the RNA is contained rigidly inside the virion. At more elevated temperatures, the NMR line width sharpens more than expected on the basis of viscosity changes and the RNA exhibits internal mobility. The occurrence of internal mobility is paralleled by an increased internal mobility of the N-terminal part of the coat protein, as could be observed by 1H-NMR spectroscopy. The influence of EDTA on the 31P-NMR line width appeared to be negligible, which is in agreement with the idea that AlMV does not 'swell' like several other RNA-containing plant viruses.     

Measurements of cytoplasmic and vacuolar pH in Neurospora using nitrogen-15 nuclear magnetic resonance spectroscopy

The nitrogen-15 chemical shift of the N1 (tau)-nitrogen of 15N-labeled histidine and the half-height line widths of proton-coupled resonances of the delta- and omega,omega'-nitrogens of 15N-labeled arginine and of the alpha-nitrogens of 15N-labeled alanine and proline were measured in intact mycelia of Neurospora crassa to obtain to estimates of intracellular pH. For intracellular 15N-labeled histidine, the N1 (tau)-nitrogen chemical shift was 200.2 ppm. In vitro measurements showed that the chemical shift was slightly affected by the presence of phosphate, with which the basic amino acids may be associated in vivo. These considerations indicate a pH of 5.7-6.0 for the environment of intracellular histidine. The half-height line widths of the delta- and omega,omega'-nitrogens of [15N]arginine were 15 and 26 Hz, respectively. In vitro studies showed that these line widths also are influenced by the presence of phosphate, and, after suitable allowance for this, the line widths indicate pH 6.1-6.5 for intracellular arginine. The half-height line widths for intracellular alanine and proline were 17 and 12 Hz, respectively, which are consistent with an intracellular pH of 7.1-7.2. Pools of histidine and arginine are found principally in the vacuole of Neurospora, most likely in association with polyphosphates. Proline and alanine are cytoplasmic. The results reported here are consistent with these localizations and indicate that the vacuolar pH is 6.1 +/- 0.4 while the cytoplasmic pH is 7.15 +/- 0.10. Comparisons of these estimates with those obtained by other techniques and their implications for vacuolar function are discussed.     

Evidence implicating heterozygous deletion of chromosome 7 in the pathogenesis of familial leukemia associated with monosomy 7.

Complete or partial monosomy 7 is a recurring cytogenetic abnormality in acute myelogenous leukemia (AML) and myeloproliferative syndromes (MPS) and is particularly common in patients with Fanconi's anemia and in secondary AML. A familial form of monosomy 7 has been recognized in which two or more siblings develop MPS or AML before age 20. We tested the hypothesis that a recessive cancer susceptibility locus on chromosome 7 was important in the pathogenesis of leukemia in familial monosomy 7 by determining the parental origins of the chromosome 7 retained in the bone marrows of three pairs of affected siblings. We found no overlapping region where all three pairs retained DNA derived from the same paternal or maternal chromosome. These data suggest that inactivation of a single allele of a putative tumor-suppressor gene may be sufficient to contribute to leukemic transformation in familial monosomy 7.     

Molecular analysis of the avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum fully supports the gene-for-gene hypothesis

The interaction between the fungal pathogen Cladosporium fulvum and tomato is supposed to have a gene-for-gene basis. Races of C. fulvum which have 'overcome' the resistance gene Cf9 of tomato, lack the avirulence gene avr9 which encodes a race-specific peptide elicitor. Races avirulent on tomato genotypes carrying the resistance gene Cf9 produce the race-specific peptide elicitor, which induces the hypersensitive response (HR) on those genotypes. The causal relationship between the presence of a functional avr9 gene and avirulence on tomato genotype Cf9 was demonstrated by cloning of the avr9 gene and subsequent transformation of C. fulvum. A race virulent on tomato genotype Cf9 was shown to become avirulent by transformation with the cloned avr9 gene. These results clearly demonstrate that the avr9 gene is responsible for cultivar specificity on tomato genotype Cf9 and fully support the gene-for-gene hypothesis. The avr9 gene is the first fungal avirulence gene to be cloned.     

Nuclear magnetic resonance studies of cis-syn, trans-syn, and 6-4 photodimers of thymidylyl(3'-5')thymidine monophosphate and cis-syn photodimers of thymidylyl(3'-5')thymidine cyanoethyl phosphotriester

Three out of four possible photodimers of thymidylyl(3'-5')thymidine monophosphates (i.e., cis-syn, 6-4, and one of the trans-syn) and two structural isomers (i.e., R and S forms) of cis-syn-thymidylyl(3'-5')thymidine cyanoethyl phosphotriester have been isolated and purified from the reaction mixtures after UV irradiation and studied by multinuclear magnetic resonance Spectroscopy. All five inter thymine base linked photodimers have grossly similar structures which are quite different from those of the parent thymidylyl(3'-5')thymidine. The base of Tp- is in the syn conformation, and that of -pT it is in the anti conformation. The sugar puckering of Tp- is dominated by the 2E conformer, but in -pT it is in 4E; except for the conformer around C5'-O5' bond, the 6-4 isomer is very similar to those of cis-syn and trans-syn conformation. As expected, there are sugar-phosphate backbone distortions in the phosphotriesters, due to the neutralization of the negative charge of the phosphate. In general the structures of all five photodimers are very close to those of the cis-syn photodimer of thymidylyl(3'-5')thymidine monophosphate cyanoethyl ester as studied by X-ray diffraction [Cadet, J., Voituriez, L., Hruska, F. E., & Grand, A. (1985) Biopolymers 24, 897-903; Hruska, F. E., Voituriez, L., Grand, A., & Cadet, J. (1986) Biopolymers 25, 1401-1417]. While the trans-syn photodimer has two structural isomers, only one [C6(of Tp-)-R] was produced by the UV irradiation and studied.     

Glutamate biosynthesis in Bacillus azotofixans. 15N NMR and enzymatic studies

Pathways of ammonia assimilation into glutamic acid in Bacillus azotofixans, a recently characterized nitrogen-fixing species of Bacillus, were investigated through observation by NMR spectroscopy of in vivo incorporation of 15N into glutamine and glutamic acid in the absence and presence of inhibitors of ammonia-assimilating enzymes, in combination with measurements of the specific activities of glutamate dehydrogenase, glutamine synthetase, glutamate synthase, and alanine dehydrogenase. In ammonia-grown cells, both the glutamine synthetase/glutamate synthase and the glutamate dehydrogenase pathways contribute to the assimilation of ammonia into glutamic acid. In nitrate-grown and nitrogen-fixing cells, the glutamine synthetase/glutamate synthase pathway was found to be predominant. NADPH-dependent glutamate dehydrogenase activity was detectable at low levels only in ammonia-grown and glutamate-grown cells. Thus, B. azotofixans differs from Bacillus polymyxa and Bacillus macerans, but resembles other N2-fixing prokaryotes studied previously, as to the pathway of ammonia assimilation during ammonia limitation. Implications of the results for an emerging pattern of ammonia assimilation by alternative pathways among nitrogen-fixing prokaryotes are discussed, as well as the utility of 15N NMR for measuring in vivo glutamate synthase activity in the cell.     

Estimation of symbiotically fixed nitrogen in field grown soybeans: An application of natural15N/14N abundance and a low level15N-tracer technique

Summary Plants from agricultural and natural upland ecosystem were investigated for¹⁵N content to evaluate the role of symbiotic N₂-fixation in the nitrogen nutrition of soybean. Increased yields and lower δ¹⁵N values of nodulating soybeansvs, non-nodulating isolines gave semi-quantitative estimates of N₂ fixation. A fairly large discrepancy was found between estimations by δ¹⁵N and by N yield at 0 kg N/ha of fertilizer. More precise estimates were made by following changes in plant δ¹⁵N when fertilizer δ¹⁵N was varied near¹⁵N natural abundance level. Clearcut linear relationships between δ¹⁵N values of whole plants and of fertilizer were obtained at 30 kg N/ha of fertilizer for three kinds of soils. In experimental field plots, nodulating soybeans obtained 13±1% of their nitrogen from fertilizer, 66±8% from N₂ fixation and 21±10% from soil nitrogen in Andosol brown soil; 30%, 16% and 54% in Andosol black soil; 7%, 77% and 16% in Alluvial soil, respectively. These values for N₂ fixation coincided with each corresponding estimation by N yield method. Other results include: 1)¹⁵N content in upland soils and plants was variable, and may reflect differences in the mode of mineralization of soil organics, and 2) nitrogen isotopic discrimination during fertilizer uptake (δ¹⁵N of plant minus fertilizer) ranged from −2.2 to +4.9‰ at 0–30 kg N/ha of fertilizer, depending on soil type and plant species. The proposed method can accurately and relatively simply establish the importance of symbiotic nitrogen fixation for soybeans growing in agricultural settings.     

epsilon-Crystallin, a novel avian and reptilian eye lens protein

Gel filtration of Peking duck eye lens proteins reveals a component eluting just behind delta-crystallin and comprising approximately 10% of the total soluble protein. The native Mr of this additional component is estimated to be 120000; it appears to be composed of three identical chains of Mr 38000 and pI 7.5. Circular dichroic spectroscopy showed a relatively high alpha-helical content. No immunological cross-reactivity is found with alpha-, beta-, gamma- or delta-crystallins, and partial amino acid sequence determinations likewise failed to reveal any similarity with other known crystallins. We conclude that this protein represents another and novel family of eye lens proteins, for which we propose the designation epsilon-crystallin. epsilon-Crystallin is translated from a 1450-base mRNA, which has been partially purified. epsilon-Crystallin is found scattered among avian and reptilian taxa, but not in other vertebrates. Its rate of evolutionary change seems to be as slow as that of alpha- and beta-crystallins.     

Molecular cloning of cDNA coding for human preprourokinase

A cDNA library was constructed in pBR322 from 18S to 20S mRNA that was extracted from human kidney cells, fractionated on oligo(dT)-cellulose column and sucrose-density gradient, and confirmed for urokinase production in Xenopus laevis oocytes. The Escherichia coli RR1 transformants were hybridized to synthetic oligonucleotide probe prepared according to the known amino acid sequence, Glu 73 to Glu 77 of human urinary urokinase chain B. The entire cloned cDNA covers a 2250-bp region, wherein the 1293-bp sequence codes for preprourokinase consisting of 431 amino acids, with the first 20 residues being a signal peptide. The 5'-untranslated region is at least 80 bp long and the 3'-untranslated region is longer than 850 bp.     

Isolation and purification of deoxyribonucleosides from 90% 13C-enriched DNA of algal cells and their characterization by 1H and 13C NMR.

13C-enriched deoxyribonucleosides have been isolated from the DNA of Algal cells grown in an atmosphere of 90% 13C-labelled carbon dioxide. The 13C enriched DNA was quantitatively hydrolysed with DNase I, snake venom phosphodiesterase I and alkaline phosphatase of intestinal mucosa. The resulting deoxyribonucleosides were separated by preparative reversed-phase high pressure liquid chromatography in 60 minutes with detection by ultraviolet absorption at 254 nm. The final products were obtained in milligram quantities in high purity and in high yield. The 1H resonances of the base and sugar protons of these deoxyribonucleosides appear as well resolved multiplets in the 600 MHz NMR spectrum, due to the extensive 1H-13C couplings. Similarly, the 13C resonances of these deoxyribonucleosides appear as multiplets in the 75.5 MHz 13C NMR spectrum, due to 13C-13C couplings. The 1H-13C and 13C-13C coupling constants were also measured and tabulated. The isotopic enrichment of 13C these deoxyribonucleosides was obtained by integration of the 1H and/or 13C NMR spectra. It was found that the enrichment varied from carbon to carbon and species to species in the range of 70-89%, suggesting differential uptake and assimilation of 90% 13CO2 during metabolism pathways. This protocol provides experimentally useful quantities of 13C-enriched deoxyribonucleosides, which may be incorporated into site-specifically labeled oligonucleotides by chemical synthesis.