Although use of cytochromeb has some pitfalls, the cytb gene still is the most widely used gene for phylogenetic analysis for taxonomic, especially are good tools for studying phylogenetics of closely re- lated species . Within species, control region se- quences usually are a better choice, because more re- laxed structural and functional constraints lead to a faster average substitution rate. In this study, the molecular phylogenetic trees were reconstructed based on cytb data of Takydromus. The result sup- ported that the Platyplacopus merged into Takydromus and negated the validity of Platyplacopus. It is also consistent with the opinions of Arnold et al. [21, 22] and Lin et al. [8, 23], whom supported the combina- tion of subgenera Platyplacopus and Takydromus into Takydromus.
Mitochondrial cytochromeb (cytb) genes of 42 strains representing 23 species of the genus Trichosporon were partially sequenced to determine their molecular phylogenetic relationships. Almost half of the 22 strains investigated (from 11 different species) contained introns in their sequences. Analysis of a 396-bp coding sequence from each strain of Trichosporon under investigation showed a total of 141 (35.6%) variable nucleotide sites. A phylogenetic tree based on the cytb gene sequences revealed that all species of Trichosporon except Trichosporon domesticum and Trichosporon montevideense had species-specific cytb genes. Trichosporon sp. strain CBS 5581 was identified as Trichosporon pullulans, and one clinical isolate, IFM 48794, was identified as Trichosporon faecale. Analysis of 132-bp deduced amino acid sequences showed a total of 34 (25.75%) variable amino acid sites. T. domesticum and T. montevideense, Trichosporon asahii and Trichosporon asteroides, and Trichosporon gracile and Trichosporon guehoae had identical amino acid sequences. A phylogenetic tree con- structed with the ascomycetes Saccharomyces douglasii and Candida glabrata taken as outgroup species and including representative species from closely related genera species of Trichosporon clustered with other basidiomycetous yeasts that contain xylose in their cell wall compositions. These results indicate the effec- tiveness of mitochondrial cytb gene sequences for both species identification and the phylogenetic analysis of Trichosporon species.
Mitochondrial cytochromeb genes (cytb) of 40 strains of Cryptococcus neoformans were partially sequenced to determine the genetic relations. With the exception of the type strain of C. neoformans var. neoformans, all strains contained introns in their sequences. Analysis of 386 bp of coding sequence from each strain under investigation revealed a total of 27 (6.99%) variable nucleotide sites and categorized isolates of C. neoformans into nine cytb types. C. neoformans var. gattii included cytb types I to V, and C. neoformans var. neoformans comprised types VI to IX. cytb types were correlated with serotypes. All strains with cytb types I, IV, and V were serotype B. All other strains except IFM 5878 (serotype B) with cytb types II and III were serotype C. Serotype D strains had cytb types VI and IX, and serotype A strains were cytb type VIII. Of four serotype AD strains, one was cytb type VII and the remaining three were type VIII. The phylogenetic tree based on deduced amino acid sequences divided the strains only into C. neoformans var. neoformans and C. neoformans var. gattii. These results indicate that cytb sequences are effective for DNA typing as well as phylogenetic analysis of C. neoformans.
The phylogeny and taxonomy of mammalian species were originally based upon shared or derived morphological characteristics. However, genetic analyses have more recently played an increasingly important role in confirming existing or establishing often radically different mammalian groupings and phylogenies. The two most commonly used genetic loci in species identification are the cytochrome oxidase I gene (COI) and the cytochromeb gene (cytb). For the first time this study provides a detailed comparison of the effectiveness of these two loci in reconstructing the phylogeny of mammals at different levels of the taxonomic hierarchy in order to provide a basis for standardizing methodologies in the future. Interspecific and intraspecific variation is assessed and for the first time, to our knowledge, statistical confidence is applied to sequence comparisons. Comparison of the DNA sequences of 217 mammalian species reveals that cytb more accurately reconstructs their phylogeny and known relationships between species based on other molecular and morphological analyses at Super Order, Order, Family and generic levels. Cytb correctly assigned 95.85% of mammal species to Super Order, 94.31% to Order and 98.16% to Family compared to 78.34%, 93.36% and 96.93% respectively for COI. Cytb also gives better resolution when separating species based on sequence data. Using a Kimura 2-parameter p-distance (x100) threshold of 1.5–2.5, cytb gives a better resolution for separating species with a lower false positive rate and higher positive predictive value than those of COI.
Abstract: The study was conducted to adopt PCR based technique for identification of species origin from meat samples of cattle and buffalo using mitochondrial cytochromeb (Cytb) gene fragment. A total of 42 ear tissue and meat samples were collected from different slaughterhouses and farms of Mymensingh, Bogra and Rangpur districts and stored in 96% ethanol at room temperature. Genomic DNA was extracted from all samples using GeNet Bio genomic DNA isolation kit. The average DNA yield of considered samples was found 204.57 ng/µl where the purity ranged from 1.82–1.99. Two (2) pair species-specific primers were used to amplify Cytb gene fragments of 472 bp and 124 bp for cattle and buffalo, respectively. The PCR results revealed different species specific amplified fragments which could discriminate between cattle (472 bp) and buffalo (124 bp) species precisely from pure and mixed samples of those species. This study suggests an accurate molecular technique for identification of cattle and buffalo species meat origin and differentiates species present in adulterated meat samples. In conclusion, this DNA based technique could be utilized for prevention of malpractice in slaughterhouse and chain shops and thereby to protect consumer’s right.
(PCR), using primer sets (L14724 and H15149 and; the internal primers, L15162 and H15915) designed by Irwin et al (1991). All PCRs 10 µl of reaction mixture contained approximately 100ng of genomic DNA, 0.5 µM of each primer, 0.2 mM dNTPs, 1.5 mM MgCl, and 2.5 units of Tag DNA polymerase. PCR thermal cycle profiles were as follows: a pre-denaturing step of 94 0 C for 5 min; denaturing step of 94 0 C for 1 min, annealing at 50 0 C for 1 min, and extension at 72 0 C for 2 min (35 cycles); plus a final extension at 72 0 C for 10 min. The PCR products were stored at 4 0 C. To remove primers and unincorporated nucleotides, amplified products were purified using QIAquick PCR purification kit (QIAGEN, USA). For sequencing, the purified PCR products were analyzed with an automated DNA sequencing machine (ABI 3770) in Korea. We used Network 4.6 version to identify the number of haplotypes from our new sequences. Published sequences for cytochromeb were retrieved from Genbank (Table 1) and aligned to our new sequences using the Codon code aligner and MEGA (version 5.05).
We compared trees obtained by UPGMA and other meth- ods (NJ, ML, and MP methods) assuming no constancy of the evolutionary rate. We preferred the tree obtained by UPGMA for two reasons. First, mt DNA is a favored molecule to be used as a molecular clock for molecular phylogenetic studies (32, 37). The rate of substitution of bases in cytochromeb genes is in proportion to evolutionary time. If the distance measure used is exactly linear with evolutionary time, that is, the evolutionary rate is constant or mt is used as a molecular clock, UPGMA gives the correct topology and correct branch lengths. Therefore, in this case, UPGMA was advocated for use in reconstructing phylogenetic trees (25, 26, 28). Second, by UPGMA, section Fumigati constituted a distinct cluster from A. flavus, A. niger, A. terreus, and A. nidulans. A. flavus and A. niger were very closely related in the tree obtained by UPGMA. Samson (33) suggested that these two species are also morphologically closely related and that they could be
F igure 5.—Northern analysis of COB mRNA in a Dcbt1 strain with no precursor form of COB. Approximately 5 mg of mitochondrial RNA was loaded onto a 1.25% agarose gel, electrophoresed, blotted onto Nytran, and probed sepa- rately with the various probes. Blots were analyzed on a Phos- phorimager. The ATP9 signal was used as a loading control for quantitation purposes.TG955 strains have cytochromeb mRNA precursor sequence deleted from ÿ1098 to ÿ956 (where the A of the initiating ATG is 11). Levels of COB mRNA were quan- tified relative to LL20 as follows: LL20, 100%; LL20Dcbt1, 16.4%; TG955, 116%; and TG955 D cbt1, 2%.
Affinity-purified rabbit anti-neutrophil cytochromeb light or heavy chain antibodies were used to immunocytochemically and biochemically localize cytochromeb in neutrophils and eosinophils. The antibodies were monospecific, recognizing polypeptides of 91 and 22 kD, respectively, on Western blots of whole neutrophil extracts. The antibodies were used in Western blot analysis of subcellular fractions of purified neutrophils to confirm that the distribution of cytochromeb spectral absorbance matched that of the two subunits. Thin sections of cryofixed, molecular distillation-dried granulocytes were labeled with the anti- cytochromeb antibodies, followed by incubation with biotin-conjugated secondary antibody, and final labeling with streptavidin-conjugated colloidal gold. Electron microscopy revealed that the cytochromeb light and heavy chains were localized primarily (80%) to 0.1-0.2- micron round or elliptical granule-like structures in neutrophils and 0.4-0.5-micron granules in eosinophils. Approximately 20% of the cytochromeb was localized to the surface,
(Criado et al., 2006) which makes sensitivity of the assay low. Therefore, the present study was carried out to develop PCR assay by targeting cytochromeb of B. gibsoni as a diagnostic target, which is present in more copies in an organism. The primers were designed based on available sequences and PCR was first standardized with microscopic positive B. gibsoni samples. The sensitivity of the cytb gene based PCR to detect B. gibsoni was 10 -2 (0.5ng/µl) for blood (50µl) with a parasitaemia of 3.17%. Whereas, with 18S PCR assay the specific amplification was observed at lower template dilution of 10 -1 (2.5ng/µl), which confirming the greater sensitivity of the cytb based PCR method than 18S. Similar results have been reported for B. bovis and B. bigemina (Salem et al., 1999) and for Theileria annulata detected to the dilution representing a parasitaemia of 10 -4 based on PCR of cytb gene (Bilgic et al., 2010).
A membrane-bound cytochromeb, a heterodimer formed by a 91-kD glycoprotein and a 22- kD polypeptide, is a critical component of the phagocyte NADPH-oxidase responsible for the generation of superoxide anion. Mutations in the gene for the 91-kD chain of this cytochrome result in the X-linked form of chronic granulomatous disease (CGD), in which phagocytes are unable to produce superoxide. Typically, there is a marked deficiency of the 91-kD subunit and the cytochrome spectrum is absent (X- CGD). In a variant form of CGD with X-linked inheritance, affected males have a normal visible absorbance spectrum of cytochromeb, yet fail to generate superoxide (X+ CGD). The size and abundance of the mRNA for the 91-kD subunit and its encoded protein were examined and appeared normal. To search for a putative mutation in the coding sequence of the 91-kD subunit gene, the corresponding RNA from an affected X+ male was amplified by the polymerase chain reaction and sequenced. A single nucleotide change, a C----A transversion, was identified that predicts a nonconservative Pro----His substitution at residue 415 of the encoded
Using a molecular clock equation ( λ = d/2t); where λ = substitution rate/site/year, d = mean distance between individuals within groups or mean distance between groups and t = divergence time), the above distances and employing the substitution rate (1 × 10 −8 substitution/site/year) for cytochromeb in mammalian species . The European-Asian wild boar divergence is estimated to have occurred 500000 YBP  or 746000 YBP . Here we found that the Vietnamese wild boars of group II have latest diverged from Asia wild boars (28000 YBP). The Vietnamese domestic pigs and Vietnamese wild boars of group I were earlier diverged than Viet- namese wild boars of group II (70500 YBP and 385500 YBP). The genetic diversity between Vietnamese wild boars of group I and group II and domestic pigs would reflect genetic differences of the ancestor population. Moreover, the divergence time between Vietnamese wild boar of group I and European wild boar is 421500 YBP. Therefore, the European wild boars and the Vietnamese wild boars of group I could have common ances- tor.
Plasmodium falciparum in vitro resistance to atovaquone has been associated with specific point mutations in the cytochromeb gene (Pfcytb) in the region spanning codons 271–284 [7,12]. A high frequency of recrudescence was observed in patients receiving atovaquone as a single drug therapy against P. falciparum [13,14]. A Y268S point mutation in the Pfcytb gene, distinct from the mutations observed in the lines selected in vitro for atovaquone resistance, was detected in the recrudescing parasites . Codon 268 polymorphism was used as marker for a molecular surveillance of atovaquone-proguanil resist- ance [15-19]. AP treatment failures were increasingly reported a few years after its introduction, with recrudesc- ing parasites presenting a markedly increased IC 50 for atovaquone [15,20,21]. In most cases, recrudescence was associated with a mutant 268 codon, either a Y268S [3,12,20,22-26], a Y268N  or a Y268C mutation . However, the presence of a mutant 268 codon was not observed in all cases of AP failure [21,27].
BIRD, which could be solved using primers designed for bird species, was not successful. For the mammal spe- cies, the BLAST analysis did not match a reference sequence based in our threshold (≥98%). At the time of the manuscript writing, Genbank and BOLD did not contain COI reference sequences for capybara or for Pampas deer. Although the COI gene is considered the DNA barcoding gene , reference sequences for several species are still unavailable in these genetic databases. Researchers have used the cytochromeb gene for species identifications [5,19] since it is one of the better repre- sented genes in GenBank  and has superior ability for separating species when compared with COI [20,21]. Table 1 Cyt- b reference sequences of species that would likely be hunted or consumed at the seizure region
The natural infection of phlebotomine sand flies by Leishmania parasites was surveyed in a desert area of Pakistan where cutaneous leishmaniasis is endemic. Out of 220 female sand flies dissected, one sand fly, Phlebotomus kazer- uni, was positive for flagellates in the hindgut. Analyses of cytochromeb (cytb), glycosomal glyceraldehyde phos- phate dehydrogenase (gGAPDH) and small subunit ribosomal RNA (SSU rRNA) gene sequences identified the parasite as a Trypanosoma species of probably a reptile or amphibian. This is the first report of phlebotomine sand flies naturally infected with a Trypanosoma species in Pakistan. The possible infection of sand flies with Trypano- soma species should be taken into consideration in epidemiological studies of vector species in areas where leish- maniasis is endemic.
rearrangements); this should be considered in the design of studies. The lizard genus Liolaemus is widely distributed in southern South America and includes more than 250 described species. The number of taxa and the distribution of Liolaemus species/populations makes them a good model for testing different hypotheses in systematics. Methods: We studied two Liolaemus species, Liolaemus nigroviridis and L. monticola as focal species to evaluate their monophyly and the influence of adding new samples from related taxa in the resulting phylogenies. We performed phylogenetic analyses (maximum likelihood and Bayesian inference) using 141 sequences of the mitochondrial DNA Cytochromeb (cyt-b) of 11 Liolaemus species.
Fig. 1. enzymatic activity of NaDh-cytochromeb 5 reductase in microsomal fraction of Guerin’s carcinoma under administration of ω-3 PUFAs. Hereinafter: I – animals inoculated with Guerin’s carcinoma (experi- mental control); II – animals that were administered ω-3 PUFAs prior to and post-Guerin’s carcinoma injec- tion; III – animals that were administered ω-3 PUFAs prior to Guerin’s carcinoma injection; IV – animals that were adminis tered ω-3 PUFAs post-Guerin’s carcinoma injection. * Denotes differences significant in comparison to group I (P ≤ 0.05); # denotes differences significant in comparison to the previous stage of carcinoma development in the same group (P ≤ 0.05)
Results: The complete mt genome of B. orientalis (Wuhan strain) was sequenced and characterized. The entire mt genome is 5996 bp in length with a linear form, containing three protein-coding genes including cytochrome c oxidase I (cox1), cytochromeb (cob) and cytochrome c oxidase III (cox3) and six rRNA large subunit gene fragments. The gene arrangement in B. orientalis mt genome is similar to those of B. bovis, B. gibsoni and Theileria parva, but different from those of T. orientalis, T. equi and Plasmodium falciparum. Comparative analysis indicated that cox1 and cob genes were more conserved than cox3. Phylogenetic analysis based on amino acid sequences of cox1, cob and cox1 + cob, respectively, revealed that B. orientalis fell into Babesia clade with the closest relationship to B. bovis. Conclusions: The availability of the entire mt genome sequences of B. orientalis provides valuable information for future phylogenetic, population genetics and molecular epidemiological studies of apicomplexan parasites.