Additional Nuclear Genes Research Articles

An Evaluation of Cryptic Lineages of Idotea Balthica (Isopoda: Idoteidae): Morphology and Microsatellites

Idotea balthica has long been studied for intraspecific variation at the phenotypic and ecological level. Recent work found deep genetic divergences among several regional populations of this species in the North Atlantic. Here we report development of microsatellite loci for Idotea and use these newly developed markers to evaluate population structure among the mitochondrial clades. We also provide a detailed morphological evaluation, including SEM description of relevant traits in one of the more divergent groups. Phenotypically, the examined clades do not differ significantly, though individuals from populations in Virginia (U.S.A.) have more setose pereiopods than European and other museum specimens. The preliminary microsatellite data presented here suggest that these markers should be of utility for a variety of small-scale ecological and behavioral analysis in some related Idotea species. Although microsatellites are inappropriate for species designations, there is significant differentiation among mitochondrial lineages indicating that nuclear loci may show similar differentiation as mtDNA among regions. Together the subtle morphological variation and microsatellite data suggest that additional nuclear gene sequencing and reproductive studies are warranted to determine the taxonomic status of these regional forms.

Evolutionary patterns in the antR-Cor gene in the dwarf dogwood complex (Cornus, Cornaceae)

The evolutionary pattern of the myc-like anthocyanin regulatory gene antR-Cor was examined in the dwarf dogwood species complex (Cornus Subgenus Arctocrania) that contains two diploid species (C. canadensis and C. suecica), their putative hybrids with intermediate phenotypes, and a tetraploid derivative (C. unalaschkensis). Full-length sequences of this gene ( approximately 4 kb) were sequenced and characterized for 47 dwarf dogwood samples representing all taxa categories from 43 sites in the Pacific Northwest. Analysis of nucleotide diversity indicated departures from neutral evolution, due most likely to local population structure. Neighbor-joining and haplotype network analyses show that sequences from the tetraploid and diploid intermediates are much more strongly diverged from C. suecica than from C. canadensis, and that the intermediate phenotypes may represent an ancestral group to C. canadensis rather than interspecific hybrids. Seven amino acid mutations that are potentially linked to myc-like anthocyanin regulatory gene function correlate with petal colors differences that characterize the divergence between two diploid species and the tetraploid species in this complex. The evidence provides a working hypothesis for testing the role of the gene in speciation and its link to the petal coloration. Sequencing and analysis of additional nuclear genes will be necessary to resolve questions about the evolution of the dwarf dogwood complex.

Dissecting the ancient rapid radiation of microgastrine wasp genera using additional nuclear genes

Previous estimates of a generic level phylogeny for the ubiquitous parasitoid wasp subfamily Microgastrinae (Hymenoptera) have been problematic due to short internal branches deep in the phylogeny. These short branches might be attributed to a rapid radiation among the taxa, the use of genes that are unsuitable for the levels of divergence being examined, or insufficient quantity of data. We added over 1200 nucleotides from four nuclear genes to a dataset derived from three genes to produce a dataset of over 3000 nucleotides per taxon. While the number of well-supported short branches in the phylogeny increased, we still did not obtain strong bootstrap support for every node. Parametric and nonparametric bootstrap simulations projected that an enormous, and likely unobtainable, amount of data would be required to get bootstrap support greater than 50% for every node. However, a marked increase in the number of well-supported nodes was seen when we conducted a Bayesian analysis of a combined dataset generated from morphological characters added to the seven gene dataset. Our results suggest that, in some cases, combining morphological and genetic characters may be the most practical way to increase support for short branches deep in a phylogeny.

Terrestrial vertebrates have two keratin gene clusters; striking differences in teleost fish

Keratins I and II form the largest subgroups of mammalian intermediate filament (IF) proteins and account as obligatory heteropolymers for the keratin filaments of epithelia. All human type I genes except for the K18 gene are clustered on chromosome 17q21, while all type II genes form a cluster on chromosome 12q13, that ends with the type I gene K18. Highly related keratin gene clusters are found in rat and mouse. Since fish seem to lack a keratin II cluster we screened the recently established draft genomes of a bird (chicken) and an amphibian ( Xenopus). The results show that keratin I and II gene clusters are a feature of all terrestrial vertebrates. Because hair with its multiple hair keratins and inner root sheath keratins is a mammalian acquisition, the keratin gene clusters of chicken and Xenopus tropicalis have only about half the number of genes found in mammals. Within the type I clusters all genes have the same orientation. In type II clusters there is a rare gene of opposite orientation. Finally we show that the genes for keratins 8 and 18, which are the first expression pair in embryology, are not only adjacent in mammals, but also in Xenopus and three different fish. Thus neighboring K8 and K18 genes seem a feature shared by all vertebrates. In contrast to the two well defined keratin gene clusters of terrestrial vertebrates, three teleost fish show an excess of type I over type II genes, the lack of a keratin type II gene cluster and a striking dispersal of type I genes, that are probably the result of the teleost-specific whole genome duplication followed by a massive gene loss. This raises the question whether keratin gene clusters extend beyond the ancestral bony vertebrate to cartilage fish and lamprey. We also analyzed the complement of non-keratin IF genes of the chicken. Surprisingly, an additional nuclear lamin gene, previously overlooked by cDNA cloning, is documented on chromosome 10. The two splice variants closely resemble the lamin LIII a+b of amphibia and fish. This lamin gene is lost on the mammalian lineage.

The continuing search: low–copy nuclear sequences for lower–level plant molecular phylogenetic studies

A recent survey by Alvarez & Wendel (2003) revealed that during a five-year period over 65% of all molecular phylogenetic studies of plants at the generic level or lower included sequences of the internal transcribed spacer region of nuclear ribosomal DNA (ITS), and more than one third of the studies used this region exclusively for reconstructing phylogeny. Why the immense popularity of ITS among plant systematists? ITS sequences have achieved popularity for the same two reasons that any method or approach becomes popular in plant systematics: it is easy to generate data and the data seem to work. There are universal primers (with rare exceptions) for the ITS region (Baldwin, 1992), and therefore, it is easy for systematists to generate data rapidly because they do not have to design primers. Also, because there are many copies of the ITS repeat per genome, it is relatively easy to amplify the region. The ITS region has worked in the sense that it has proven sufficiently variable to resolve relationships in many different plant groups at the generic level or lower, which are the levels at which many plant systematists conduct research. Several studies have also demonstrated the value of ITS sequences at higher taxonomic levels (Hershkovitz & Lewis, 1996; Hershkovitz & Zimmer, 1996; Goertzen & al., 2003). If the ITS region has served plant systematists so well, why even look for another region to sequence? It is beyond the scope of this column to discuss the potential limitations of the ITS region, and we refer readers to the insightful and comprehensive critique of these limitations provided by Alvarez & Wendel (2003). We hasten to add, however, that while there certainly are limitations in using ITS sequences for phylogenetic reconstruction, it remains a genomic region that has proven and likely will continue to prove immensely useful to plant systematists. After all, without the insights that sequences of the ITS region have provided, we would be limited in our ability to judge the utility of other nuclear sequences that we will discuss here. For example, Tank & Sang (2001) discuss the importance of having a well-established phylogeny for Paeonia (based on morphology and molecular data, including the ITS region) as a framework for judging the utility of their sequences of GPAT (Table 1). On the other hand, it is becoming increasingly clear that some of the advantages of the ITS region may also make this region problematic. For example, universal primers may facilitate PCR amplification of contaminants. Although most of the copies of ITS per genome are identical, there may be rare copies in an individual that are not amplified due to selective amplification of only one copy. Concerted evolution often homogenizes copy types, and together, selective amplification and concerted evolution could obscure processes (e.g., hybridization) in the history of an organism. Another compelling reason to search for alternative low-copy nuclear sequences is that, despite the utility of ITS sequences for resolving relationships within genera, there are many examples, too numerous to cite, where resolution is poor because of the relatively short sequence length of the ITS region and the lack of sufficient variation. Thus, including additional nuclear gene sequence data has the potential to provide a more robust estimate of phylogenetic relationships. Another motivation for seeking additional nuclear sequences is that any inferred organismal phylogeny becomes much more compelling when it is based on more than one region because it becomes more than just a single gene tree (Cronn & al., 2003). However, few nuclear genes exist as single copies in most plant species, which complicates generating DNA sequence data from the nuclear genome. Generating sequence data from multiple gene regions in the chloroplast is easily accomplished because many different regions have been well characterized (e.g., matK, trnL-trnF, psbA-trnH, atpB) and most of these regions have universal primers available for amplification and sequencing. Employing sequences of multiple cpDNA gene regions often helps to resolve relationships with more support, but since the chloroplast genome is generally inherited as a single unit and lacks recombination, phylogenies based on multiple cpDNA regions effectively represent a single gene tree (Doyle, 1992). Given that it is desirable to include low-copy nuclear

Mdv1p is a WD repeat protein that interacts with the dynamin-related GTPase, Dnm1p, to trigger mitochondrial division.

Mitochondrial fission is mediated by the dynamin-related GTPase, Dnm1p, which assembles on the mitochondrial outer membrane into punctate structures associated with sites of membrane constriction and fission. We have identified additional nuclear genes required for mitochondrial fission, termed MDV (for mitochondrial division). MDV1 encodes a predicted soluble protein, containing a coiled-coil motif and seven COOH-terminal WD repeats. Genetic and two-hybrid analyses indicate that Mdv1p interacts with Dnm1p to mediate mitochondrial fission. In addition, Mdv1p colocalizes with Dnm1p in fission-mediating punctate structures on the mitochondrial outer membrane. Whereas localization of Mdv1p to these structures requires Dnm1p, localization of Mdv1p to mitochondrial membranes does not. This indicates that Mdv1p possesses a Dnm1p-independent mitochondrial targeting signal. Dnm1p-independent targeting of Mdv1p to mitochondria requires MDV2. Our data indicate that MDV2 also functions separately to regulate the assembly of Dnm1p into punctate structures. In contrast, Mdv1p is not required for the assembly of Dnm1p, but Dnm1p-containing punctate structures lacking Mdv1p are not able to complete division. Our studies suggest that mitochondrial fission is a multi-step process in which Mdv2p regulates the assembly of Dnm1p into punctate structures and together with Mdv1p functions later during fission to facilitate Dnm1p-dependent mitochondrial membrane constriction and/or division.

Onchocerca volvulus: Limited Heterogeneity in the Nuclear and Mitochondrial Genomes

Keddie, E. M., Higazi, T., Boakye, D., Merriweather, A., Wooten, M. C., and Unnasch, T. R. 1999. Onchocerca volvulus: Limited heterogeneity in the nuclear and mitochondrial genomes. Experimental Parasitology93, 198–206. West African populations of Onchocerca volvulus endemic to the rain forest and savanna bioclimes of West Africa differ in their ability to induce ocular disease in infected individuals. In recent years, both clinical- and animal-model-based studies have implicated particular parasite antigens in the development of ocular onchocerciasis. To test the hypothesis that the difference in pathogenic potential of blinding and nonblinding parasites might be reflected in qualitative differences in antigens that have been implicated in the development of ocular onchocerciasis, we compared the sequences of two parasite antigens implicated in the development of ocular disease in blinding- and nonblinding-strain parasites. The results demonstrated a high level of homogeneity between the parasite strains in these genes. The study was extended to include additional nuclear genes encoding antigens that are commonly recognized by individuals infected with O. volvulus and to the mitochondrial genome of the parasite. The results demonstrate a high degree of homogeneity in both the nuclear and the mitochondrial genomes among O. volvulus isolates collected from several different sites in Africa and in the Americas. This high degree of genetic homogeneity may reflect the passage of the parasite through a recent genetic bottleneck.

A Molecular Phylogeny of Reptiles

The classical phylogeny of living reptiles pairs crocodilians with birds, tuataras with squamates, and places turtles at the base of the tree. New evidence from two nuclear genes, and analyses of mitochondrial DNA and 22 additional nuclear genes, join crocodilians with turtles and place squamates at the base of the tree. Morphological and paleontological evidence for this molecular phylogeny is unclear. Molecular time estimates support a Triassic origin for the major groups of living reptiles.

Protein synthesis in mitochondria from yeast strains carrying nam and mim suppressor genes

Yeast mitochondria isolated from two different wild type strains ( gal + and gal −), whether grown on galactose or glucose, synthesize all mitochondrial polypeptides with similar efficiencies and in proportions approximating those detected in vivo. Mitochondria isolated from mit − mutants synthesize in vitro a mutant pattern of mitochondrial proteins, indistinguishable from the in vivo products. The mutant pattern is restored to the wild type one in mitochondria isolated from pseudorevertant strains carrying an additional nuclear ( nam3-1 and R705) or mitochondrial ( mim3-1) informational suppressor gene. Suppression is expressed in isolated mitochondria without the obligatory presence of cytosol at the level of both respiratory control and specific polypeptide synthesis. Translation in isolated mitochondria is sensitive to paromomycin. The antibiotic differentiates between translation in mitochondria from wild type strains and that in nam-type gene carrying strains. This strongly suggests that nam-type mutations affect the mitoribosoome enhancing ambguity of translation, thus allowing for the pseudoreversion of mit − phenotypes.

Treponemal serology on Bali Island, Indonesia.

As part of a multidisciplinary study of the population of Bali, Indonesia, treponemal serology was carried out on 2452 serum samples from subjects of both sexes. Sera reactive to the Treponema pallidum immobilisation test (TPI) were found in 81 (3.3%) subjects with a male prevalence of 4% and a female prevalence of 2%. All the reactive sera were from villagers. Of 1118 students sampled in various towns, none had reactive TPI tests. The prevalence of reactive sera varied greatly from one village to another; up to 50% of the sera examined were reactive. Geographical and socioeconomic analyses of the data show a strict correlation between poor socioeconomic status and high reactivity rates to the TPI test. Fifty-seven per cent of all the reactive sera originated from subjects living in two districts where yaws had recently been reported. Only three of the 1406 subjects, aged 15-29 years, had reactive sera. The reactivity rate steadily increased in the age groups 30-44, 45-59, and 60 years and over. Biological false-positive reactions occurred in 3.8% of the sera tested.