Kinetoplast DNA permits characterization of pathogenic plant trypanosomes of economic importance

Summary— Twelve Phytomonas isolates were obtained from different plants originating from several countries and cultured in vitro in complex media. The kinetoplast DNA (kDNA) was purified and observed by electron microscopy. The structure of kDNA from all isolates appeared as a large network of interlocked minicircles with some maxicircles extruding from the network, as has often been shown for Trypanosomatidae. Topoisomerase II resolved the kDNA network into free minicircles which were then analyzed by electron microscopy and by electrophoresis in agarose gel. The minicircle sizes varied from 1.3 to 2.8 kilobase pairs according to the Phytomonas isolate. The analysis by restriction endonucleases revealed a base sequence heterogeneity in the minicircles of 10 of these Phytomonas isolates. By contrast, in 2 Phytomonas isolates, more than 90% of their minicircle content was found to be homogeneous. Most interestingly, the minicircle cleavage patterns were found to be different between Phytomonas isolates and thus could be used to distinguish them.     

Kinetoplast DNA in the insect trypanosomes Crithidia luciliae and Crithidia fasciculata: II. Sequence evolution of the minicircles

Minicircle sequence evolution has been studied by comparison of the minicircles from Crithidia fasciculata and C. luciliae (C. fasciculata, var. luciliae) by restriction enzyme analysis and hybridization experiments. In contrast to the maxicircle sequence, the minicircle sequence of these trypanosomes evolves very rapidly. No conservation of restriction fragments has been observed and cross-hybridization of minicircles, minicircle fragments, and total kDNA is relatively weak. We conclude that no fragment larger than 550 bp is perfectly conserved between all minicircles of the two trypanosomes. Alterations in the minicircle fragment patterns of our stocks of trypanosomes were even apparent in a cultivation period of 1.5 to 2 years. The alterations suggest a random drift of the sequence which supports a noncodogenic function for the minicircles. Double restriction enzyme digestion experiments show that primary fragments are homogeneous with respect to cleavage by the second enzyme. This suggests that sequence rearrangements, rather than point mutations are the basis of the minicircle sequence heterogeneity.     

Minicircle variation in Beta mitochondrial DNA

Summary Mitochondrial DNAs from nine male fertile and eight cytoplasmic male sterile (cms) accessions of wild and cultivated Beta beets were investigated for the presence of low molecular weight DNA molecules. Five different supercoiled DNA molecules were detected, varying in size from 1.33 to 1.63 kb. Southern hybridizations revealed multimeric forms and sequence homologies between the minicircles. The occurrence of the different minicircles among the 17 accessions was investigated by agarose gel electrophoresis and Southern hybridization using minicircle specific probes. The 1.33 and 1.63 kb minicircles were found in most accessions, the other three minicircles were found in one or two of the wild Beta beet accessions. The presence of a low number of small, more or less homologous, minicircles in all investigated plants makes these molecules a general characteristic of Beta mtDNA. No association is found between the presence or absence of specific minicircles and the expression of male sterility. Neither does the distribution of the different minicircles in Beta beets indicate any essential biological role of these minicircles.     

Characterization of Phytomonas isolated from fruits by electrophoretic isoenzymes and kinetoplast-DNA analysis

Abstract Two flagellates of the family trypanosomatidae were isolated from the fruits of Lycopersicon esculentum (tomato) and Annona cherimolia (cherimoya) in the southeastern region of Spain. The isolates were characterized by isoenzyme analysis using nine different isoenzymes and by analysis of kinetoplast DNA (kDNA) restriction fragment length polymorphism using four different restriction endonucleases. Most of the isoenzymes were unable to distinguish between the two fruit isolates, while they were all able to distinguish these two from four other Phytomonas isolates, three of which were from laticiferous plants i.e. Euphorbia characias E. hirta and E. hyssopifolia , and one was a phloem-restricted isolate associated with Hartrot disease. Only the enzyme Superoxide dismutase was able to differentiate between the two fruit isolates. Electrophoretic and restriction endonuclease analysis of kDNA minicircles, using four restriction enzymes, showed similar if not identical restriction cleavage patterns of the minicircles of the two isolates from fruits, while the patterns were different for the other isolates. These results confirm the hypothesis that the two isolates from fruits constitute a group of trypanosomatids that are the same or closely related and that this group can parasitize more than one host plant.     

Observation of Flagellate Protozoa (Phytomonas sp.) in Cocos nucifera, Cecropia palmata and Euphorbia hirta

Trypanosomatid flagellates (Phytomonas sp.) were detected in the sieve tubes of ‘hartrot'-diseased coconut palms (Cocos nucifera) and in the laticifers of Cecropia palmata and Euphorbia hirta plants occurring as weeds in and around coconut plantations with diseased palms. The possible interrelationship and transmission by insect vectors of these protozoa is discussed.     

Diversity of Vicia faba circular mtDNA in whole plants and suspension cultures

Summary A comparative analysis of the Vicia faba mitochondrial genome in whole plants and in longterm suspension culture has been conducted. Restriction fragment patterns of the mtDNA isolated from these two sources were notably different. Electronmicroscopic analysis also revealed significant differences. Large circular mtDNA patterns shifted from a 37–80 kb subpopulation, which was predominant in whole plants, to 18–34 kb subpopulations although in both classes notable quantities of circular molecules of 80 to 120 kb and more were also found. Both in whole plant and suspension culture cells very large circular DNAs were observed. Some of them had lengths nearly 290 kb and could be considered as evidence of the existence of master chromosomes. The minicircular DNA population was also altered. In the suspension culture we observed a notable increase of percentage of minicircles with sizes near 1 kb. Simultaneously, the percentage of minicircles with sizes near 3.5–10 kb significantly increased in suspension culture cells. In addition, a new peak (10–12 kb) of minicircles appeared. Copy number alterations for some sequences homologous to CCC1A, CCC1B and CCC2 (Negruk et al. 1982, 1985) were shown. Southern hybridization revealed the existence of a family of minicircles having sizes 1.4–2 kb with predominance of CCC1A, CCC1B and CCC2. The copy numbers of CCC1B and some minor minicircles was changed in the suspension culture when compared with the whole plants.     

Kinetoplast DNA of Trypanosoma brucei: Physical map of the maxicircle

Trypanosoma brucei maxicircle DNA in kinetoplast DNA (kDNA) networks was characterized with restriction endonucleases. The data allow the construction of a circular map of a 22.2-kb molecule. Based on these and previous data each T. brucei kDNA network contains about 45 maxicircles which probably have the same sequence. The maxicircle of strain 164 used in this study was slightly larger and had three EcoRI sites compared to two found in other strains. Fragments generated by digestion with BamHI were largely singly cleaved maxicircles that had a density of 1.681 g/cm³ compared to 1.693 g/cm³ for the intact network. This suggests that maxicircles have a higher A + T content than minicircles. Minicircles in the kDNA network were also characterized with restriction endonucleases. Each enzyme cleaved a specific subset of minicircles from the network. However, no single restriction endonuclease or combination of up to three of these enzymes cleaved all molecules in the network. These results are consistent with earlier results of renaturation kinetic experiments and indicate that there are many different sequence classes of mini-circle DNA.     

The HLA-AW24 gene: Sequence,surroundings and comparison with the HLA-A2 and HLA-A3 genes

A cosmid clone containing two class I sequences was found to cause expression of the HLA-AW24 protein after transfection into mouse L cells. The restriction map of this cosmid shows extensive homology over 26 kb with the map of the HLA-A3 region obtained from cosmids of the same library, constructed with DNA from an HLA-A3/HLA-AW24 heterozygote, but diverges over the remaining 14 kb. The HLA-AW24 gene was subcloned from this cosmid and its nucleotide sequence was determined. Amino acid and, more strikingly, nucleotide sequence comparisons with other HLA alleles indicate that the A locus alleles are more closely related to each other than to alleles from other HLA loci. A very skewed distribution of silent substitutions is apparent, and the occurrence of clustered multiple substitutions hints at gene-conversion-like events.     

Cloning of histidine genes of Azospirillum brasilense: organization of the ABFH gene cluster and nucleotide sequence of the hisB gene

Summary A cluster of four Azospirillum brasilense histidine biosynthetic genes, hisA, hisB, hisF and hisH, was identified on a 4.5 kb DNA fragment and its organization studied by complementation analysis of Escherichia coli mutations and nucleotide sequence. The nucleotide sequence of a 1.3 kb fragment that complemented the E. coli hisB mutation was determined and an ORF of 624 nucleotides which can code for a protein of 207 amino acids was identified. A significant base sequence homology with the carboxyterminal moiety of the E. coli hisB gene (0.53) and the Saccharomyces cerevisiae HIS3 gene (0.44), coding for an imidazole glycerolphosphate dehydratase activity was found. The amino acid sequence and composition, the hydropathic profile and the predicted secondary structures of the yeast, E. coli and A. brasilense proteins were compared. The significance of the data presented is discussed.Abbreviations IGP imidazole glycerolphosphate - HP histidinolphosphate     

Axenic Cultivation of Phytomonas (Trypanosomatidae) Associated with Laticiferous Plants in Suriname1

Using variously enriched media, axenic cultures were obtained of Phytomonas spp. associated with Allamanda cathartica, Mandevilla scabra, and Rhabdadenia biflora (Apocynaceae), and Blepharodon nitidus (Asdepiadaceae) and Euphorbia hirta (Euphor-biaceae). Cultures were usually subcultured once every week for many weeks in succession. Attempts to grow the flagellates from Bonafousia tetrastachya (Apocynaceae), Cecropia palmata (Cecropiaceae), and Euphorbia hyssopifolia (Euphorbiaceae) were unsuccessful. The “phytomonads” in latex of different host plants showed morphological differences, whereas the cultured flagellates were similar in morphology whatever the host plant source or culture medium.     

Expression of the linear DNA plasmid pRS64 in the plant pathogenic fungus Rhizoctonia solani

The plant pathogenic isolate RI-64 of anastomosis group 4 of Rhizoctonia solani possesses three linear DNA plasmids (pRS64-1, -2, and -3). Unique poly(A)^– RNA, 0.5 kb in length and hybridizable with the pRS64 DNAs was found in mycelial cells of the isolate RI-64. The overall homology at the nucleotide level between pRS64-1, -2, and -3, and the cDNA prepared from the poly(A)^– RNA was 100%, 73%, and 84%, respectively. The open reading frames found in pRS64-1, -2, and -3 (ORF1-1, ORF2-1, and ORF3-1) are 68 amino acids long. The amino acids sequence showed no significant homology with known proteins. Extracts from Escherichia coli cells expressing ORF1-1 contain a specific protein of 7 kDa. Antisera raised against the ORF1-1 product obtained from E. coli cells cross-reacted with the specific proteins found in the mycelia. The results indicate that the DNA plasmids found in R. solani contain a sequence that encodes a specific protein which may be involved in determination of plant pathogenicity.     

Comparative Analysis of the Fine Structure of Four Isolates of Trypanosomatids of the Genus Phytomonas1

The fine structure of trypanosomatids from the plants Euphorbia hyssopifolia, E. characias, E. pinea, and Manihot esculenta, cultivated under axenic conditions, was compared. All parasites had a structural organization characteristic of members of the Trypanosomatidae. The organization of the membranes of the endoplasmic reticulum (ER) varied according to the isolate. In Phytomonas francai (isolated from M. esculenta), the ER cisternae form parallel rows. In Phytomonas sp. from E. characias, a para-crystalline array of the ribosomes attached to the ER membranes was observed. The ER membranes found in Phytomonas sp. from E. pinea seemed to originate from the central portion of the protozoon, branching in all directions. The peroxisome-like organelle (glycosome) found in Phytomonas sp. from E. hyssopifolia and E. characias occasionally showed an organized array. Taken together, the morphological observations make it possible to distinguish the four isolates of Phytomonas.     

Incompatibility and molecular relationships between small bacteriocinogenic plasmids of Staphylococcus aureus

The sequence relations between small bacteriocinogenic plasmids (pRJ6, pRJ9, pRJ10 and pRJ11) of Staphylococcus aureus were investigated by comparing restriction maps and by hybridization. Plasmids pRJ10 and pRJ11 showed identical restriction maps, similar to that of pRJ9. The restriction map of pRJ6 differed from those of pRJ9 and pRJ10/pRJ11. Both groups of plasmids were shown to share a region of homology of at least 2.6 kb. The incompatibility relationships between them were also investigated by using plasmid derivatives tagged with transposon Tn551. Plasmids pRJ6 and pRJ9 proved to belong to different incompatibility groups.     

Physical map of Aspergillus nidulans mitochondrial genes coding for ribosomal RNA: an intervening sequence in the large rRNA cistron

Summary A detailed map of the 32 kb mitochondrial genome of Aspergillus nidulans has been obtained by locating the cleavage sites for restriction endonucleases Pst I, Bam H I, Hha I, Pvu II, Hpa II and Hae III relative to the previously determined sites for Eco R I, Hind II and Hind III. The genes for the small and large ribosomal subunit RNAs were mapped by gel transfer hybridization of in vitro labelled rRNA to restriction fragments of mitochondrial DNA and its cloned Eco R I fragment E3, and by electron microscopy of RNA/DNA hybrids.The gene for the large rRNA (2.9 kb) is interrupted by a 1.8 kb insert, and the main segment of this gene (2.4 kb) is separated from the small rRNA gene (1.4 kb) by a spacer sequence of 2.8 kb length.This rRNA gene organization is very similar to that of the two-times larger mitochondrial genome of Neurospora crassa, except that in A. nidulans the spacer and intervening sequences are considerably shorter.     

An analysis of trypanosomatids kDNA minicircle by absolute dinucleotide frequency

Trypanosomatid mitochondrial DNA (kDNA) possesses thousands of copies of small circular molecules called minicircles. Due to a high level of nucleotide polymorphism among copies, sequence alignment for species or strain characterization is not appropriate. In this work we report dinucleotide absolute frequency as a method to analyze minicircle sequences heterogeneity in trypanosomatids. Using Trypanosoma rangeli and Leishmania guyanensis minicircles as example of sequence length heterogeneity, we show that dinucleotide frequency of minicircles whose length variation is less than to 10% is relatively constant. Dinucleotide frequencies in Leishmania genus point out three clusters of predominant dinucleotide profiles: GG/TT/TG for Old World species; ii) TT/AA/TA for New World species and iii) TT/GG(AA) TA(AT) for Sauroleishmania. Trypanosoma species displayed broad range composition and the highest frequency values. Their dinucleotide profile appears to be species specific, except for African trypanosomes which exhibit similar composition. The low number of sequences from Crithidia, Herpetomonas, Phytomonas and Wallaceina did not allow a generalized analysis, however some species present highly similar compositional profile, e.g., Wallaceina species. Distinct signatures for Trypanosomatidae family members can be generated by using values of absolute frequencies, range and composition of most/least frequent dinucleotides from minicircles. Each species can be graphically represented by a diagram of frequencies along with a box plot of summary statistics.     

A region flanking H-2K is duplicated to a distant site in most mouse t haplotypes

Mouse t haplotypes contain at least one inversion, which encompasses the major histocompatibility complex, relative to their wild-type counterparts. A DNA probe for a single copy sequence which flanks the H-2K region in inbred strains was found to have undergone further rearrangements in the t haplotypes. In most t haplotypes, this sequence is duplicated at a distant site, and the two regions show 1 % recombination. The length of homology shared by the two sites is likely to be at least 10–15 kb. Three different alleles, as defined by restriction fragment length polymorphisms, were found for each of the two sites among different t haplotypes. These may reveal evolutionary relationships among these chromosomes.     

Molecular cloning and sequence of theBacillus stearothermophilus translational initiation factor IF2 gene

Summary The structural gene for theBacillus stearothermophilus initiation factor IF2 was localized to a 6 kbHindIII restriction fragment by cross-hybridization with theSstI-SmaI fragment of theEscherichia coli infB gene. This fragment corresponds to the central region of the molecule containing the GTP-binding domain which is homologous inE. coli IF2, EF-Tu, EF-G and the humanras1 oncogene protein. After cloning into pACYC177, theHindIII fragment was further analysed by restriction mapping and cross-hybridization. A smaller (2.2 kb)SphI-HindIII fragment, which showed cross-hybridization, was subcloned into M13 phage and sequenced by the dideoxy chain-terminating method. This fragment was found to contain the entire IF2 gene except for the region coding for the N-terminus. This remaining region, coding for 45 amino acids, was located by homologous hybridization on an overlappingClaI-SstI fragment which was also subcloned and sequenced. Overall, theB. stearothermophilus IF2 gene codes for a protein of 742 amino acids (M_r=82,043) whose primary sequence displays extensive homology with the C-terminal two-thirds (but little or no homology with the N-terminal one-third) of the correspondingE. coli IF2 molecule. When cloned into an expression vector under the control of the λP_L promoter, theB. stearothermophilus IF2 gene, reconstituted by ligation of the two separately cloned pieces, could be expressed at high levels inE. coli cells.     

Human cellular sequences detectable with adenovirus probes

Previous studies suggesting homology between human cellular DNA and the DNAs from adenovirus types 2 and 5 are extended in the present paper. A clone (ChAd^h), isolated from a human genomic DNA library using an adenovirus probe, hybridized to discrete regions of adenovirus 2 DNA, including part of the transforming genes E1a and E1b, as well as to repeated sequences within human DNA. The E1a and E1b genes both hybridize to the same 300 base pair Sau3AI fragment within ChAd^h although there is no obvious homology between E1a and E1b. The Ad 2 E1a gene was also used as a probe to screen other cellular DNAs to determine whether repeated sequences detectable with Ad 2 DNA probes were conserved over long evolutionary periods. Hybridization was detected to the genomes of man, rat, mouse and fruit fly, but not to those of yeast and bacteria. In addition to a smear hybridization, discrete fragments were detected in both rodent and fruit fly DNAs. The experiments reported suggest the existence of two different types of cellular sequences detected by Ad 2 DNA: (1) repeated sequences conserved in a variety of eukaryote genomes and (2) a possible unique sequence detected with an E1a probe different from that responsible for hybridization to repeated sequences. This unique sequence was detected as an EcoRI fragment in mouse DNA and had a molecular size of about 8.8 kb.