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De novo synthesis of total and ribosomal ribonucleic acids has been studied during the early stages of Artemia sp. development. By in vivo incorporation studies of [14C]HCO3- an increase has been found in both total and ribosomal RNA synthesis post hatching, with a similar distribution of radioactivity and base composition.
16 strains of the genus Prototheca do not produce extracellular amylolytic enzymes. The base composition of their DNA shows rather continuous values from 62% to 78% GC (guanine + cytosine). Their assignment to four species and their possible relationship with Chlorella protothecoides are discussed.
Deoxyribonucleic acid (DNA) base composition, as measured by guanine plus cytosine (G + C) content, was determined in 17 strains of Sporothrix schenckii and Ceratocystis species. The average G + C content for S. schenckii was 54.7 +/- 0.2 mol%. No strain of Ceratocystis, with the exception of C. minor, gave values for G + C content similar to those obtained for S. schenckii. All DNA samples from strains of S. schenckii cross-hybridized with high percentages of relative binding. DNA samples from C. stenoceras and C. pilifera showed low degrees of homology with S. schenckii DNA. The DNA from C. minor hybridized with 75% binding with that from a strain of S. schenckii, as compared with the homologous reaction.
The article analyses the method of topical open treatment of postoperative pyothorax in 17 patients with the use of biologically active helevin-based compositions. The control group was made up of 11 patients with a similar suppurative process who received topical treatment by the same method but with the use of ointments with antibiotics and antiseptics on a fat base. With the use of biologically active compositions (BAC) on a helevin-base, the empyema cavity was completely freed from the pyonecrotic masses and its walls filled with granulation tissue on day 7 of treatment. In the control group the cavity was completely cleansed only on day 19 of treatment. It is shown that BAC possesses a high antibacterial effect not only due to the antibacterial components but because it is based on the laws of physical antiseptics.
The B-A transition of DNA in oriented films of DNA-drug complexes is more or less restricted as a consequence of drug binding as revealed by infrared linear dichroism. A fraction of DNA is irreversibly locked into the B form. This behavior is described by the number of DNA base pairs "frozen" in the B form by one drug molecule. This quantity is dependent on the DNA sequence the drug is attached to. In this paper, drug complexes of oriented films of NaDNA with a GC content of 42% from calf thymus and a GC-rich DNA from Micrococcus lysodeikticus were compared. The restriction of the B-A transition of DNA complexes with two intercalating antibiotics, aclacinomycin A and violamycin BI, is not severely influenced by the base composition of DNA. By contrast, the strong groove binding oligopeptide antibiotics netropsin and distamycin A are much less effective to restrict the B-A transition of GC-rich DNA than of AT-rich DNA. This finding is in agreement with previous results by other methods which support a model based upon a strong preference of AT clusters by these two non-intercalating drugs.
The mean base compositions of DNA from 27 strains of Pseudomonas putrefaciens, P. rubescens and P. piscicida ranged from 43-4 to 53-2 mol% GC with genome sizes from 3.04 X 10(9) to 4.23 X 10(9) daltons. On the basis of in vitro DNA-DNA binding, estimated spectrophotometrically from initial renaturation rates, P. putrefaciens strains were heterogenous in the extent to which they shared similar nucleotide sequences, and were divided into four DNA homology groups. The DNA characteristics of strains in these groups correlated with several biochemical characteristics that facilitated identification of clinical isolates of P. putrefaciens. The two species P. putrefaciens and P. rubescens appear to be synonymous and none of the four groups of P. putrefaciens was related in DNA sequences to P. pisicida. Pseudomonas putrefaciens should theretofore be retained as a single species and characteristics for identifying the various groups within the species are listed.
We have examined the thermodynamics of binding of a series of oligolysines (net charge z = +2 to +10) containing one, two, or three tryptophans to several single-stranded (ss) homo-polynucleotides [poly(A), poly(C), poly(I), poly(dU), poly(dT)] and duplex (ds) DNA in order to investigate the effects of peptide charge, tryptophan content, and polynucleotide base and sugar type. Equilibrium association constants, Kobs, were measured as a function of monovalent salt concentration (KCH3CO2) and temperature by monitoring the quenching of the peptide tryptophan fluorescence upon interaction with the polynucleotides, from which the dependence of delta G(o)obs, delta H(o)obs, and delta S(o)obs on [KCH3CO2] was obtained. As observed previously with poly(U) [Mascotti, D.P., & Lohman, T.M. (1992) Biochemistry 31, 8932], the dependence of delta G(o)obs on [K+] for peptide binding to each polynucleotide is entirely entropic in origin (i.e., delta H(o)obs is independent of [K+]), consistent with the conclusion that Kobs increases with decreasing salt concentration due to the favorable increase in entropy resulting from the displacement of bound cations (K+) from the nucleic acid upon formation of the complex. For each ss polynucleotide, we find that significantly less than one potassium ion is released thermodynamically per net positive peptide charge, as determined from the value of delta log Kobs/delta log [K+]. Interestingly, (-delta log Kobs/delta log [K+])/z decreases with increasing peptide charge for poly(A), poly(C), and poly(dT), contrary to the behavior observed with poly(U) and ds-DNA, which may reflect a significant release of bound water upon formation of peptide complexes with these ss homo-polynucleotides or an increased binding of K+ to the ss polynucleotide with increasing [K+]. Alternatively, there may be conformational differences between the bound states of oligolysines of low charge, relative to oligolysines of higher charge. However, in all cases, peptides with z < +4 display different thermodynamics of binding than peptides with z > +4. The presence of tryptophan (Trp) within these peptides does not influence the salt dependence of Kobs for binding to poly(A), poly(C), or poly(dT). However, the Trp content of the peptide does contribute significantly to the thermodynamics of these interactions: Trp interactions result in a favorable contribution to delta H(o)obs, but an unfavorable contribution to delta S(o)obs, with little effect on delta G(o)obs due to entropy-enthalpy compensations. Oligolysines containing Trp also display a small, but significant, dependence of Kobs on base composition, with Kobs decreasing in the order poly(I) >> poly(dT) approximately poly(U) approximately poly(A) >> poly(C).
The binding of the alkaloid sanguinarine to natural DNAs of differing GC content has been studied by spectrophotometry and viscometry techniques. Binding parameters determined from spectrophotometric measurements by Scatchard analysis, according to an excluded-site model, indicate a very high specificity of sanguinarine binding to GC rich DNA. In the strong binding region, the increase of contour length of DNA depends strongly on its base composition, being larger with GC rich DNA than with AT rich DNA. It is concluded that the alkaloid binds preferentially to the GC pairs in DNA template.
The deoxyribonucleic acid base composition (percent guanine + cytosine [GC]) was determined for 29 strains, representing 18 species of the genus Kluyveromyces. It was concluded that on the basis of GC content (47.4%) and other properties K. veronae occupies an uncertain position in the genus Kluyveromyces. The GC content of the remaining 17 species ranged from 35.3 to 43.4%, and three groups of species were recognized. The GC content of the first ranged from 35.3 to 38.0%; that of the second group from 39.5 to 41.7%; that of the third group from 42.4 to 43.4%. Several species revealed a nearly identical GC content. The GC contents do not correspond in all instances with the five groups of species proposed by van der Walt.
A key problem in antisense therapeutics is the relatively poor cell uptake of oligonucleotides and subsequent transport to the cytoplasm and nucleus. Although the chemical characteristics of oligonucleotides seem likely to affect their uptake by cells, little is known about this issue. In this article we explore the effect of base composition on oligonucleotide uptake. We show that phosphorothioate homo-G oligomers have a distinctly greater cellular uptake than other phosphorothioate homooligomers. This is probably due to a greater initial association with the plasma membrane, because homo-G oligomers show the greatest binding to liposome membranes, when tested at physiological ionic strength. Under different buffer conditions appreciable differences in membrane binding to liposomes were detected for the various homooligonucleotides.
Because of a subtle anomaly we encountered upon an analytical gel while characterizing a point mutation in an exon of a patient, we decided to perform expensive and time-consuming procedures to characterize the anomaly. Although initial and subsequent Southern blots and PCR analyses of this patient's mutation suggested that his mutation lay directly within a TaqI recognition site, further characterization revealed that the mutation actually lay in a base immediately outside the recognition site. Had we included an appropriate double-stranded DNA control in the restriction enzyme digestion of this patient's PCR-amplified exon, we could have arrived at the correct conclusion as to the location of the mutation without incurring high costs and time loss. This brief report depicts the use of DNA controls of appropriate length and base composition as a means of avoiding erroneous conclusions and expense in routine mutational analyses in the clinical setting.
A method is described for determination of the base composition (as guanine+cytosine or adenine+thymine content) of DNA by accurate measurement of the adenine/guanine ratio. The DNA is hydrolysed with 0.03n-hydrochloric acid for 40min. to release the purines. The hydrolysate is subjected to ion-exchange chromatography on Zeo-Karb 225. Apurinic acids are eluted with 0.03n-hydrochloric acid and then guanine and adenine are eluted separately with 2n-hydrochloric acid. Guanine and adenine are each collected as a single fraction, and the amount of base in each case is determined by measuring the volume and the extinction at suitable wavelengths. For use in the calculations, millimolar extinction coefficients in 2n-hydrochloric acid of 12.09 for adenine at 262mmu, and 10.77 for guanine at 248mmu, were determined with authentic samples of bases. The method gives extremely reproducible results: from 12 determinations with calf thymus DNA the adenine/guanine molar ratio had a standard deviation of 0.011; this corresponds to a standard deviation in guanine+cytosine content of 0.2% guanine+cytosine.
Hybrids formed between human alpha and beta globin cDNA and total human cellular DNA have been studied by thermal denaturation and cesium chloride density gradient centrifugation. From these studies, the weight average G + C content of human alpha globin cDNA has been determined to be 62% +/- 2% and that of human beta globin cDNA 51% +/- 2%. These values correlate well with the results of G + C content of the human alpha and beta globin cDNAs as determined by direct nucleotide sequence analysis of the cDNAs. Thermal denaturation and cesium chloride density gradient centrifugation of DNA-cDNA hybrids can therefore provide accurate information on the base composition of mRNA related sequences of any single copy gene for which a relatively pure cDNA can be obtained, without the necessity for direct nucleotide sequence analysis.
The authors studied the possible relationship between a genetic characteristic, like DNA base composition, and certain phenotypic characteristics, i.e., sensitivity to lytic agents, morphology of colonies, and biochemical reactions in 34 strains of spore-bearing bacilli. From the results obtained two groups of bacilli have been identified. The first group includes the species B. subtilis, B. pumilus, B. licheniformis, and B. firmus and one strain of B. megaterium. The mean value of the GC% of the DNA is 44.22 +/- 1.76. All the strains examined are highly sensitive to lysozyme and resistant to sodium lauryl sulphate (S.L.S.); the surface colonies have a "rhizoid" appearance and the microcolonies on slide microculture are star-shaped. The second group includes the species B. cereus, B. cereus var. mycoides, B. anthracis, and B. thuringiensis. The mean value of the GC% of the DNA is 33.65 +/- 0.59. All the strains belonging to this group are resistant to both lysozyme and S.L.S., and the surface macro-colonies and the microcolonies have a "medusae head" appearance. The two groups also have certain different biochemical reactions; e.g., anaerobic growth and the egg yolk reaction, with few exception, are negative for the first group and positive for the second; furthermore, the strains in the first group (with rare exceptions) cause fermentation in the three carbohydrates, glucose, arabinose, and xylose, while glucose only is fermented by all strains with one exception in the second group. The position of B. megaterium is not yet clear, although one strain may certainly be included in the first group. Lysis by lipase is extremely variable and does not correlate with any of the other characteristics studied. The other species studied in relation to the characteristics, considered in our research (B. coagulans, B. macerans, B. polymyxa, B. laterosporus, B. alvei, B. circulans, B. stearothermophilus, and B. brevis), are not susceptible to grouping, either in the first, or in the second or even in a separate group.
Rapid identification of Fusobacterium spp. is hampered by their inability to ferment carbohydrates and the availability of relatively few useful phenotypic characters. In an attempt to identify new diagnostic markers for species, we reported recently the potential utility of glutamate dehydrogenase (GDH) electrophoretic mobilities for distinguishing eight species of Fusobacterium. We have extended these observations to include all recognised members of the genus except F. prausnitzii and F. perfoetens, and our results show that they cluster into three broad electrophoretic groups. Some species, such as F. periodonticum, F. simiae and F. necrophorum, possessed GDH with similar electrophoretic mobilities. However, within such clusters, the electrophoretic migration of 2-oxoglutarate reductase (OGR) distinguished between species. Neither GDH or OGR mobility alone clearly differentiated all species, but their combined use provided unambiguous discrimination of all species except F. varium and F. mortiferum. The DNA base compositions of all species except F. naviforme (ATCC 25832) and F. sulci, were within the range 26-34 mol% G + C, suggesting the genus may be homogeneous. However, the peptidoglycan composition divided the genus into two major groups that contained either lanthionine or diaminopimelic acid; F. mortiferum peptidoglycan contained both dibasic amino acids.
A practical and effective method for the extraction of mitochondrial DNA from Candida species was developed. Zymolyase was used to induce yeast protoplasts, and mitochondrial DNA was extracted from DNase I-treated mitochondrial preparations. Restriction endonuclease analyses of mitochondrial DNAs from 19 isolates representing seven species of Candida (C. albicans, C. kefyr, C. lusitaniae, C. maltosa, C. parapsilosis, C. shehatae, and C. tropicalis) and Lodderomyces elongisporus revealed different cleavage patterns that appeared to be specific for the species. Few common restriction fragments were evident. The genome sizes of the mitochondrial DNAs ranged from 26.4 to 51.4 kilobase pairs, and the guanine-plus-cytosine contents ranged from 20.7 to 36.8 mol%. There was no correlation between the base compositions of nuclear and mitochondrial DNAs. Eight isolates of C. parapsilosis, including the type culture, and an ascosporogenous strain of L. elongisporus, which was once proposed as the teleomorph of C. parapsilosis, had similar mitochondrial DNA molecular sizes (30.2 and 28.8 kilobase pairs); however, restriction endonuclease patterns of these organisms were distinct. These data provide additional support for discrimination of these two species. The results of our experiments demonstrate that mitochondrial DNA analyses may provide useful criteria for the differentiation of yeast species.
The correlation between the melting temperature of intracellular DNA, determined by differential scanning calorimetry (DSC) of whole bacteria, and its guanine + cytosine (G + C) content, was examined for 58 species of bacteria. Samples of vegetative cells were heated in a Perkin-Elmer DSC-2C at 10 degrees C min-1 from 5 to 130 degrees C, cooled to 5 degrees C and then re-heated as before. Literature values for the mole fraction of G + C, XGC, were linearly related to the temperature, Tmax, at which the reversible peak, pr, observed on the second heating run was at a maximum, via the equation XGC = (Tmax -73.8)/41.0. This equation accounted for 91.9% of the variance in XGC with 95% confidence limits of +/- 7.3%, approximately 1.6 times the corresponding uncertainty (+/- 4.5%) quoted by De Ley (Journal of Bacteriology 101, 738-754, 1970) for estimates based on the spectroscopically determined melting temperature of purified DNA. Random errors of measurement of Tmax did not greatly limit the precision of the prediction and it was concluded that factors additional to base composition affected the temperature of DNA melting within the bacterial cell. Displacement of Tmax values from the fitted line was particularly noticeable in Campylobacter, Corynebacterium and Bacterionema species and part of the residual variation appeared to be species specific, possibly caused by differences in intracellular solute concentration.
Using a novel chemical tritium derivative method, we have determined the base composition of 4S RNA isolated from an RNA tumor virus, the avian myeloblastosis virus, and from normal and neoplastic host cells. Extensive differences were detected, particularly with respect to the amount of methylated bases in the viral RNA. The viral 4S RNA, which fulfills the criteria for designation as transfer RNA, appears to be derived from a precursor pool that is different from the precursor population of host-cell 4S RNA. These results are discussed in regard to the possible relationship between transfer RNA of avian mycoblastosis virus and cellular transfer RNA.