Enzyme variation among clones of Trypanosoma cruzi

The genetic relationships within and between stocks of Trypanosoma cruzi were studied by the in vitro isolation of clones and sub-clones and by the comparison of their isoenzyme patterns in thin-layer starch-gel electrophoresis. Altogether 13 clones and 36 sub-clones were isolated from stocks CL89 and Y207 of T. cruzi. Employing the enzymes L-alanine aminotransferase (ALAT), L-aspartate aminotransferase (ASAT), glucose phosphate isomerase (GPI), malic enzyme (ME), malate dehydrogenase (MDH), glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), and phosphoglucomutase (PGM), two zymodemes, B and C, emerged among the clones with distinct banding patterns. These zymodemes were consistently distinguished by ALAT, ASAT, GPI, G6PD, 6PGD, and PGM and the differences in enzyme profiles were simultaneously reflected in all six enzyme systems. That the enzymic characters as genetic determinants are stable was demonstrated after recloning and successive replica-platings, i.e., the distinct enzyme patterns remained consistent and homogeneous, the siblings retained the enzyme profiles of their parental clones, and no segregation of the enzyme patterns was observed. Our data from clone and sub-clone examinations show that the isoenzymes act as labels to characterize T. cruzi stocks. Furthermore, enzyme variation was demonstrated among clones isolated from stock CL89.     

Broad-scale latitudinal patterns of genetic diversity among native European and introduced house sparrow (Passer domesticus) populations

Introduced species offer unique opportunities to study evolution in new environments, and some provide opportunities for understanding the mechanisms underlying macroecological patterns. We sought to determine how introduction history impacted genetic diversity and differentiation of the house sparrow (Passer domesticus), one of the most broadly distributed bird species. We screened eight microsatellite loci in 316 individuals from 16 locations in the native and introduced ranges. Significant population structure occurred between native than introduced house sparrows. Introduced house sparrows were distinguished into one North American group and a highly differentiated Kenyan group. Genetic differentiation estimates identified a high magnitude of differentiation between Kenya and all other populations, but demonstrated that European and North American samples were differentiated too. Our results support previous claims that introduced North American populations likely had few source populations, and indicate house sparrows established populations after introduction. Genetic diversity also differed among native, introduced North American, and Kenyan populations with Kenyan birds being least diverse. In some cases, house sparrow populations appeared to maintain or recover genetic diversity relatively rapidly after range expansion (<50 years; Mexico and Panama), but in others (Kenya) the effect of introduction persisted over the same period. In both native and introduced populations, genetic diversity exhibited large-scale geographic patterns, increasing towards the equator. Such patterns of genetic diversity are concordant with two previously described models of genetic diversity, the latitudinal model and the species diversity model.     

Phylogeography of the Angolan black and white colobus monkey,Colobus angolesnsis palliatus,in Kenya and Tanzania

Little is known about genetic variation in the 6–8 subspecies of Colobus angolensis, currently distinguished by pelage differences. We present a comparative genetic analysis of one of these subspecies, C. a. palliatus, in Kenya and Tanzania that assesses evolutionary relationships and patterns of mitochondrial genetic diversity in 103 individuals across its geographic range. Fecal samples from approximately 156 individuals were collected in four localities: (1) Diani Forest, Kenya; (2) Shimoni, Kenya; (3) Udzungwa Mountains National Park, Eastern Arc Mountains, Tanzania; and (4) Mount Rungwe, Southern Highlands, Tanzania. These samples represent at least six groups, with 5–15 samples from each. Comparative sequence analysis of a 1,795 base pair mtDNA fragment revealed 19 unique haplotypes in four populations. Phylogenetic analyses suggest that sampled Kenyan haplotypes are paraphyletic, with one Kenyan haplotype basal to all other sampled haplotypes. Analysis of molecular variance (AMOVA) suggests high levels of genetic variation among populations (Φ_ST 0.72, P<0.001). Genetic data are concordant with a subspecies level differentiation between C. a. palliatus populations in Kenya and those in Central and southern Tanzania, as earlier suggested based on pelage differences. This study highlights the evolutionary distinctiveness of Kenyan populations of C. a. palliatus relative to Tanzanian populations. Although C. a. palliatus habitat in Tanzania is currently better protected than in Kenya, our results suggest Kenyan and Tanzanian populations should be considered distinct units, and the protection of C. a. palliatus habitat in Kenya, as well as habitat connectivity between Kenyan populations, should be prioritized for conservation and management. Am. J. Primatol. 72:715–724, 2010. © 2010 Wiley‐Liss, Inc.     

Analysis of genetic heterogeneity among five gynogenetic clones of silver crucian carp, Carassius auratus gibelio Bloch, based on detection of RAPD molecular markers

The gynogenetic silver crucian carp, Carassius auratus gibelio, is a unique model system for studying evolutionary genetics and selective breeding, owing to its specific genetic background and reproductive modes. Five gynogenetic clones were analyzed by the random amplified polymorphic DNA (RAPD) technique, using 30 10-nucleotide-long primers. Twenty-six primers produced well-amplified DNA fragments with reproducible banding patterns, and 24 primers were polymorphic. Nearly identical banding patterns were observed among individuals within each clone, suggesting that each clone might possess a specific pattern owing to its gynogenesis. In contrast, the RAPD patterns of the five clones differed from each other. A phylogenetic tree was constructed using UPGMA cluster analysis based on a total of 3,744 distinguishable fragments (156 per individual). Average genetic distances within and among the five clones clearly indicated their intraclonal homogeneity, interclonal heterogeneity, and phylogenetic relationships. Clones A and P were the most closely related, whereas the most divergence was seen between clone D and clone E or F. A total of 88 polymorphic fragments were scored from 24 primers after excluding bands that were monomorphic for the five clones. Most primers corresponding to the polymorphic fragments amplified reproducible markers specific for one clone or that were shared by two, three, or four clones. Several primers (e.g., Opj-1, Opj-7, and Opp-10) produced abundant banding patterns that could be used to discriminate between the five clones. Markers specific for one or two clones were also identified. The RAPD markers identified in this study will likely benefit evolutionary genetics and selective breeding studies.     

Malic enzyme type VII isoenzyme as an indicator of suramin resistance in Trypanosoma evansi

This study analysed the suramin sensitivity of 29 stocks of Trypanosoma evansi isolated from Egypt, Sudan, and Indonesia and compared the results with the isoenzyme banding patterns of 20 soluble enzymes in these stocks of T. evansi. The results showed that the type VII banding pattern of malic enzyme was found only in T. evansi stocks which were highly resistant to suramin.     

Evidence that Kenyan House Sparrows Passer domesticus invaded from multiple sites

Here we characterize genetic patterns across the range of House Sparrows in Kenya using six microsatellite markers. We screened House Sparrows from two remote locations in northern Kenya, Marsabit (n = 24) and Wajir (n = 27), which are separated from other colonized areas in Kenya by minimally developed, arid habitat, and then compared these birds with House Sparrows in 10 more central and longer established Kenyan cities (n = 233) in this range. House Sparrows from Marsabit and Wajir originated from a separate source, probably Somalia and/or Ethiopia, from other Kenyan House Sparrows, probably Mombasa. Furthermore, the genetic characteristics of northern and southern populations indicate that they have not yet mixed, supporting a hypothesis that the large, minimally (human) developed, arid landscape spanning nearly all of northern Kenya, including the 100 000 km² Chalbi Desert, is a barrier to dispersal for House Sparrows.     

Trypanosoma brucei rhodesiense: characterisation of stocks from Zambia, Kenya, and Uganda using repetitive DNA probes

We have previously described a system for characterising the relationships between trypanosome stocks of the T.brucei group based on Southern blotting with repetitive DNA probes followed by cluster analysis of resultant banding patterns (G. Hide et al. Molec. Bioch. Parasitol. 39, 213-226, 1990). In this study, we extend this analysis to examine the relationships between trypanosome stocks isolated from major sleeping sickness foci in Zambia, Kenya, and Uganda. We show that the trypanosome strains responsible for disease in Zambia are quite distinct from those sampled from the Kenya/Uganda foci. Furthermore, the human serum resistant stocks isolated from the Kenya/Uganda foci which were isolated from man (or from animals) were found to form a tight group in the cluster analysis, while stocks isolated from nonhuman sources in the same area or stocks from elsewhere were found in separate groups. Thus, the human infective trypanosome strains found in these foci may have common origins and have, perhaps, arisen by clonal selection from a common source.     

Diversity and genetic differentiation among populations of Indian and Kenyan tea (Camellia sinensis (L.) O. Kuntze) revealed by AFLP markers

AFLP markers were successfully employed to detect diversity and genetic differentiation among Indian and Kenyan populations of tea (Camellia sinensis (L.) O. Kuntze). Shannon's index of diversity was used to partition the total phenotypic variation into between and within population components. On average, most of the diversity was detected within populations, with 79% of the variation being within and 21% being between populations of Indian and Kenyan tea. A dendrogram constructed on the basis of band sharing distinctly separated the three populations of tea into China type (sinensis), Assam type (assamica) and Cambod type (assamica ssp. lasiocalyx) in a manner consistent with the present taxonomy of tea, the known pedigree of some of the genotypes and their geographical origin. Principal coordinate (PCO) analysis grouped Assam genotypes both from India and Kenya supporting the suggestion that the Kenyan clones have been derived from collections made in this region. The China types were more dispersed on the PCO plot which is a reflection of wider genetic variation. As would be expected, clones collected from the same region exhibited less overall genetic variation. AFLP analysis discriminated all of the tested genotypes from India and Kenya, even those which cannot be distinguished on the basis of morphological and phenotypic traits. Received: 2 May 1996 / Accepted: 14 June 1996     

Variability of Colletotrichum kahawae in Relation to Other Colletotrichum Species from Tropical Perennial Crops and the Development of Diagnostic Techniques

Twenty‐six isolates of Colletotrichum kahawae, the causal agent of coffee berry disease, from coffee in Africa, and 25 isolates, mostly of Colletotrichum gloeosporioides, from coffee and other tropical perennial crops, were examined for the ability to metabolize citrate and tartrate and their molecular genetic variability was assessed using restriction fragment length polymorphisms (RFLP) and variable number tandem repeats (VNTR). Twenty‐four isolates of C. kahawae were also assessed using amplified fragment length polymorphisms (AFLP). Vegetative compatibility within a collection of nine isolates, including two of C. gloeosporioides was also assessed. All isolates of C. kahawae from across Africa failed to metabolize citrate or tartrate, but all other isolates metabolized one or both. Colletotrichum kahawae isolates also showed minimal variability using the molecular techniques with two isolates from Cameroon showing slightly different banding patterns in RFLP analysis. All other isolates had variable VNTR and RFLP banding patterns. AFLP analysis failed to detect variability within 12 isolates from Kenya, but did detect differences between isolates from other countries. Five isolates from Kenya were vegetatively compatible but differed from two from Cameroon and from two C. gloeosporioides isolates. Results demonstrate some geographic variability within C. kahawae isolates, although this is small, probably due to the relatively young age of C. kahawae populations. The biochemical and molecular techniques used showed clear differences from other Colletotrichum isolates, and can be used to distinguish the species. Lack of citrate and tartrate metabolism provides a readily applicable diagnostic method.     

5S ribosomal gene clusters in wheat: pulsed field gel electrophoresis reveals a high degree of polymorphism

Summary The long-range structure of 5S rRNA gene clusters has been investigated in wheat (Triticum aestivum L.) by means of pulsed field gel electrophoresis. Using aneuploid stocks, 5S rRNA gene clusters were assigned to sites on chromosomes 1B, 1D, 513 and 5D. Cluster sizes were evaluated and the copy number of 5S DNA repeats was estimated at 4700-5200 copies for the short repeating unit (410 bp) and about 3100 copies for the long repeat (500 bp) per haploid genome. A comparison of wheat cultivars revealed extremely high levels of polymorphism in the 5S rRNA gene clusters. With one restriction enzyme digest all varieties tested gave unique banding patterns and, on a per fragment basis, 21-fold more polymorphism was detected among cultivars for 5S DNA compared to standard restriction fragment length polymorphisms (RFLPs) detected with single copy clones. Experiments with aneuploid stocks suggest that the 5S rRNA gene clusters at several chromosomal sites contribute to this polymorphism. A number of previous reports have shown that wheat cultivars are not easily distinguished by isozymes or RFLPs. The high level of variation detected in 5S rRNA gene clusters therefore offers the possibility of a sensitive fingerprinting method for wheat. 5S DNA and other macro-satellite sequences may also serve as hypervariable Mendelian markers for genetic and breeding experiments in wheat.     

Molecular and morphologic approaches to discrimination of variability patterns in chub mackerel, Scomber japonicus

The systematic status and the evolutionary biology of chub mackerel (Scomber japonicus) in the South West Atlantic Ocean is confusing with an unknown degree of genetic differentiation and reproductive isolation between units. Simultaneous genetic and morphologic analyses were made on 227 fish collected from two areas of the South West Atlantic Ocean and one from the Mediterranean Sea. The genetic analysis was based on 36 protein-coding loci, 16 of which were variable. The morphologic analyses include six morphometric length measurements and a meristic character. Correspondence between genetic and morphologic variability patterns indicates isolated Mediterranean and Southwest Atlantic subgroups of S. japonicus and, less clearly, possible additional divergence in two regional stocks within the latter group. The most conservative approach to management is to manage the stocks independently of one another.     

Strain selection and genetic variation in Gracilaria chilensis (Gracilariales, Rhodophyta)

Strain selection processes in seaweed often have assumed that sterile clones could be maintained for long periods in a diversity of environments without major genetic changes. However, clonal species such as Gracilaria chilensis exhibit intra-clonal variation in performance and ongoing studies suggest such changes may be due to rapid changes in DNA composition associated with growth, via mitotic recombinations. Therefore performance of a given ramet in this type of seaweed should be understood as the dynamic outcome of rapid reactions between the environment and the changing genotype of the selected strain. To evaluate this idea, we measured changes in genetic variability, as detected by DNA-fragment polymorphism using RAPDs-PCR, exhibited by clones of G. chilensis after two transfers to different environmental conditions (from field to controlled laboratory conditions and from the laboratory to large-scale tank culture). The transfer to laboratory conditions reduced the frequency of low similarity values and increased the frequency of intermediate similarity values in DNA banding patterns, suggesting the branchlets produced under controlled laboratory conditions have less genetic variability (evaluated as total DNA polymorphism) than plants recently collected in the field. Tank incubation reduced the total range of similarity and significantly increased the frequency of high similarity values. Results thus suggest the dynamic of genetic changes in vegetative clones of Gracilaria chilensis that is fast and strongly affected by the external environment. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic Diversity of Human Enterovirus 68 Strains Isolated in Kenya Using the Hypervariable 3′- End of VP1 Gene

Reports of increasing worldwide circulation of human enterovirus-68 (EV68) are well documented. Despite health concerns posed by resurgence of these viruses, little is known about EV68 strains circulating in Kenya. In this study, we characterized 13 EV68 strains isolated in Kenya between 2008 and 2011 based on the Hypervariable 3′- end of the VP1 gene. Viral RNA was extracted from the isolates and partial VP1 gene amplified by RT-PCR, followed by nucleotide sequencing. Alignment of deduced amino acid sequences revealed substitutions in Kenyan EV68 isolates absent in the prototype reference strain (Fermon). The majority of these changes were present in the BC and DE-loop regions, which are associated with viral antigenicity and virulence. The Kenyan strains exhibited high sequence homology with respect to those from other countries. Natural selection analysis based on the VP1 region showed that the Kenyan EV68 isolates were under purifying selection. Phylogenetic analysis revealed that majority (84.6%) of the Kenyan strains belonged to clade A, while a minority belonged to clades B and C. Overall, our results illustrate that although EV68 strains isolated in Kenya were genetically and antigenically divergent from the prototype strain (Fermon), they were closely related to those circulating in other countries, suggesting worldwide transmissibility. Further, the presence of shared mutations by Kenyan EV68 strains and those isolated in other countries, indicates evolution in the VP1 region may be contributing to increased worldwide detection of the viruses. This is the first study to document circulation of EV68 in Kenya.     

INTRASPECIFIC VARIATION IN THE DINOFLAGELLATE GAMBIERDIZSCUS TOXICUS (DINOPHYCEAE). I. ISOZYME ANALYSIS

Intraspecific variation among 19 isolates of the ciguatera-causing dinoflagellate Gambierdiscus toxicus Adachi & Fukuyo (Dinophyceae) collected from French Polynesia, New Caledonia, and the French West Indies was investigated by isozyme analysis. Comparison of their cell sizes and growth rates revealed that significant variation exists among these clones. Comparison of electrophoretic patterns for seven enzyme systems indicated that G. toxicus is comprised of numerous biochemically distinct strains. Isolates from Tubuai and Hao appeared to be the most distantly related. Tahitian strains of G. toxicus also showed a remarkably low degree of similarity with the Tubuai isolates. The latter, which were taken from the same locale in Tubuai, also exhibited highly heterogeneous electrophoretic Profiles when compared to each other, suggesting a multiclonal origin. The single isolate analyzed from the Atlantic Ocean was most closely related to Tahitian isolates, despite their geographic separation. Finally, no clear relationship was found between the electrophoretic profiles of these isolates and their capacity to produce ciguatoxic compounds .     

Characterization by electrophoretic zymograms of 19 Trypanosoma cruzi clones derived from two chronic chagasic patients

1. Electrophoretic patterns of aspartate aminotransferase, glucose-6-phosphate dehydrogenase, phosphoglucomutase, glucose-phosphate isomerase, malic enzyme and alcohol dehydrogenase have been analyzed in extracts from Trypanosoma cruzi, Tulahuén strain, 19 clones derived from isolates obtained from two chronic chagasic patients from Argentina and from Brazilian stocks Silvio X10/1 (zymodeme 1), Esmeraldo/1 (zymodeme 2), and CAN-III/1 (zymodeme 3). 2. The clones isolated from one of the patients were genetically heterogeneous. 3. Phosphoglucomutase and glucose phosphate isomerase patterns for the clones analyzed clearly differ from those of the Brazilian stocks. 4. Grouping of clones on the basis of isozyme patterns showed some correlation with that based on total DNA per organism. 5. Under the experimental conditions used, the polyacrylamide gel electrophoresis micromethod employed was advantageous over starch gel electrophoresis.     

Phylogenetic relationships between cultivated and wild species of the genusBeta revealed by DNA “fingerprinting”

Forty-one accessions of the genusBeta representing wild and cultivated species of all sections were analyzed by DNA fingerprinting. Four sugar beet minisatellite DNA probes revealed characteristic banding patterns with Southern-hybridizedBeta DNA restricted withHindIII. A total of 111 polymorphic RFLP bands were scored across all accessions. Cluster analysis based on genetic similarity estimates for all 820 combinations of accessions revealed the following results. (1) All accessions could unambiguously be identified by a characteristic RFLP banding pattern. (2) The sugar beet cultivars examined displayed a low level of genetic diversity; they showed high similarity toB. Vulgaris ssp.maritima but low genetic similarity to the other wild species of section I. (3) In most cases, the present taxonomic classification of the genusBeta was confirmed. Species of sections II, III, and IV were clearly distinguishable from those of section I except forB. Macrocarpa, which showed high similarity to wild species of section II. In a second experiment, 108 single-copy RFLP probes from sugar beet were Southern hybridized withB. procumbens DNA. A surprisingly low degree of homology (34%) was found. The results are discussed with regard to the taxonomic classification of the genusBeta.     

Isozyme electrophoresis patterns of the liver fluke, Clonorchis sinensis from Kimhae, Korea and from Shenyang, China

An enzyme analysis of the liver fluke, Clonorchis sinensis from Kimhae, Korea and from Shenyang, China was conducted using a horizontal starch gel electrophoresis in order to elucidate their genetic relationships. A total of eight enzymes was employed from two different kinds of buffer systems. Two loci from each enzyme of aconitase and esterase (alpha-Na and beta-Na); and only one locus each from six enzymes, glucose-6-phosphate dehydrogenase (G6PD), alpha-glycerophosphate dehydrogenase (GPD), 3-hydroxybutyrate dehydrogenase (HBDH), malate dehydrogenase (MDH), phosphoglucose isomerase (PGI), and phosphoglucomutase (PGM) were detected. Most of loci in two populations of C. sinensis showed homozygous monomorphic banding patterns and one of them, GPD was specific as genetic markers between two different populations. However, esterase (alpha-Na), GPD, HBDH and PGI loci showed polymorphic banding patterns. Two populations of C. sinensis were more closely clustered within the range of genetic identity value of 0.998-1.0. In summarizing the above results, two populations of C. sinensis employed in this study showed mostly monomorphic enzyme protein banding patterns, and genetic differences specific between two populations.     

Variation between human and animal isolates of Giardia as demonstrated by DNA fingerprinting

Five Giardia stocks collected from animals and man in Alberta, Canada, were compared by DNA fingerprinting. Although many DNA bands were common to all stocks, differences in the DNA banding patterns were seen. These same stocks had previously been shown to be identical by restriction enzyme cleavage of genomic DNA.     

Evaluation of ribosomal RNA gene restriction patterns for the classification of Bacillus species and related genera

AIMS: To identify Bacillus species and related genera by fingerprinting based on ribosomal RNA gene restriction patterns; to compare ribosomal RNA gene restriction patterns-based phylogenetic trees with trees based on 16S rRNA gene sequences; to evaluate the usefulness of ribosomal RNA gene restriction patterns as a taxonomic tool for the classification of Bacillus species and related genera. METHODS AND RESULTS: Seventy-eight bacterial species which include 42 Bacillus species, 31 species from five newly created Bacillus-related genera, and five species from five phenotypically related genera were tested. A total of 77 distinct 16S rRNA gene hybridization banding patterns were obtained. The dendrogram resulting from UPGMA analysis showed three distinct main genetic clusters at the 75% banding pattern similarity. A total of 77 distinct 23S and 5S rRNA genes hybridization banding patterns were obtained, and the dendrogram showed four distinct genetic clusters at the 75% banding pattern similarity. A third dendrogram was constructed using a combination of the data from the 16S rRNA gene fingerprinting and the 23S and 5S rRNA genes fingerprinting. It revealed three distinct main phylogenetic clusters at the 75% banding pattern similarity. CONCLUSIONS: The Bacillus species along with the species from related genera were identified successfully and differentiated by ribosomal RNA gene restriction patterns, and most were distributed with no apparent order in various clusters on each of the three dendrograms. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data indicate that ribosomal RNA gene restriction patterns can be used to reconstruct the phylogeny of the Bacillus species and derived-genera that approximates, but does not duplicate, phylogenies based on 16S rRNA gene sequences.