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Caudal polymorphism and cephalic morphology among first-stage larvae of Parelaphostrongylus odocoilei (Protostrongylidae: Elaphostrongylinae) in Dall's sheep from the Mackenzie Mountains, Canada.

We demonstrate polymorphism in the structure of the tail among first-stage larvae of Parelaphostrongylus odocoilei (Protostrongylidae). Two distinct larvae, both with a characteristic dorsal spine, include (1) a morphotype with a kinked conical tail marked by 3 distinct transverse folds or joints and a symmetrical terminal tail spike and (2) a morphotype with a digitate terminal region lacking folds or joints and with an asymmetrical, subterminal tail spike. These divergent larval forms had been postulated as perhaps representing distinct species of elaphostrongyline nematodes. Application of a multilocus approach using ITS-2 sequences from the nuclear genome and COX-II sequences from the mitochondrial genome confirmed the identity of these larvae as P. odocoilei. Additionally, based on scanning electron microscopy (low-temperature field emission), the cephalic region of these larvae consisted of a cuticular triradiate stoma surrounded by 6 single circumoral papillae of the inner circle, 10 papillae of the outer circle (4 paired and 2 single), and 2 lateral amphids. Our's is the first demonstration of structural polymorphism among larval conspecifics in the Metastrongyloidea and Strongylida. The basis for this polymorphism remains undetermined, but such phenomena, if discovered to be more widespread, may contribute to continued confusion in discriminating among first-stage larvae for species, genera, and subfamilies within Protostrongylidae.

Animals↗

Synonymy of Calyptogena solidissima with Calyptogena kawamurai (Bivalvia: Vesicomyidae) and its population structure revealed by mitochondrial DNA sequences.

Nucleotide sequences of part (1,101 bp) of the mitochondrial cytochrome oxidase c subunit I (COI) gene were determined for two specimens of Calyptogena kawamurai collected in Kashima Nada and Suruga Bay, respectively. These sequences were identical to each other and to those from many individuals of Calyptogena solidissima, i.e., 11 of 12 specimens from a seep area in Nankai Trough, two of 20 from hydrothermal-vent fields in Okinawa Trough, and one of 14 from a seep area on Kuroshima Knoll. The nucleotide sequences of the 5' part (about 700 bp) of the first internal transcribed spacer (ITS-1) also showed a close relationship between C. kawamurai and C. solidissima. The radiating threads on the shell surface that were emphasized in describing C. solidissima are not consistent throughout these local populations. Variation in cardinal dentition was confirmed to be intraspecific by observations of a series of specimens. The shell length-height and shell length-width relationships of both species all fit a single regression line. These results suggest that C. solidissima is a junior synonym of C. kawamurai. The populations of Nankai Trough, Okinawa Trough, and Kuroshima Knoll were shown to be diverging genetically from each other. Populations of Okinawa Trough and Kuroshima Knoll are suggested to have derived independently from the most common haplotype of Nankai Trough.

Animals↗

Biogeography of two species of Symbiodinium (Freudenthal) inhabiting the intertidal sea anemone Anthopleura elegantissima (Brandt).

We have analyzed the genetic profiles of dinoflagellate populations obtained from the Pacific coast sea anemone Anthopleura elegantissima (Brandt) at collection sites from Washington to California. Genetic differences within the symbiont populations of California anemones have been uncovered by restriction length polymorphism (RFLP) analysis of the small subunit (SSU) and large subunit (LSU) ribosomal RNA genes, and by denaturing gradient gel electrophoresis (DGGE) of the internal transcribed spacer region 2 (ITS 2). The existence of two Symbiodinium species is substantiated by sequence analysis of the variable regions V1, V2, and V3 of the SSUrDNA, which also establishes their phylogenetic relatedness to other members of the genus Symbiodinium. Anemones from Washington and Oregon harbor a single dinoflagellate species, for which we propose the name S. muscatinei sp. nov. At these northern locations, S. muscatinei either exists alone or co-occurs with the Chlorella-like green algal symbiont. Our results indicate that S. muscatinei co-occurs with a second dinoflagellate, S. californium, in mixed populations in central and southern California. We suggest that the geographic distribution of these dinoflagellates is related to the temperature cline created by latitude.

Animals↗

[Molecular biological analysis of Tinea faciei caused by a clinically anomalous strain of Trichophyton mentagrophytes].

We report a case of Trichophyton mentagrophytes infection. A 27-year-old female dairy worker visited our hospital in August 2001 for evaluation of an erythematous lesion near her right eye. The patient had been treated with antibiotics and an anti-allergic drug at another clinic with no effect on the lesion. KOH preparation of the scale revealed mycelia and T. mentagrophytes was isolated from the culture. The isolated pathogen was identified as T. mentagrophytes animal type 1 by the classification system based on the cluster analysis of nuclear ribosomal internal transcribed spacer 1 (ITS1) DNA sequences. The oral administration of terbinafine at 125 mg/day and antifungal cream was effective. T. mentagrophytes is one of the most common dermatophytes isolated from humans and animals. We should be aware that animals may carry this kind of fungal pathogen.

Administration, Oral↗

Primer on medical genomics part II: Background principles and methods in molecular genetics.

The nucleus of every human cell contains the full complement of the human genome, which consists of approximately 30,000 to 70,000 named and unnamed genes and many intergenic DNA sequences. The double-helical DNA molecule in a human cell, associated with special proteins, is highly compacted into 22 pairs of autosomal chromosomes and an additional pair of sex chromosomes. The entire cellular DNA consists of approximately 3 billion base pairs, of which only 1% is thought to encode a functional protein or a polypeptide. Genetic information is expressed and regulated through a complex system of DNA transcription, RNA processing, RNA translation, and posttranslational and cotranslational modification of proteins. Advances in molecular biology techniques have allowed accurate and rapid characterization of DNA sequences as well as identification and quantification of cellular RNA and protein. Global analytic methods and human genetic mapping are expected to accelerate the process of identification and localization of disease genes. In this second part of an educational series in medical genomics, selected principles and methods in molecular biology are recapped, with the intent to prepare the reader for forthcoming articles with a more direct focus on aspects of the subject matter.

Animals↗

Molecular epidemiology of Pseudomonas aeruginosa from cystic fibrosis in Sicily: genome macrorestriction analysis and rapid PCR-ribotyping.

This study addresses the epidemiologic relatedness of a collection of Pseudomonas aeruginosa isolates from cystic fibrosis patients attending the Pediatric Clinic, Catania, Sicily. Genome macrorestriction analysis after pulsed field gel electrophoresis (PFGE) was used to characterise all strains. Furthermore, a rapid typing procedure, developed in this study, based on polymerase chain reaction amplified ribosomal DNA spacer polymorphisms (PCR-ribotyping), straight from bacterial cultures, was used. On the basis of macrorestriction analysis after PFGE, persistence of infection was shown in all patients; two cross-transmission episodes were identified in the nosocomial as well as in the familiar environment. PCR-ribotyping proved to be useful for a DNA-based identification test, suitable for screening purposes. The rapid amplification protocol here tested is proposed to evaluate the discriminatory power of other specific target sequences in PCR-based typing assays, for epidemiologic purposes.

Adolescent↗

An engineered lox sequence containing part of a long terminal repeat of HIV-1 permits Cre recombinase-mediated DNA excision.

In our previous report, one 34-bp sequence from a long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1) clone, loxLTR-1, was proposed as a target site for site-specific excision by modified Cre recombinase. To support this suggestion, an engineered lox sequence, designated loxIL1, was made. This variant lox has the corresponding sequence of loxLTR-1 at the spacer region and the last two bases of inverted repeat sequence. Through in vitro recombination assay, loxIL1 also allowed the wild-type Cre to specifically recombine the sequence. An in vitro DNA binding experiment with mutants CreK244R and CreK244L revealed that lysine 244 of Cre plays an important role in interaction with the engineered lox. This result suggests that loxLTR-1 would be a candidate for antiviral strategy using site-specific recombinase.

DNA, Intergenic↗

Molecular phylogeography, reticulation, and lineage sorting in Mediterranean Senecio sect. Senecio (Asteraceae).

The Mediterranean species complex of Senecio serves to illustrate evolutionary processes that are likely to confound phylogenetic inference, including rapid diversification, gene tree-species tree discordance, reticulation, interlocus concerted evolution, and lack of complete lineage sorting. Phylogeographic patterns of chloroplast DNA (cpDNA) haplotype variation were studied by sampling 156 populations (502 individuals) across 18 species of the complex, and a species phylogeny was reconstructed based on sequences from the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. For a subset of species, randomly amplified polymorphic DNAs (RAPDs) provided reference points for comparison with the cpDNA and ITS datasets. Two classes of cpDNA haplotypes were identified, with each predominating in certain parts of the Mediterranean region. However, with the exception of S. gallicus, intraspecific phylogeographic structure is limited, and only a few haplotypes detected were species-specific. Nuclear sequence divergence is low, and several unresolved phylogenetic groupings are suggestive of near simultaneous diversification. Two well-supported ITS clades contain the majority of species, amongst which there is a pronounced sharing of cpDNA haplotypes. Our data are not capable of diagnosing the relative impact of reticulation versus insufficient lineage sorting for the entire complex. However, there is firm evidence that S. flavus subsp. breviflorus and S. rupestris have acquired cpDNA haplotypes and ITS sequences from co-occurring species by reticulation. In contrast, insufficient lineage sorting is a viable hypothesis for cpDNA haplotypes shared between S. gallicus and its close relatives. We estimated the minimum coalescent times for these haplotypes by utilizing the inferred species phylogeny and associated divergence times. Our data suggest that ancestral cpDNA polymorphisms may have survived for ca. 0.4-1.0 million years, depending on molecular clock calibrations.

Biological Evolution↗

[Phylogeny of the Malus (apple tree) species, inferred from its morphological traits and molecular DNA analysis].

Some debated issues of the genus Malus (apple) taxonomy were examined using a variety of species from the collection of the Maikop Experimental Station, Vavilon Research Institute of Plant Industry (Krasnodar krai). Phylogenetic relationships among these species were studied using traditional analysis of morphological traits, RAPD, and complete sequencing of the 5'-internal transcribed spacer (ITS1), 5.8S rRNA, 3'-internal transcribed spacer (ITS2) (constituting a cluster of the rRNA genes), and the terminal fragment of the matK gene encoding chloroplast maturase. The results showed that the Sorbomalus section was polyphyletic; the American apple M. fusca was closely related to the species contributing to the East Asian center of the genus origin, and the American species M. angustifolia, M. coronaria, and M. ioensis were closely related to the M. trilobata relict species, whose assignment to the genus Malus is debated by some authors. Molecular analysis of the species relationships showed that the Middle Asian apple M. sieversii is the species from which apple domestication started.

Asia↗

[rDNA its sequencing of Herba Dendrobii (Huangcao)].

AIM: To study the genetic diversity of ITS sequences of Herba Dendrobii (Huangcao) and analyze the utility of ITS sequences in molecular authentication of Herba Dendrobii (Huangcao) and phylogenetic of Dendrobium. METHODS: The ITS gene fragment was amplified using a pair of primers. The PCR products were purified and sequenced by the methods of Sanger Dideoxy. RESULTS: The DNA sequence of 228-233 bp ITS 1, 242-247 bp ITS 2 gene fragments and 5.8S rDNA were obtained from 14 samples of Dendrobium. The interspecific substitution varies from 11.79% to 31.58% at ITS 1 and 10.29% to 25.30% at ITS 2. The intraspecific substitution of D. nobile is 0.87% at ITS 1 and without difference at ITS 2. The substitution between Dendrobium and outgroup varies from 23.56% to 36.89% at ITS 1 and 26.52% to 33.31% at ITS 2. The phylogenetic tree based on ITS 1 and ITS 2 data was set up. CONCLUSION: The ITS 1 and ITS 2 gene fragments were highly conservative at intraspecific level in Dendrebium, while they were less conservative at interspecific level in D. nobile. They were least conservative between Dendrobium and outgroup. Hence, the sequence of this fragment is a good molecular marker for authentication of the Huangcao. But, further study is necessary for the phylogenetic of Dendrobium.

Base Sequence↗

Distributions of exons and introns in the human genome.

The human genome is revisited using exon and intron distribution profiles. The 26,564 annotated genes in the human genome (build October, 2003) contain 233,785 exons and 207,344 introns. On average, there are 8.8 exons and 7.8 introns per gene. About 80% of the exons on each chromosome are < 200 bp in length. < 0.01% of the introns are < 20 bp in length and < 10% of introns are more than 11,000 bp in length. These results suggest constraints on the splicing machinery to splice out very long or very short introns and provide insight to optimal intron length selection. Interestingly, the total length in introns and intergenic DNA on each chromosome is significantly correlated to the determined chromosome size with a coefficient of correlation r = 0.95 and r = 0.97, respectively. These results suggest their implication in genome design.

Alternative Splicing↗

The Hyrcanus group of Anopheles (Anopheles) in China (Diptera: Culicidae): species discrimination and phylogenetic relationships inferred by ribosomal DNA internal transcribed spacer 2 sequences.

The Hyrcanus group comprises many closely related species with wide distributions in the Oriental and Palaearctic regions. The sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA were determined for 12 species in China--An. crawfordi, An. hyrcanus, An. junlianensis, An. kunmingensis, An. kweiyangensis, An. lesteri, An. liangshanensis, An. peditaeniatus, An. pullus, An. sinensis, and two unknown species within the group. The length of the ITS2 ranged from 436 bp in An. hyrcanus to 469 bp in An. crawfordi, with GC contents of 44.9-46.8%. Intraspecific variation was found in three species (An. junlianensis, An. liangshanensis, and An. pullus) at the level of 0.0-0.4%, whereas interspecific differences ranged from 1.6% between An. liangshanensis and An. kunmingensis to 50.8% between An. peditaeniatus and sp. 1. The ITS2 comparisons revealed two unknown species, verified the valid species status for An. kunmingensis, and found An. pullus in China. We agree that An. anthropophagus is a junior synonym of An. lesteri. The validation of An. junlianensis awaits recognition of the molecular identity of the entity identified as An. yatsushiroensis. The ITS2 divergences were used for inferring phylogenetic relationships among 12 species in China. The estimation revealed close relationships among An. liangshanesis, An. kunmingensis, An. kweiyangensis, An. lesteri, and An. sinensis. Our study emphasizes the need for the molecular identity of the species members in integrated studies in systematics, bionomics, and population genetics for the Hyrcanus group.

Animals↗

Identification of novel indigenous Bacillus thuringiensis isolates.

Internally Transcribed Spacers (ITS) characterization and distribution of Repetitive Extragenic Palindromic (REP) sequences were studied in the genome of 223 field isolates of Bacillus thuringiensis from Madurai, India. They were characterized by morphological, biochemical and molecular methods. One hundred and twenty four of a total 223 isolates fitted ITS characterization of B. thuringiensis varieties known. Significant genomic variation was observed among seven isolates using REP primers. The ITS PCR product (EMBL accession number AJ639659) exhibited 98% nucleotide sequence homology with B. thuringiensis and placed the origin of indigenous isolate LDC-7 closer to B. thuringiensis on the basis of phylogenetic analysis.

Bacillus thuringiensis↗

u-Genome: a database on genome design in unicellular genomes.

Unicellular eukaryotes were among the first ones to be selected for complete genome sequencing because of the small size of their genomes and their interactions with humans and a broad range of animals and plants. Currently, ten completely sequenced unicellular genome sequences have been publicly released and as the number of available unicellular genomes increases, comparative genomics analysis within this group of organisms becomes more and more instructive. However, such an analysis is difficult to carry out without a suitable platform gathering not only the original annotations but also relevant information available in public databases or obtained by applying common bioinformatics methods. With the aim of solving these difficulties, we have developed a web-accessible database named u-Genome, the unicellular genome design database. The database is unique in featuring three datasets namely (1) orthologous proteins (2) paralogous proteins and (3) statistical distributions on exons, introns, intergenic DNA and correlations between them. A tool, Uniview, designed to visualize the gene structures for individual genes in the genome is also integrated. This database is of importance in understanding unicellular genome design and architecture and evolution related studies. The database is available through a web interface at http://sege.ntu.edu.sg/wester/ugenome.

Animals↗

Hybridization, polyploidy, and the evolution of sexual systems in Mercurialis (Euphorbiaceae).

Hybridization and polyploidy are widely believed to be important sources of evolutionary novelty in plant evolution. Both can lead to novel gene combinations and/or novel patterns of gene expression, which in turn provide the variation on which natural selection can act. Here, we use nuclear and plastid gene trees, in conjunction with morphological data and genome size measurements, to show that both processes have been important in shaping the evolution of the angiosperm genus Mercurialis, particularly a clade of annual lineages that shows exceptional variation in the sexual system. Our results indicate that hexaploid populations of M. annua, in which the rare sexual system androdioecy is common (the occurrence of males and hermaphrodites) is of allopolyploid origin involving hybridization between an autotetraploid lineage of M. annua and the related diploid species M. huetii. We discuss the possibility that androdioecy may have evolved as a result of hybridization between dioecious M. huetii and monoecious tetraploid M. annua, an event that brought together the genes for specialist males with those for hermaphrodites.

Biological Evolution↗

Structure and evolution of the intergenic region in a ribosomal DNA repeat unit of wheat.

The complete nucleotide sequence of the intergenic region between the 25 S and 18 S wheat ribosomal RNA genes has been determined from a 4.6 kb EcoRI-BamHI fragment (1 kb = 10(3) bases or base-pairs) subcloned from the plasmid pTa71. Within this subclone the intergenic DNA is flanked by the 3' end of the 25 S and the 5' end of the 18 S ribosomal RNA sequences. Four repeat families are present within the intergenic region. The major repeat family A, consists of 12 direct repeat units of 135 or 136 base-pairs (bp) flanked by diverged truncated copies. Within each A repeat a subrepeat structure has been revealed. Family B, which is localized to the 5' side of the A repeats, contains three repeat units, one of 152 bp, the second of 150 bp and a truncated unit of 107 bp. Family C, which is localized in the transcribed rRNA precursor, consists of two direct repeat units of 172 and 174 bp and possesses some short subrepeat motifs. The C repeats may have evolved by and diverged from one another by the insertion of short transposable sequences. Family D consists of two direct repeat units of 30 bp located 5' to the start of transcription. Statistical analysis of repeat family A showed that there is a significant association between the similarity of any two repeat units and their distance apart in the array. The near identity of members of the A family is maintained presumably by processes such as unequal crossing over and gene conversion, but the members at each end of the array show more divergence. Sequence motifs in the A and C repeat families and in other regions including the 5' end of 18 S RNA are related, implying that much of the intergenic DNA may have evolved from a few short ancestral sequences. The B and D repeats or their equivalent are not found in a maize ribosomal DNA repeat unit. The DNA in the external transcribed spacer DNA 5' to the 18 S RNA sequence is longer in wheat than in maize. This is due principally to two duplications and insertion of a sequence with dyad symmetry in the wheat gene.

Base Sequence↗

Characterization of natural populations of Nitrobacter spp. using PCR/RFLP analysis of the ribosomal intergenic spacer.

DNA sequences from the intergenic spacer (IGS) region of the ribosomal operon were amplified by the polymerase chain reaction (PCR) technique using two primers derived from 16S and 23S rRNA conserved sequences. The PCR products, cleaved by 4 base cutting restriction enzymes, were used to differentiate Nitrobacter strains. This method offered a convenient alternative to serological testing for characterization of Nitrobacter isolates and enabled a large number of strains to be genotypically characterized easily and rapidly. This method was successfully used to characterize natural populations of Nitrobacter from various soils and a lake. A diversity was demonstrated in various soils, and in a lake both in freshwater and in sediments. Strains closely related to both WL and LL were found in these ecosystems. It seems that the diversity of Nitrobacter populations was not associated with global environments but may be related to the presence of locally coexisting niches.

Base Sequence↗

[Restriction fragment length polymorphisms of chloroplast DNA and the intergenic spacer of rRNA gene in cultivated barley of China].

A sample of 80 accessions collected from several major barley growing areas in China was assayed for restriction fragment length polymorphisms at two regions of the chloroplast DNA and the intergenic spacer of the ribosomal RNA gene (rDNA). The results showed that the spacer length was highly polymorphic and 8 spacer length variants constituting 8 phenotypes were observed in this sample. It is also clear that the spacer length variants and the phenotypes were differentially distributed in different geographical regions, probably due to adaptation of the genotypes to the local environments. We did not detect variation in the two chloroplast fragments, indicating that the variability of chloroplast DNA is low in cultivated barley.

Chloroplasts↗