Soil microbial community analysis using denaturing gradient gel electrophoresis

The most biological diversity on this planet is probably harbored in soils. Understanding the diversity and function of the microbiological component of soil poses great challenges that are being overcome by the application of molecular biological approaches. This review covers one of many approaches being used: separation of polymerase chain reaction (PCR) amplicons using denaturing gradient gel electrophoresis (DGGE). Extraction of nucleic acids directly from soils allows the examination of a community without the limitation posed by cultivation. Polymerase chain reaction provides a means to increase the numbers of a target for its detection on gels. Using the rRNA genes as a target for PCR provides phylogenetic information on populations comprising communities. Fingerprints produced by this method have allowed spatial and temporal comparisons of soil communities within and between locations or among treatments. Numerous samples can be compared because of the rapid high throughput nature of this method. Scientists now have the means to begin addressing complex ecological questions about the spatial, temporal, and nutritional interactions faced by microbes in the soil environment.

Soil microbial community analysis using terminal restriction fragment length polymorphisms

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a polymerase chain reaction (PCR)-fingerprinting method that is commonly used for comparative microbial community analysis. The method can be used to analyze communities of bacteria, archaea, fungi, other phylogenetic groups or subgroups, as well as functional genes. The method is rapid, highly reproducible, and often yields a higher number of operational taxonomic units than other, commonly used PCR-fingerprinting methods. Sizing of terminal restriction fragments (T-RFs) can now be done using capillary sequencing technology allowing samples contained in 96- or 384-well plates to be sized in an overnight run. Many multivariate statistical approaches have been used to interpret and compare T-RFLP fingerprints derived from different communities. Detrended correspondence analysis and the additive main effects with multiplicative interaction model are particularly useful for revealing trends in T-RFLP data. Due to biases inherent in the method, linking the size of T-RFs derived from complex communities to existing sequence databases to infer their taxonomic position is not very robust. This approach has been used successfully, however, to identify and follow the dynamics of members within very simple or model communities. The T-RFLP approach has been used successfully to analyze the composition of microbial communities in soil, water, marine, and lacustrine sediments, biofilms, feces, in and on plant tissues, and in the digestive tracts of insects and mammals. The T-RFLP method is a user-friendly molecular approach to microbial community analysis that is adding significant information to studies of microbial populations in many environments.

Stable Isotope Probing:Linking Functional Activity to Specific Members of Microbial Communities

Linking organisms or groups of organisms to specific functions within natural environments is a fundamental challenge in microbial ecology. Advances in technology for manipulating and analyzing nucleic acids have made it possible to characterize the members of microbial communities without the intervention of laboratory culturing. Results from such studies have shown that the vast majority of soil organisms have never been cultured, highlighting the risks of culture-based approaches in community analysis. The development of culture-independent techniques for following the flow of substrates through microbial communities therefore represents an important advance. These techniques, collectively known as stable isotope probing (SIP), involve introducing a stable isotope-labeled substrate into a microbial community and following the fate of the substrate by extracting diagnostic molecular species such as fatty acids and nucleic acids from the community and determining which specific molecules have incorporated the isotope. The molecules in which the isotope label appears provide identifying information about the organism that incorporated the substrate. Stable isotope probing allows direct observations of substrate assimilation in minimally disturbed communities, and thus represents an exciting new tool for linking microbial identity and function. The use of lipids or nucleic acids as the diagnostic molecule brings different strengths and weaknesses to the experimental approach, and necessitates the use of significantly different instrumentation and analytical techniques. This short review provides an overview of the lipid and nucleic acid approaches, discusses their strengths and weaknesses, gives examples of applications in various settings, and looks at prospects for the future of SIP technology.

Expression of muscle-related genes and two MyoD genes during amphioxus notochord development

The notochord is one of the diagnostic features of the phylum Chordata. Despite the similarities in the early morphogenetic patterns of the notochords of various chordates, they are strikingly distinct from one another at the histological level. The amphioxus notochord is one example of an evolutionary novelty because it is made up of muscle cells. Our previous expressed sequence tag analysis, targeting messenger RNAs expressed in the adult amphioxus notochord, demonstrated that many muscle-related genes are expressed there. To characterize amphioxus notochord cells and to gain insights into the myogenic program in the notochord, we determined the spatial and temporal expression patterns of these muscle-related genes during amphioxus development. We found that BbNA1 (notochord actin), Amphi-Trop I (troponin I), Amphi-TPmyosin (tropomyosin), Amphi-MHC2 (myosin heavy chain), Amphi-nMRLC (notochord-specific myosin regulatory light chain), AmphinTitin/MLCK (notochord-specific titin/myosin light chain kinase), Amphi-MLP/CRP3 (muscle LIM protein), and Amphi-nCalponin (notochord-specific calponin) are expressed with characteristic patterns in notochord cells, including the central cells, dorsally located cells, and ventrally located cells, suggesting that each notochord cell has a unique molecular architecture that may reflect its function. In addition, we characterized two MyoD genes (Amphi-MyoD1 and Amphi-MyoD2) to gain insight into the genetic circuitry governing the formation of the notochord muscle. One of the MyoD genes (Amphi-MyoD2) is expressed in the central notochord cells, and the coexistence of Amphi-MyoD2 transcripts along with the Amphi-MLP/CRP3 transcripts implies the participation of Amphi-MyoD2 in the myogenic program in the notochord muscle.

An estimate of divergence time of parazoa and eumetazoa and that of cephalochordata and vertebrata by aldolase and triose phosphate isomerase clocks

Previously we suggested that four proteins including aldolase and triose phosphate isomerase (TPI) evolved with approximately constant rates over long periods covering the whole animal phyla. The constant rates of aldolase and TPI evolution were reexamined based on three different models for estimating evolutionary distances, It was shown that the evolutionary rates remain essentially unchanged in comparisons not only between different classes of vertebrates but also between vertebrates and arthropods and even between animals and plants, irrespective of the models used, Thus these enzymes might be useful molecular clocks for inferring divergence times of animal phyla, To know the divergence time of Parazoa and Eumetazoa and that of Cephalochordata and Vertebrata, the aldolase cDNAs from Ephydatia fluviatilis, a freshwater sponge, and the TPI cDNAs from Ephydatia fluviatilis and Branchiostoma belcheri an amphioxus, have been cloned and sequenced, Comparisons of the deduced amino acid sequences of aldolase and TPI from the freshwater sponge with known sequences revealed that the Parazoa-Eumetazoa split occurred about 940 million years ago (Ma) as determined by the average of two proteins and three models, Similarly, the aldolase and TPI clocks suggest that vertebrates and amphioxus last shared a common ancestor around 700 Ma and they possibly diverged shortly after the divergence of deuterostomes and protostomes.

Topographic changes in nascent and early mesoderm in amphioxus embryos studied by Dil labeling and by in situ hybridization for a Brachyury gene

In amphioxus embryos, the nascent and early mesoderm (including chorda-mesoderm) was visualized by expression of a Brachyury gene (AmBra-2). A band of mesoderm is first detected encircling the earliest (vegetal plate stage) gastrula sub-equatorially. Soon thereafter, the vegetal plate invaginates. resulting in a cap-shaped gastrula with the mesoderm localized at the blastoporal lip and completely encircling the blastopore. As the gastrula stage progresses, DiI (a vital dye) labeling demonstrates that the entire mesoderm is internalized by a slight involution of the epiblast into the hypoblast all around the perimeter of the blastopore. Subsequently. during the early neurula stage, the internalized mesoderm undergoes anterior extension mid-dorsally (as notochord) and dorsolaterally (in paraxial regions when segments will later form). By the late neurula stage, AmBra-2 is no longer transcribed throughout the mesoderm as a whole; instead. expression is detectable only in the posterior mesoderm and in the notochord, but not in par axial mesoderm where definitive somites have formed.

Lineage-specific domain fusion in the evolution of purine nucleotide cyclases in cyanobacteria

Cyclic nucleotides (both cAMP and cGMP) play extremely important roles in cyanobacteria, such as regulating heterocyst formation, respiration, or gliding. Catalyzing the formation of cAMP and cGMP from ATP and GTP is a group of functionally important enzymes named adenylate cyclases and guanylate cyclases, respectively. To understand their evolutionary patterns, in this study, we presented a systematic analysis of all the cyclases in cyanobacterial genomes. We found that different cyanobacteria had various numbers of cyclases in view of their remarkable diversities in genome size and physiology. Most of these cyclases exhibited distinct domain architectures, which implies the versatile functions of cyanobacterial cyclases. Mapping the whole set of cyclase domain architectures from diverse prokaryotic organisms to their phylogenetic tree and detailed phylogenetic analysis of cyclase catalytic domains revealed that lineage-specific domain recruitment appeared to be the most prevailing pattern contributing to the great variability of cyanobacterial cyclase domain architectures. However, other scenarios, such as gene duplication, also occurred during the evolution of cyanobacterial cyclases. Sequence divergence seemed to contribute to the origin of putative guanylate cyclases which were found only in cyanobacteria. In conclusion, the comprehensive survey of cyclases in cyanobacteria provides novel insight into their potential evolutionary mechanisms and further functional implications.

Characterization of amphioxus GDF8/11 gene, an archetype of vertebrate MSTN and GDF11

MSTN, also known as growth and differentiation factor 8 (GDF8), and GDF11 are members of the transforming growth factor-beta (TGF-beta) subfamily. They have been thought to be derived from one ancestral gene. In the present study, we report the isolation and characterization of an invertebrate GDF8/11 homolog from the amphioxus (Branchiostoma belcheri tsingtauense). The amphioxus GDF8/11 gene consists of five exons flanked by four introns, which have two more exons and introns than that of other species. In intron III, a possible transposable element was identified. This suggested that this intron might be derived from transposon. The amphioxus GDF8/11 cDNA encodes a polypeptide of 419 amino acid residues. Phologenetic analysis shows that the GDF8/11 is at the base of vertebrate MSTNs and GDF11s. This result might prove that the GDF8/11 derived from one ancestral gene and the amphioxus GDF8/11 may be the common ancestral gene, and also the gene duplication event generating MSTN and GDF11 occurred before the divergence of vertebrates and after or at the divergence of amphioxus from vertebrates. Reverse transcriptase polymerase chain reaction results showed that the GDF8/11 gene was expressed in new fertilized cell, early gastrulation, and knife-shaped embryo, which was different from that in mammals. It suggested that the GDF8/11 gene might possess additional functions other than regulating muscle growth in amphioxus.

Population genetics of Crassostrea ariakensis in Asia inferred from microsatellite markers

Crassostrea ariakensis is an important aquacultured oyster species in Asia, its native region. During the past decade, consideration was given to introducing C. ariakensis into Chesapeake Bay, in the United States, to help revive the declining native oyster industry and bolster the local ecosystem. Little is known about the ecology and biology of this species in Asia due to confusion with nomenclature and difficulty in accurately identifying the species of wild populations in their natural environment. Even less research has been done on the population genetics of native populations of C. ariakensis in Asia. We examined the magnitude and pattern of genetic differentiation among 10 wild populations of C. ariakensis from its confirmed distribution range using eight polymorphic microsatellite markers. Results showed a small but significant global theta (ST) (0.018), indicating genetic heterogeneity among populations. Eight genetically distinct populations were further distinguished based on population pairwise theta (ST) comparisons, including one in Japan, four in China, and three populations along the coast of South Korea. A significant positive association was detected between genetic and geographic distances among populations, suggesting a genetic pattern of isolation by distance. This research represents a novel observation on wild genetic population structuring in a coastal bivalve species along the coast of the northwest Pacific.

Origin of mitochondrial DNA diversity of domestic yaks

Background: The domestication of plants and animals was extremely important anthropologically. Previous studies have revealed a general tendency for populations of livestock species to include deeply divergent maternal lineages, indicating that they were domesticated in multiple, independent events from genetically discrete wild populations. However, in water buffalo, there are suggestions that a similar deep maternal bifurcation may have originated from a single population. These hypotheses have rarely been rigorously tested because of a lack of sufficient wild samples. To investigate the origin of the domestic yak (Poephagus grunnies), we analyzed 637 bp of maternal inherited mtDNA from 13 wild yaks (including eight wild yaks from a small population in west Qinghai) and 250 domesticated yaks from major herding regions.Results: The domestic yak populations had two deeply divergent phylogenetic groups with a divergence time of > 100,000 yrs BP. We here show that haplotypes clustering with two deeply divergent maternal lineages in domesticated yaks occur in a single, small, wild population. This finding suggests that all domestic yaks are derived from a single wild gene pool. However, there is no clear correlation of the mtDNA phylogenetic clades and the 10 morphological types of sampled yaks indicating that the latter diversified recently. Relatively high diversity was found in Qinghai and Tibet around the current wild distribution, in accordance with previous suggestions that the earliest domestications occurred in this region. Conventional molecular clock estimation led to an unrealistic early dating of the start of the domestication. However, Bayesian estimation of the coalescence time allowing a relaxation of the mutation rateConclusion: The information gathered here and the previous studies of other animals show that the demographic histories of domestication of livestock species were highly diverse despite the common general feature of deeply divergent maternal lineages. The results further suggest that domestication of local wild prey ungulate animals was a common occurrence during the development of human civilization following the postglacial colonization in different locations of the world, including the high, arid Qinghai-Tibetan Plateau.