Trypanosoma (Nannomonas) congolense: molecular characterization of a new genotype from Tsavo, Kenya

Parasitology ◽  
1993 ◽  
Vol 106 (2) ◽  
pp. 151-162 ◽  
Author(s):  
P. A. O. Majiwa ◽  
M. Maina ◽  
J. N. Waitumbi ◽  
S. Mihok ◽  
E. Zweygarth

SUMMARYTrypanosoma (Nannomonas) congolense comprises morphologically identical but genetically heterogeneous parasites infective to livestock and other mammalian hosts; three different genotypes of this parasite have been described previously. Restriction enzyme fragment length polymorphisms (RFLPs) in both kinetoplast DNA minicircle and nuclear DNA sequences, and randomly amplified polymorphic deoxyribonucleic acid (RAPD) patterns have been used here to demonstrate the existence of another type of T. (N.) congolense that is genotypically distinct from those that have so far been characterized at the molecular level. A highly repetitive, tandemly arranged DNA sequence and oligonucleotide primers, for use in polymerase chain reaction (PCR) amplification are described, which can be used for specific identification of the trypanosome and its distinction from others within the Nannomonas subgenus.

Parasitology ◽  
1989 ◽  
Vol 99 (1) ◽  
pp. 57-66 ◽  
Author(s):  
D. R. Moser ◽  
G. A. Cook ◽  
Diane E. Ochs ◽  
Cheryl P. Bailey ◽  
Melissa R. McKane ◽  
...  

SUMMARYThe nuclear DNA ofTrypanosoma congolensecontains a family of highly conserved 369 base pair (bp) repeats. The sequences of three cloned copies of these repeats were determined. An unrelated family of 177 bp repeats has previously been shown to occur in the nuclear DNA ofTrypanosoma brucei brucei(Sloofet al.1983a). Oligonucleotides were synthesized which prime the specific amplification of each of these repetitive DNAs by the polymerase chain reaction (PCR). Amplification of 10% of the DNA in a single parasite ofT. congolenseorT. bruceispp. produced sufficient amplified product to be visible as a band in an agarose gel stained with ethidium bromide. This level of detection, which does not depend on the use of radioactivity, is about 100 times more sensitive than previous detection methods based on radioactive DNA probes. The oligonucleotides did not prime the amplification of DNA sequences in other trypanosome species nor inLeishmania, mouse or human DNAs. Amplification of DNA from the blood of animals infected withT. congolenseand/orT. bruceispp. permitted the identification of parasite levels far below that detectable by microscopic inspection. Since PCR amplification can be conducted on a large number of samples simultaneously, it is ideally suited for large-scale studies on the prevalence of African trypanosomes in both mammalian blood and insect vectors.


Parasitology ◽  
1995 ◽  
Vol 111 (3) ◽  
pp. 301-312 ◽  
Author(s):  
L. H. Garside ◽  
W. C. Gibson

SUMMARYRestriction fragment length polymorphism (RFLP) analysis of both genomic and kinetoplast DNA from representative stocks from 3 Trypanosoma congolense subgroups (Savannah, Forest, and Kilifi), T. simiae and T. godfreyi, was used to investigate the relatedness of the different groups within subgenus Nannomonas, DNA probes for β-tubulin and the ribosomal DNA (rDNA) locus were isolated from a T. congolense Savannah genomic library; additional probes were generated by PCR amplification of mini-exon and glutamate and alanine rich protein (GARP) gene sequences. Our results provide evidence that at the molecular level the T. congolense Savannah and Forest groups are the most closely related groups within the subgenus Nannomonas: the Savannah and the Forest groups had mini-exon gene repeats of identical size, which shared homology, had mini-circles of the same size and had a high level of similarity (63%) when the banding patterns produced with a tubulin and rDNA probe were subjected to numerical analysis. All other pairwise combinations of groups have very low percentage similarities of < 10%, suggesting that the Kilifi group trypanosomes, are as distantly related to the T. congolense Savannah and Forest groups as they are to T. simiae or T. godfreyi. The conservation of the GARP gene between the Savannah, Forest and Kilifi groups provides the only evidence linking the Kilifi trypanosomes to the other groups in T. congolense. We find no evidence for the presence of the GARP gene in the T. simiae or T. godfreyi group trypanosomes.


Parasitology ◽  
1992 ◽  
Vol 104 (1) ◽  
pp. 67-74 ◽  
Author(s):  
W. T. Artama ◽  
M. W. Agey ◽  
J. E. Donelson

SUMMARYTwo clones from separate isolates of Trypanosoma evansi in Indonesia were found by polymerase chain reaction (PCR) analyses to contain 3 different repeated nuclear DNA sequences of Trypanosoma brucei spp: the consensus sequence for a highly repetitive 177 base pairs and the gene repeats encoding procyclin and the spliced leader. In addition, the 994 bp minicircle sequence of one of the clones was determined, and PCR amplification primers specific for minicircles of T. evansi were identified that do not amplify minicircle sequences in the T. brucei spp. clones tested.


1995 ◽  
Vol 74 (04) ◽  
pp. 1079-1087 ◽  
Author(s):  
Klaus-P Radtke ◽  
José A Fernández ◽  
Bruno O Villoutreix ◽  
Judith S Greengard ◽  
John H Griffin

SummarycDNAs for protein C inhibitor (PCI) were cloned from human and rhesus monkey 1 liver RNAs by reverse transcription and polymerase chain reaction (PCR) amplification. Sequencing showed that rhesus monkey and human PCI cDNAs were 93% identical. Predicted amino acid sequences differed at 26 of 387 residues. Pour of these differences (T352M, N359S, R362K, L3631) were in the reactive center loop that is important for inhibitory specificity, and two were in the N-terminal helix (M8T, E13K) that is implicated in glycosaminoglycan binding. PCI in human or rhesus monkey plasma showed comparable inhibitory activity towards human activated protein C in the presence of 10 U/ml heparin. However, maximal acceleration of the inhibition of activated protein C required 5-fold lower heparin concentration for rhesus monkey than for human plasma, consistent with the interpretation that the additional positive charge (E13K) in a putative-heparin binding region increased the affinity for heparin.


2021 ◽  
Vol 11 (4) ◽  
pp. 1943
Author(s):  
Joo-Young Kim ◽  
Ju Yeon Jung ◽  
Da-Hye Kim ◽  
Seohyun Moon ◽  
Won-Hae Lee ◽  
...  

Analytical techniques such as DNA profiling are widely used in various fields, including forensic science, and novel technologies such as direct polymerase chain reaction (PCR) amplification are continuously being developed in order to acquire DNA profiles efficiently. However, non-specific amplification may occur depending on the quality of the crime scene evidence and amplification methods employed. In particular, the ski-slope effect observed in direct PCR amplification has led to inaccurate interpretations of the DNA profile results. In this study, we aimed to reduce the ski-slope effect by using dimethyl sulfoxide (DMSO) in direct PCR. We confirmed that DMSO (3.75%, v/v) increased the amplification yield of large-sized DNA sequences more than that of small-sized ones. Using 50 Korean buccal samples, we further demonstrated that DMSO reduced the ski-slope effect in direct PCR. These results suggest that the experimental method developed in this study is suitable for direct PCR and may help to successfully obtain DNA profiles from various types of evidence at crime scenes.


Author(s):  
Danny A. Humphreys-Pereira ◽  
Taeho Kim ◽  
Joong-Ki Park

Abstract This chapter presents procedures on polymerase chain reaction (PCR) amplification, protocols for PCR, cloning and sequencing, and mitochondrial genome annotation and gene identification for the characterization of nematodes.


Parasitology ◽  
1994 ◽  
Vol 109 (4) ◽  
pp. 423-433 ◽  
Author(s):  
S. Eresh ◽  
S. M. McCallum ◽  
D. C. Barker

SUMMARYFollowing cloning of Leishmania (L.) amazonensis kinetoplast DNA two recombinant clones were identified: one specific for L. (L.) amazonensis and the other specific for L. (L.) amazonensis and closely related isolates. DNA sequences from these clones were compared with those of other kinetoplastids and oligonucleotide primers were designed to be used in the polymerase chain reaction. A pair of these primers has been shown not only to be highly specific for L. mexicana complex isolates but can also be used to distinguish between L. (L.) mexicana and L. (L.) amazonensis isolates. These primers have been tested with water-lysed cultures, crude DNA extracts from human patients, potential host reservoirs, sandfly vectors and with cell pellets after isoenzyme characterization. The results of these tests indicate that the primers can be used specifically in the presence of excess host DNA originating from the majority of South American countries.


The Auk ◽  
2002 ◽  
Vol 119 (3) ◽  
pp. 695-714 ◽  
Author(s):  
I. J. Lovette ◽  
E. Bermingham

AbstractThe wood-warblers (family Parulidae) fall within a radiation of passerine birds commonly known as the New World nine-primaried oscines. Defining familial relationships within that radiation has previously been challenging because of its extremely high diversity, a paucity of phylogenetically informative morphological characters, and an apparent high rate of cladogenesis early in the radiation's history. Here, analyses of mitochondrial and nuclear DNA sequences demonstrate that the 25 extant genera traditionally placed in the Parulidae do not form a monophyletic group. Instead, all reconstructions identify a well-resolved clade of 19 genera (Vermivora, Parula, Dendroica, Catharopeza, Mniotilta, Setophaga, Protonotaria, Helmitheros, Limnothlypis, Seiurus, Oporornis, Geothlypis, Wilsonia, Cardellina, Ergaticus, Myioborus, Euthlypis, Basileuterus, and Phaeothlypis) that are all morphologically typical wood-warblers traditionally placed in the Parulidae. Six genera traditionally assigned to the Parulidae—Microligea, Teretistris, Zeledonia, Icteria, Granatellus, and Xenoligea—fall outside this highly supported clade in all mtDNA-based and nuclear DNA-based reconstructions, and each is probably more closely allied to taxa traditionally placed in other nine-primaried oscine families. The long, well-supported, and independently confirmed internode at the base of this wood-warbler clade provides the opportunity to define a monophyletic Parulidae using several complementary molecular phylogenetic criteria. Support for those relationships comes from reconstructions based on a range of nucleotide-intensive (from 894 to 3,638 nucleotides per taxon) and taxon-intensive (45 to 128 species) analyses of mtDNA sequences, as well as independent reconstructions based on nucleotide substitutions in the nuclear-encoded c-mos gene. Furthermore, the 19 typical wood-warbler genera share a synapomorphic one-codon c-mos deletion not found in other passerines. At a slightly deeper phylogenetic level, our mtDNA-based reconstructions are consistent with previous morphologic and genetic studies in suggesting that many nine-primaried oscine taxa have unanticipated affinities, that many lineages arose during an early and explosive period of cladogenesis, and that the generation of a robust nine-primaried oscine phylogeny will require robust taxonomic sampling and extensive phylogenetic information.


2005 ◽  
Vol 71 (11) ◽  
pp. 7224-7228 ◽  
Author(s):  
Tina Lütke-Eversloh ◽  
Gregory Stephanopoulos

ABSTRACT In order to get insights into the feedback regulation by tyrosine of the Escherichia coli chorismate mutase/prephenate dehydrogenase (CM/PDH), which is encoded by the tyrA gene, feedback-inhibition-resistant (fbr) mutants were generated by error-prone PCR. The tyrA fbr mutants were selected by virtue of their resistance toward m-fluoro-d,l-tyrosine, and seven representatives were characterized on the biochemical as well as on the molecular level. The PDH activities of the purified His6-tagged TyrA proteins exhibited up to 35% of the enzyme activity of TyrAWT, but tyrosine did not inhibit the mutant PDH activities. On the other hand, CM activities of the TyrAfbr mutants were similar to those of the TyrAWT protein. Analyses of the DNA sequences of the tyrA genes revealed that tyrA fbr contained amino acid substitutions either at Tyr263 or at residues 354 to 357, indicating that these two sites are involved in the feedback inhibition by tyrosine.


2008 ◽  
Vol 98 (5) ◽  
pp. 509-518 ◽  
Author(s):  
W.T. Tay ◽  
G.T. Behere ◽  
D.G. Heckel ◽  
S.F. Lee ◽  
P. Batterham

AbstractApplying microsatellite DNA markers in population genetic studies of the pest mothHelicoverpa armigerais subject to numerous technical problems, such as the high frequency of null alleles, occurrence of size homoplasy, presence of multiple copies of flanking sequence in the genome and the lack of PCR amplification robustness between populations. To overcome these difficulties, we developed exon-primed intron-crossing (EPIC) nuclear DNA markers forH. armigerabased on ribosomal protein (Rp) and the Dopa Decarboxylase (DDC) genes and sequenced alleles showing length polymorphisms. Allele length polymorphisms were usually from random indels (insertions or deletions) within introns, although variation of short dinucleotide DNA repeat units was also detected. Mapping crosses demonstrated Mendelian inheritance patterns for these EPIC markers and the absence of both null alleles and allele ‘dropouts’. Three examples of allele size homoplasies due to indels were detected in EPIC markers RpL3, RpS6 and DDC, while sequencing of multiple individuals across 11 randomly selected alleles did not detect indel size homoplasies. The robustness of the EPIC-PCR markers was demonstrated by PCR amplification in the related species,H. zea,H. assultaandH. punctigera.


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