Homologous transformation of the lignin-degrading basidiomycete Phanerochaete chrysosporium

Summary A clone containing the Phanerochaete chrysosporium ade1 gene was isolated from a EMBL3 genomic library using the ade5 gene encoding aminoimidazole ribonucleotide synthetase, from Schizophyllum commune, as a probe. A 6.0 kb fragment incorporating the ade1 gene was subcloned into pUC18 (pADE1) and used to transform the P. chrysosporium ade1 auxotrophic strain. Transformation frequencies were similar to those obtained previously with the S. commune ade5 gene; however, homologous transformants arose earlier than heterologous transformants. The transformants were mitotically and meiotically stable and Southern blot analysis indicated that the plasmid, pADE1, integrated ectopically in single or multiple copies. The pADE1 insert was mapped for restriction sites and the approximate location of the ade1 gene within the insert was determined.     

The nature of extra-chromosomal maintenance of transforming plasmids in the filamentous basidiomycete Phanerochaete chrysosporium

Summary The nature of extra-chromosomal maintenance of the transforming plasmid p12-6 in Phanerochaete chrysosporium was studied. Our results indicate that the plasmid is maintained in the fungal transformants extra-chromosomally as part of a larger endogenous plasmid (designated pME) of P. chrysosporium. Using the total DNA of p12-6 fungal transformants, p12-6, as well as a larger plasmid, p511, were recovered in recA ^– E. coli strains while only p12-6 was recovered in recA ⁺ E. coli strains. The results also showed that the cytosine methylation system has no apparent effect on the strain-dependent recovery of p12-6 and p511 in E. coli from the total DNA of fungal transformants.     

Transformation of Phanerochaete chrysosporium and Neurospora crassa with adenine biosynthetic genes from Schizophyllum commune

Summary Protoplasted basidiospores of two different adenine auxotrophs of the lignin-degrading basidiomycete Phanerochaete chrysosporium were transformed to prototrophy using plasmids containing genes encoding adenine biosynthetic enzymes from Schizophyllum commune. Fragments containing these genes were subcloned into pUC18 and P. chrysosporium transformants obtained with these subclones were analyzed. The subclones were mapped for restriction sites and the approximate locations of the complementing genes were determined. One of these plasmids was used to transform the Neurospora crassa auxotrophic strain ade2, thereby identifying the S. commune ade5 biosynthetic gene as encoding phosphoribosylaminoimidazole synthetase.     

Differentiation of species and strains among filamentous fungi by DNA fingerprinting

Summary We have analyzed 11 strains and clones, representing five species (Penicillium janthinellum, P. citrioviridae, P. chrysogenum, Aspergillus niger, Trichoderma harzianum) and three genera of filamentous fungi, for the presence of hypervariable loci in their genomes by hybridization with simple repeat oligonucleotides and the DNA of phage M13. The oligonucleotide probes (CT)₈, (GTG)₅ and (GACA)₄, as well as M13 DNA, are informative probes for fingerprinting in all genera and species tested. The probe (GATA)₄ produced informative fingerprints only with the genomic DNA of A. niger. There was no similarity between the fingerprints originating from fungi of different genera and also little similarity between the fingerprints of different species belonging to the same genus. Fingerprints of strains of the same species differed only slightly from each other. Fingerprints of clones originating from one strain were identical. The results indicate that DNA fingerprinting is a powerful method to differentiate species and strains of filamentous fungi.     

The genomic synteny at DNA level between human and chimpanzee chromosomes

The evolutionary relationship between human (Homo sapiens) and chimpanzee (Pan troglodytes) has been the subject of debate and scrutiny for over two decades. The close relationship established by numerous parameters may or may not reflect homology at the DNA level. The recent advent of a molecular method termed the chromosomein situ suppression hybridization (CISS)-technique has prompted us to explore the phylogenetic relationship at the DNA sequence level. Cross-hybridization data using human-derived whole chromosome paints (WCPs) suggests an apparent genomic synteny with chimpanzee chromosomes at the DNA level, thus providing a better understanding of an evolutionary relationship between humans and chimpanzees.     

Analysis of extrachromosomal DNA from normal and tumor cells

Two independent methods (pulse-electrophoresis of intact DNA isolated from agarose-encapsulated cells and differential extraction of intact DNA from Celite-immobilized nucleoproteins) yielded evidence of the presence of free (extrachromosomal) DNA in cells of various origin, both malignant and normal, cultured and isolated from animal tissues. Free DNA from all cell types studied form three discrete electrophoretic bands of 400, 250–300, and 50 thousand base pairs. According to the data on³H-thymidine incorporation, free and chromosomal DNA differ in their metabolic properties. By means of the polymerase chain reaction using c-myc and L-myc primers it is established that free DNAs of HT1080 cells contain structural genes. Application of the method of conformational polymorphism of single-strand fragments revealed their identity to the corresponding genes localized in the chromosomes. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 119, No 5, pp. 533–536, May, 1995 Presented by I. P. Ashmarin, Member of the Russian Academy of Medical Sciences     

Comparison of the blood level of endogenous paramagnetics in health and pathology using proton magnetic relaxation

An experimental study is made of the longitudinal proton relaxation timeT ₁ in human serum in health and pathology as a function of the protein concentration using partially lyophilized samples. A comparison of the results with those obtained with modeled aqueous protein solutions and extrapolation of the dependences to the zero concentration demonstrate that the concentration of paramagnetic admixtures is the same in groups of healthy subjects and patients with malignant neoplasms who have reliably differentT ₁ values in native samples. There is no difference in the longitudinal proton relaxation time of the nonaqueous component of the serum between these groups. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 11, pp. 557–560, November, 1995     

Effect of a new antitumor drug cycloplatam on DNA structure and production

The new antitumor drug cycloplatam [amine (cyclopentylamine)-S-(-)-malatoplatinum(II)] caused dose-dependent formation of interstrand DNA ligationsin vitro depending on the duration of incubation. Like cisplatin, cycloplatam caused a deep and long inhibition of DNA production in tumor cells. The effect of cycloplatam in bone marrow and small intestine epithelium cells was weaker and shorter than that of cisplatin. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 125, No. 2, pp. 197–199, February, 1998     

Population variation in the A chromosome distribution of satellite DNA and ribosomal DNA in the grasshopper Eyprepocnemis plorans

The double FISH analysis of two repetitive DNAs (a satellite DNA and ribosomal DNA) in 12 natural populations of the grasshopper Eyprepocnemis plorans collected at the south (Granada and Málaga provinces) and south-east (Albacete and Murcia provinces) of the Iberian Peninsula has shown their widespread presence throughout the whole genome as well as extensive variation among populations. Both DNAs are found in most A chromosomes. Regularly, both DNAs occurred in the S₁₁ and X chromosomes, rDNA in the S₁₀ and satDNA in the L₂ and M₃. No correlation was found between the number of satDNA and rDNA clusters in the A genomes of the 12 populations analysed, and both figures were independent of the presence of B chromosomes. The genomic distribution of both DNAs showed no association with the geographical localization of the populations analysed. Finally, we provide evidence that the supernumerary chromosome segment proximally located on the S₁₁ chromosome is, in most cases, the result of satDNA amplification but, in some cases, it might also derive from amplification of both satDNA and rDNA.     

Phologeny of Alternaria fungi known to produce host-specific toxins on the basis of variation in internal transcribed spacers of ribosomal DNA

The internal transcribed spacer regions (ITS1 and ITS2) of ribosomal DNA from Alternaria species, including seven fungi known to produce host-specific toxins, were analyzed by polymerase chain reaction-amplification and direct sequencing. Phylogenetic analysis of the sequence data by the Neighbor-joining method showed that the seven toxin-producing fungi belong to a monophyletic group together with A. alternata. In contract, A. dianthi, A. panax, A. dauci, A. bataticola, A. porri, A. sesami and A. solani, species that can be morphologically distinguished from A. alternata, could be clearly separated from A. alternata by phylogenetic analysis of the ITS variation. These results suggest that Alternaria pathogens which produce host-specific toxins are pathogenic variants within a single variable species, A. alternata.This paper is dedicated to the late Syoyo Nishimura     

Comparison of the level of mitotic activity and duration of mitosis in normal and neoplastic mouse tissues during the 24-hour period

Diurnal rhythms of cell division in the epithelium of the forestomach and in a transplantable carcinoma of the forestomach were found to be largely similar and the duration of mitosis in both these tissues varied during the course of the 24-hour period. The mean diurnal mitotic activity in the tumor was twice as high as in the normal forestomach. By contrast, in the course of 24 h colchamine (colcemid) led to the accumulation of 121.1 of mitoses, compared with only 83.8 in the carcinoma. The larger number of mitoses in the tumor when counted in the ordinary way can be explained by the 2.7 times greater mean diurnal duration of mitosis in carcinoma of the forestomach than in the normal epithelium of the forestomach.Laboratory of Chronobiology, Scientific-Research Center, N. I. Pirogov Second Moscow Medical Institute. (Presented by Adademician of the Academy of Medical Sciences of the USSR V. V. Kupriyanov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 11, pp. 1363–1365, November, 1976.     

Effect of exogenous DNA on content and biosynthesis of endogenous DNA in rat bone marrow

The effect of exogenous DNA on the content and rate of biosynthesis of endogenous DNA in rat bone marrow was studied. After injection of high-polymer homologous DNA into intact rats the endogenous DNA content per gram bone marrow was found to be reduced for the first 3 days (the decrease was most marked after 3 days, namely by 36%) and it returned to normal by the 6th day. The rate of DNA biosynthesis in rat bone marrow was increased after 18 h (when it was twice as high as in the control) and after 6 days (by 58%), and it was close to normal again 1–3 days after injection of DNA.(Presented by Academician of the Academy of Medical Sciences of the USSR P. D. Gorizontov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 8, pp. 952–953, August, 1976.     

Divergence of satellite DNA and interspersion of dispersed repeats in the genome of the wild beet Beta procumbens

Several repetitive sequences of the genome of Beta procumbens Chr. Sm., a wild beet species of the section Procumbentes of the genus Beta have been isolated. According to their genomic organization, the repeats were assigned to satellite DNA and families of dispersed DNA sequences.The tandem repeats are 229–246 bp long and belong to an AluI restriction satellite designated pAp11. Monomers of this satellite DNA form subfamilies which can be distinguished by the divergence or methylation of an internal restriction site. The satellite is amplified in the section Procumbentes, but is also found in species of the section Beta including cultivated beet (Beta vulgaris). The existence of the pAp11 satellite in distantly related species suggests that the AluI sequence family is an ancient component of Beta genomes and the ancestor of the diverged satellite subfamily pEV4 in B. vulgaris. Comparative fluorescent in-situ hybridization revealed remarkable differences in the chromosomal position between B. procumbens and B. vulgaris, indicating that the pAp11 and pEV4 satellites were most likely involved in the expansion or rearrangement of the intercalary B. vulgaris heterochromatin.Furthermore, we describe the molecular structure, and genomic and chromosomal organization of two repetitive DNA families which were designated pAp4 and pAp22 and are 1354 and 582 bp long, respectively. The families consist of sequence elements which are widely dispersed along B. procumbens chromosomes with local clustering and exclusion from distal euchromatic regions. FISH on meiotic chromosomes showed that both dispersed repeats are colocalized in some chromosomal regions. The interspersion of repeats of the pAp4 and pAp22 family was studied by PCR and enabled the determination of repeat flanking sequences. Sequence analysis revealed that pAp22 is either derived from or part of a long terminal repeat (LTR) of an Athila-like retrotransposon. Southern analysis and FISH with pAp4 and pAp22 showed that both dispersed repeats are species-specific and can be used as DNA probes to discriminate parental genomes in interspecific hybrids. This was tested in the sugar beet hybrid PRO1 which contains a small B. procumbens chromosome fragment.     

The complete DNA sequence of the mitochondrial genome of the dermatophyte fungus Epidermophyton floccosum

We report here the complete nucleotide sequence of the 30.9-kb mitochondrial genome of the dermatophyte fungus Epidermophyton floccosum. All genes are encoded on the same DNA strand and include seven subunits of the reduced nicotinamide adenine dinucleotide ubiquinone oxireductase (nad1, nad2, nad3, nad4, nad4L, nad5, and nad6), three subunits of cytochrome oxidase (cox1, cox2, and cox3), apocytochrome b (cob), three subunits of ATP synthase (atp6, atp8, and atp9), the small and large ribosomal RNAs (rns and rnl), and 25 tRNAs. A ribosomal protein gene (rps5) is present as an intronic ORF in the large ribosomal subunit. The genes coding for cob and cox1 carry one intron and nad5 carries two introns with ORFs. The mtDNA of E. floccosum has the same gene order as Trichophyton rubrum mtDNA, with the exception of some tRNA genes. Maximum likelihood phylogenetic analysis confirms T. rubrum as a close relative of E. floccosum. This is the first complete mitochondrial sequence of a species of the order Onygenales. This sequence is available under GenBank accession number AY916130.     

Dot hybridization and polymerase amplification of DNA in the prenatal diagnosis of cytomegalovirus infection

Molecular biology techniques (dot hybridization and polymerase amplification of DNA) are used to diagnose cytomegalovirus infection in pregnant women and newborns. It is concluded from the results that dot hybridization is a convenient tool in screening studies and that polymerase DNA amplification followed by verification of the reaction product using restriction enzymes and blot hybridization makes the diagnosis of this disease more reliable and may find wide application in practical laboratories. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 7, pp. 77–79, July, 1994 Presented by A. A. Totolyan, Member of the Russian Academy of Medical Sciences