Genetic Variation in Nacobbus aberrans: An Approach toward Taxonomic Resolution

Biochemical and molecular analyses of genetic variation were evaluated to address the taxonomic status of Nacobbus aberrans. Isolates from Mexico, Peru, and Argentina, cultured on tomato in the greenhouse, were analyzed with respect to isozyme and DNA marker variation. Although acid phosphatase and malate dehydrogenase revealed distinct profiles for each isolate, non-specific esterases revealed possible affinities between the Peruvian isolates and between the isolates from Mexico and Peru. Two of l 0 RAPD primers revealed affinities suggested by esterase profiles. RFLP analysis of the rDNA repeating unit with six restriction enzymes revealed identical cleavage patterns between the Peru isolates and a distinct profile shared by isolates from Mexico and Argentina. Nucleotide sequence analysis of the 5.8S rRNA coding region revealed differences among the four isolates at eight of 157 positions; sequences of the Peruvian isolates differed from each other at only one position, whereas the Mexican and Argentine isolates were identical and could be distinguished from the Peruvian isolates. A distance matrix from unweighted pairwise comparisons of the 5.8S rDNA revealed apparent elevated intraspecific divergence in N. aberrans comparable to intergeneric divergence between Heterodera and Globodera. Analysis of additional N. aberrans isolates from throughout the distribution range should help determine the full extent of intraspecific genetic variation that underlies the phenotypic and morphologic diversity of the genus.     

Mitochondrial gene divergence of Colombian Drosophila pseudoobscura

Isolated populations of drosophila pseudoobscura, separated from North American populations by about 2,400 km, were found in Colombia in 1960. We compared for sequences of the small ribosomal RNA (srRNA) gene on the mitochondria between North American and Colombian D. pseudoobscura in order to clarify the age of the Colombian isolates. The North American populations were not genetically different from each other but were genetically different from the Colombian populations. The Mexican strains represent the area from which the Colombian founders might have come. The estimated net nucleotide divergence between Mexican and Colombian D. pseudoobscura indicates that the Colombian population is not an ancient lineage. Phylogenies using both distance and parsimony methodologies reinforced this conclusion. The Colombian samples group together with both methods but, according to the bootstrap analysis, not significantly. It appears that the populations have not been separated long enough for their DNA sequences to show much divergence.      

Genomic DNA sequence polymorphism of Trypanosoma (Schizotrypanum) cruzi isolates with phenotypic variation

1. Single-copy DNA sequences were analyzed by molecular hybridization to determine both the extent of complementarity and time of evolutionary divergence between three isolates obtained from Argentina, Mexico or Venezuela. 2. The Mexico-Venezuela pair appeared much closer than the Mexico-Argentina pair. 3. The tentative values for time of evolutionary divergence were higher than those estimated previously by allozymic variation.     

Molecular differentiation and phylogeny of entomopathogenic nematodes (rhabditida: heterorhabditidae) based on ND4 gene sequences of Mitochondrial DNA.

We determined partial ND4 gene sequences of mitochondrial DNA from 15 heterorhabditid nematode isolates, representing 5 species collected from different regions of the world, by using polymerase chain reaction (PCR) and direct-sequencing of PCR products. Aligned nucleotide as well as amino acid sequences were used to differentiate nematode species by comparing sequence divergence and to infer phylogeny of the nematodes by using maximum parsimony and likelihood methods. Robustness of our phylogenetic trees was checked by bootstrap tests. The 15 nematode isolates can be divided into 7 haplotypes based on DNA sequences. On a larger scale, the sequence divergence revealed 4 distinct groups corresponding to 4 described species. No sequence divergence was detected from 5 isolates of Heterorhabditis bacteriophora or between Heterorhabditis marelatus to Heterorhabditis hepialius. Our sequence data yielded phylogenetic trees with identical topologies when different tree-building methods were used. Most relationships were also confirmed by using amino acid sequences in maximum parsimony analysis. Our molecular phylogeny of Heterorhabditis species support an existing taxonomy that is based largely on morphology and the sequence divergence of the ND4 gene permits species identification.     

DNA Sequence Comparison among Closely Related Drosophila Species in the MULLERI Complex

DNA hybridization was used to establish DNA sequence relationships among seven Drosophila species. Single-copy DNA was isolated from four species within the Drosophila mulleri complex, D. mojavensis, D. arizonensis, D. ritae and D. starmeri. These single-copy DNAs were used as tracers to be hybridized with each other and one additional member of the mulleri complex, D. aldrichi, a member of a closely related complex, D. hydei, and a distantly related species, D. melanogaster. Two methods have been used to determine the relatedness between these species: (1) the extent of duplex formed as measured by binding to hydroxyapatite and (2) the thermal stability of the duplexed DNA. Moderately repetitive DNA was purified from these species and used similarly to determine the divergence of this family of sequences. The rate of nucleotide substitution was estimated to be 0.2 +/-, 0.1% base pair change per million years for both single-copy and middle-repetitive DNAs. The size of the D. arizonensis genome, a representative of the mulleri complex, was calculated to be 2.2 X 10(8) base pairs from its kinetic complexity similar to that of D. hydei. The relative amounts (18%) and average reiteration frequency (100 copies) of the middle-repetitive DNA are similar for all Drosophila species studied. Finally, the data are presented in a phylogenetic tree.     

The divergence between human and baboon globin genes.

Complementary DNA (cDNA) was prepared with viral RNA-directed DNA polymerase from purified baboon globin messenger RNA (mRNA). Homologous and heterologous hybrids between human and baboon mRNAs and cDNAs were compared for extent of hybridisation and thermal stability. Higher mRNA inputs to the hybridizations were required to reach saturation in the heterologous cases. The melting temperature of the heterologous hybrid was 5 degrees C lower than the homologous hybrid. Between these two primates, divergence has occurred in the globin gene to a smaller extent than that possible from third position changes in the coding sequences of the divergence of total DNA. Globin cDNA prepared from baboon will not in general be useful as a probe for human globin mRNA or human globin gene sequences.     

Molecular cloning and physical mapping of the tupaia herpesvirus genome.

Purified virion DNA of about 200 kilobase pairs of tupaia herpesvirus strain 2 was cleaved with EcoRI or HindIII restriction endonuclease. Restriction fragments representing the complete viral genome including both termini were inserted into the EcoRI, HindIII, and EcoRI-HindIII sites of the bacterial plasmid pAT153. Restriction maps for the restriction endonucleases EcoRI and HindIII were constructed with data derived from Southern blot hybridizations of individual viral DNA fragments or cloned DNA fragments which were hybridized to either viral genome fragments or recombinant plasmids. The analysis revealed that the tupaia herpesvirus genome consists of a long unique sequence of 200 kilobase pairs and that inverted repeat DNA sequences of greater than 40 base pairs do not occur, in agreement with previous electron microscopic data. No DNA sequence homology was detectable between the tupaia herpesvirus DNA and the genome of murine cytomegalovirus, which was reported to have a similar structure. In addition, seven individual isolates of tupaia herpesvirus were characterized. The isolates can be grouped into five strains by their DNA cleavage patterns.     

Nature of the heterogeneity in mispairing of reannealed middle-repetitive fern DNA

The genome of the homosporous fern Thelypteris normalis contains a large middle-repetitive component, essentially a single second-order kinetic class, which exhibits heterogeneity in the precision of pairing of the reformed duplexes upon remelting. There are two possible models to explain this observed sequence heterogeneity. Either different families of the middle-repetitive class exhibit different degrees of sequence divergence and T_m reduction (inter familial heterogeneity), or else all are equally diverged, and the broad melt is the sum of thousands of equally broad melts for all the families (intra familial heterogeneity). To differentiate between these two hypotheses, iodinated Thelypteris DNA, reannealed through middle-repetitive C₀t, was thermally fractionated on hydroxyapatite into low (65–75° C), medium (75–85° C), and high (85–95° C) thermal stability classes. When reannealed with excess cold DNA, each class remelted over its characteristic temperature range. C₀t curves of these thermal fractions reannealed with cold driver demonstrated that all were from the middle-repetitive class. It was shown that these results were not due to G + C differences nor to artifacts of the labeling technique. Therefore it was concluded that, although all families consisted of approximately the same number of repeats, the families ranged from those with virtually no sequence divergence to those barely able to reanneal at the criterion used, in accordance with the model of inter familial heterogeneity. Though this model may have wide applicability to middle-repetitive DNAs, a different pattern appears to prevail in Selaginella, a heterosporous pteridophyte. Some evolutionary implications are discussed.     

Chromosomes,DNA sequences,and evolution in salamanders of the genus Plethodon

Chromosomes and DNA sequence homologies have been studied in 15 species of North American salamander belonging to the genus Plethodon. These include 4 Eastern small species, 5 Eastern large species, 5 Western, and 1 New Mexican species. All species have 14 metacentric or sub-metacentric chromosomes. Their karyotypes are closely similar, but their C values range from 18–69 pg. DNA:DNA molecular hybridization studies showed that salamanders belonging to the same species group had between 60 and 90% of the observed repetitive DNA sequences in common, different groups of Eastern species had between 40 and 60% in common, and Eastern and Western groups had less than 10% in common. The slowly reassociating DNA sequences were also diverse among species, but higher levels of homology were observed than in the case of repetitive sequences. The New Mexican species was exceptional in showing little homology with other species with respect to either repetitive or slowly reassociating sequences.     

Restriction fragment length polymorphism analysis of Cryptosporidium parvum isolates of bovine and human origin.

Cryptosporidium parvum oocysts isolated from different hosts and geographical areas were compared by restriction endonuclease analysis of repetitive DNA: Iowa (bovine), Florida (bovine), New York (bovine), Peru (human), Brazil (human), and Mexico (human). Southern blot hybridization analysis was performed using the restriction endonuclease enzyme Eco RI and the DNA probe pV47-2. The probe hybridized with 18 bands present in all the isolates. The Brazilian, Mexican, and Peruvian human isolates had an additional common band of 4.3 kbp that was absent in the bovine isolates. Two extra bands of 14 and 12 kbp were present in the Brazilian isolate whereas the Mexican isolate had an extra band of 14 kbp. When the Iowa and Peru C. parvum isolates were passed twice through calves, oocysts recovered from both passages showed identical banding patterns, suggesting that recombination of the repetitive sequences was not altered during sexual reproduction. The DNA digested with other restriction endonucleases were tested confirming differences between isolates. A genomic DNA library is currently being produced to better define isolate variation in C. parvum.     

Genetic analysis of Helicobacter pylori strain populations colonizing the stomach at different times postinfection

Genetic diversity of the human gastric pathogen Helicobacter pylori in an individual host has been observed; whether this diversity represents diversification of a founding strain or a mixed infection with distinct strain populations is not clear. To examine this issue, we analyzed multiple single-colony isolates from two to four separate stomach biopsies of eight adult and four pediatric patients from a high-incidence Mexican population. Eleven of the 12 patients contained isolates with identical random amplified polymorphic DNA, amplified fragment length polymorphism, and vacA allele molecular footprints, whereas a single adult patient had two distinct profiles. Comparative genomic hybridization using whole-genome microarrays (array CGH) revealed variation in 24 to 67 genes in isolates from patients with similar molecular footprints. The one patient with distinct profiles contained two strain populations differing at 113 gene loci, including the cag pathogenicity island virulence genes. The two strain populations in this single host had different spatial distributions in the stomach and exhibited very limited genetic exchange. The total genetic divergence and pairwise genetic divergence between isolates from adults and isolates from children were not statistically different. We also analyzed isolates obtained 15 and 90 days after experimental infection of humans and found no evidence of genetic divergence, indicating that transmission to a new host does not induce rapid genetic changes in the bacterial population in the human stomach. Our data suggest that humans are infected with a population of closely related strains that vary at a small number of gene loci, that this population of strains may already be present when an infection is acquired, and that even during superinfection genetic exchange among distinct strains is rare.     

Identifying a single-copy DNA sequence associated with the expression of a heterochromatic gene, the light locus of Drosophila melanogaster

Although little is known about the molecular organization of most genes within heterochromatin, the unusual properties of these chromosomal regions suggest that genes therein may be organized and expressed very differently from those in euchromatin. We report here the cloning, by P transposon tagging, of sequences associated with the expression of the light locus, an essential gene found in the heterochromatin of chromosome 2 of Drosophila melanogaster. We conclude that this DNA is either a segment of the light locus, or a closely linked, heterochromatic sequence affecting its expression. While other functional DNA sequences previously described in heterochromatin have been repetitive, light gene function may be associated, at least in part, with single-copy DNA. This conclusion is based upon analysis of DNA from mutations and reversions induced by P transposable elements. The cloned region is unusual in that this single-copy DNA is embedded within middle-repetitive sequences. The in situ hybridization experiments also show that, unlike most other sequences in heterochromatin, this light-associated DNA evidently replicates in polytene chromosomes, but its diffuse hybridization signal may suggest an unusual chromosomal organization.     

Molecular evolution of mitochondrial ribosomal DNA in the fungal genus Tricholoma: barcoding implications

The molecular evolution of the V6 and V9 domains of the mitochondrial SSU-rDNA was investigated to evaluate the use of these sequences for DNA barcodes in the Basidiomycota division. The PCR products from 27 isolates belonging to 11 Tricholoma species were sequenced. Both domains in the isolates belonging to the same species had identical sequences. All the species possess distinctive V9 sequences due to point mutations and insertion/deletion events. Secondary structures revealed that the insertion-deletion events occurred in regions not directly involved in the maintenance of the standard SSU-rRNA structure. The inserted sequences possess conserved motifs that enable their alignment among phylogenetically distant species. Hence, the V9 domain by displaying identical sequences within species, an adequate divergence level, easy amplification, and alignment represents an alternative molecular marker for the Basidiomycota division and opens the way for this sequence to be used as specific molecular markers of the fungal kingdom.     

Restriction fragment length polymorphisms in the mushrooms Agaricus brunnescens and Agaricus bitorquis

Two Agaricus species, A. brunnescens (a commercial mushroom) and A. bitorquis (a wild, edible species), were examined for restriction fragment length polymorphisms. EcoRI-digested nuclear DNA from isolates of both species were cloned in plasmid vector pUC18. Ten random recombinant clones were used in Southern DNA-DNA hybridizations to probe EcoRI-digested DNA from 11 A. brunnescens isolates (7 commercial, 2 wild type, and 2 homokaryotic) and 7 A. bitorquis isolates. Most cloned fragments were polymorphic in both species. There were fewer different genotypes than expected, however, in the sample of commercial A. brunnescens strains. DNA from homokaryotic strains showed fewer bands in most hybridizations than DNA from heterokaryotic strains. All A. bitorquis isolates could be distinguished from each other as well as from every A. brunnescens strain. Putative homokaryons were detected by the loss of polymorphic bands among protoplast regenerates from one commercial strain and two strains collected in the wild.     

Sequence homology between mitochondrial DNAs of different eukaryotes.

The sequence divergence of mitochondrial DNAs (mtDNA) from rat, mouse, guinea pig, monkey, and chicken has been examined by DNA-DNA hybridization. mtDNAs, isolated as closed circular molecules by propidium iodide-CsCl centrifugation, were labeled in vitro by use of Escherichia coli DNA polymerase I, and renatured (Tm-35 degrees) in the presence of a 2500-fold excess of heterologous mtDNA. Single-stranded and duples DNA were separated by hydroxylapatite chromatography. The thermal stability of heteroduplexes was compared to the homoduplex by thermal elution chromatography on hydroxylapatite columns. Heteroduplex fromation between the tritiated myDNAs and a 2500-fole excess of rar mtDNA were 70, 59, 37, and 22%, respectively, for mouse, guinea pig, monkey, and chicken. Similar results were obrained in reciprocal hybridizations where one of the other mtDNAs was present in excess. Considerable mismatching of sequences in all the heterohybrids was indicated by a 18-24 degrees depression in the te50 of the heteroduplexes compared with the homoduplex. There was no apparent change in heteroduplex formation when the concentration ratio of driving DNA in excess to [3H]mtDNA was varied between 1250 and 7500. Furthermore, a second renaturation with excess driving DNA after completion of the first reaction resulted in no detectable augmenting of heteroduplex formation. Similar sequences appear to be conserved preferentially in different organisms, since the presence of two of fouf different heterologous mtDNAs in excess resulted in only moderate and nonadditive increases in heteroduplex formation. Evolutionary divergence of mtDNA sequences appears to have occurred at rates similar to that for unique sequences nuclear DNA.     

DNA probes distinguish geographical isolates and identify a novel DNA molecule of Babesia bovis

A genomic DNA library of Babesia bovis was screened to identify DNA probe candidates for direct detection of the parasite. Two sequences, Bo6 and Bo25, had the highest sensitivity and further analysis revealed unique characteristics of each of these. Neither sequence hybridized detectably to bovine DNA. Bo6 detected 100 pg of both a Mexican and an Australian isolate of B. bovis, but Bo6 also detected 1.0 ng of Babesia bigemina DNA under identical conditions. A unique characteristic of Bo6 is that it hybridizes to an apparent 7.4-kilobase DNA in undigested genomic DNA of both B. bovis and B. bigemina. The sequence is well conserved between the 2 geographic isolates of B. bovis, but it is apparently divergent in B. bigemina. Bo25 did not hybridize detectably to bovine or B. bigemina DNA. This sequence detected 100 pg of homologous B. bovis Mexican isolate DNA, but the sensitivity was reduced to 1 ng for the Australian isolate DNA. The restriction enzyme profile of the Bo25 sequence in genomic DNA differed markedly in the number, size, and intensity of bands between the 2 B. bovis geographic isolates tested. Thus, the Bo25 sequence can distinguish geographic isolates of B. bovis.     

Human chromosome-specific repetitive DNA sequences: novel markers for genetic analysis

Two recombinant DNA clones that are localized to single human chromosomes were isolated from a human repetitive DNA library. Clone pHuR 98, a variant satellite 3 sequence, specifically hybridizes to chromosome position 9qh. Clone pHuR 195, a variant satellite 2 sequence, specifically hybridizes to chromosome position 16qh. These locations were determined by fluorescent in situ hybridization to metaphase chromosomes, and confirmed by DNA hybridizations to human chromosomes sorted by flow cytometry. Pulsed field gel electrophoresis analysis indicated that both sequences exist in the genome as large DNA blocks. In situ hybridization to intact interphase nuclei showed a well-defined, localized organization for both DNA sequences. The ability to tag specific human autosomal chromosomes, both at metaphase and in interphase nuclei, allows novel molecular cytogenetic analyses in numerous basic research and clinical studies.