Cross-species microarray hybridizations: a developing tool for studying species diversity

The use of cross-species hybridization (CSH) to DNA microarrays, in which the target RNA and microarray probe are from different species, has increased in the past few years. CSH is used in comparative, evolutionary and ecological studies of closely related species, and for gene-expression profiling of many species that lack a representative microarray platform. However, unlike species-specific hybridization, CSH is still considered a non-standard use of microarrays. Here, we present the recent developments in the field of CSH for cDNA and oligomer microarray platforms. We discuss issues that influence the quality of CSH results, including platform choice, experiment design and data analysis, and suggest strategies that can lead to improvement of CSH studies to investigate species diversity.     

土壤微生物多样性研究方法

概述了研究土壤微生物多样性的主要方法．传统上,土壤微生物群落的分析依赖于培养技术,使用各种培养基最大限度地培养各种微生物群体,但仍只能培养和分离出一小部分土壤微生物群落．使用Biolog分析、磷脂脂肪酸分析和核酸分析等方法,可研究和表征那些现在还不能够被培养的土壤微生物。从而获取关于土壤微生物群落多样性的更多和更完整的信息．     

OCAM: a new tool for studying the oligomeric diversity of MscL channels

We have developed a new technique to study the oligomeric state of proteins in solution. OCAM or Oligomer Characterization by Addition of Mass counts protein subunits by selectively shaving a protein mass tag added to a protein subunit via a short peptide linker. Cleavage of each mass tag reduces the total mass of the protein complex by a fixed amount. By performing limited proteolysis and separating the reaction products by size on a blue native PAGE gel, a ladder of reaction products corresponding to the number of subunits can be resolved. The pattern of bands may be used to distinguish the presence of a single homo-oligomer from a mixture of oligomeric states. We have applied OCAM to study the mechanosensitive channel of large conductance (MscL) and find that these proteins can exist in multiple oligomeric states ranging from tetramers up to possible hexamers. Our results demonstrate the existence of oligomeric forms of MscL not yet observed by X-ray crystallography or other techniques and that in some cases a single type of MscL subunit can assemble as a mixture of oligomeric states.     

Experimental model for studying the participation of bone marrow cells in antibody formation

Participation of bone marrow cells in the production of IgM antibody forming cells (AFC) in the primary immune response to sheep red blood cells (SRBC) in C57Bl/6 and BDA/2 mice was studied. The animals of this line differed in sensitivity to preoral benz(a)pyren (BP) injection. After BP injection a toxical injury of bone marrow cells was observed for two days in DBA 2 mice but was not marked in C57Bl/6 mice. In the former it was followed by a 10-fold decrease of IgMAFC, while no profound changes were noticed in the immune response of the latter. A new model is offered for the evaluation of bone marrow cell participation. A suggestion is made concerning some connection of immunodepression in the bone marrow with the change of the stem hemopoietic precursor differentiation.     

Protein dynamics and the diversity of an antibody response

The immune system is remarkable in its ability to produce antibodies (Abs) with virtually any specificity from a limited repertoire of germ line precursors. Although the contribution of sequence diversity to this molecular recognition has been studied for decades, recent models suggest that protein dynamics may also broaden the range of targets recognized. To characterize the contribution of protein dynamics to immunological molecular recognition, we report the sequence, thermodynamic, and time-resolved spectroscopic characterization of a panel of eight Abs elicited to the chromophoric antigen 8-methoxypyrene-1,3,6-trisulfonate (MPTS). Based on the sequence data, three of the Abs arose from unique germ line Abs, whereas the remaining five comprise two sets of siblings that arose by somatic mutation of a common precursor. The thermodynamic data indicate that the Abs recognize MPTS via a variety of mechanisms. Although the spectroscopic data reveal small differences in protein dynamics, the anti-MPTS Abs generally show similar levels of flexibility and conformational heterogeneity, possibly representing the convergent evolution of the dynamics necessary for function. However, one Ab is significantly more rigid and conformationally homogeneous than the others, including a sibling Ab from which it differs by only five somatic mutations. This example of divergent evolution demonstrates that point mutations are capable of fixing significant differences in protein dynamics. The results provide unique insight into how high affinity Abs may be produced that bind virtually any target and possibly, from a more general perspective, how new protein functions are evolved.     

Muscular dysgenesis: a model system for studying skeletal muscle development

Muscular dysgenesis, caused by an autosomal recessive lethal mutation (mdg) in mice, is characterized by an absence of contraction of skeletal muscle. A historical review of the investigation of this disorder is presented. The early studies of the morphological and physiological aspects of the disorder in vivo and in vitro presented evidence for dysfunction in the skeletal muscle excitation-contraction (E-C) system, and thus suggested that skeletal muscle was the primary target of dysfunction in dysgenesis. Subsequent evidence, including the phenomenon of rescue (restoration of contraction) of dysgenic muscle in culture by spinal cord cells, argued for involvement of the nervous system in the disorder. Experiments demonstrating that dysgenic muscle lacks the slow calcium current associated with E-C coupling, and the protein (the dihydropyridine receptor) also associated with such coupling, led to the discovery of the probable site of the mutation: the gene for the alpha 1 subunit of the dihydropyridine receptor. The neuronal involvement hypothesis was further countered by several lines of evidence, including the phenomenon of fusion of nonmyogenic normal cells with dysgenic myotubes in cocultures of normal cells and dysgenic muscle. The use of the mutant as a model for studying the development of normal skeletal muscle is discussed and future avenues of research are explored.     

Catecholamine systems of retina: a model for studying synaptic mechanisms

The retina contains three catecholamine neurotransmitters: dopamine (DA); norepinephrine (NE); and epinephrine (EPI). DA and EPI appear to be associated with separate amacrine neurons that directly participate in the visual process. NE, in contrast, appears to be associated primarily with the sympathetic nerves that innervate the blood vessels of the retina. We present a synopsis of the anatomy, physiology, biochemistry and pharmacology of these retinal neurons. We also suggest that some diseases usually associated with catecholamines of brain may have their counterpart in retina.     

Plant domestication: a model for studying the selection of linkage

The process of domestication leads to acquisition of traits that are often similar between plant species that belong to the same family but have different breeding systems. Hence domestication is a useful model for studying evolutionary responses to selection in plants with contrasting breeding systems. We consider a stochastic model simulating gene flow between a natural population and an initial population containing mutants with domesticated phenotypes at low frequency. We assume that a large number of loci contribute equally to the cultivated phenotype. Our results indicate that the number of loci for which the mutant (‘domestication’) allele is maintained is larger in autogamous plants than in allogamous ones and that domestication can lead to the selection of tightly linked combinations of genes in allogamous plants. This work provides a general model for the selection of gene clusters through a sieve effect and it is discussed in comparison with models proposed to explain the evolution of linkage of genes determining wing patterns in butterflies exhibiting Batesian mimicry.     

Cobblestone HUVECs: a human model system for studying primary ciliogenesis

Primary cilia are microtubule based sensory organelles that play an important role in maintaining cellular homeostasis. Malfunctioning results in a number of abnormalities, diseases (ciliopathies) and certain types of cancer. Morphological and biochemical knowledge on cilia/flagella, (early) ciliogenesis and intraflagellar transport is often obtained from model systems (e.g. Chlamydomonas) or from multi ciliary cells like lung or kidney epithelium.In this study endothelial cells in isolated human umbilical veins (HUVs) and cultured human umbilical vein endothelial cells (HUVECs) are compared and used to study primary ciliogenesis. By combining fluorescence microscopy, SEM, 2D and 3D TEM techniques we found that under the tested culturing conditions 60% of cobblestone endothelial cells form a primary cilium. Only a few of these cilia are present (protruding) on the endothelial cell surface, meaning that most primary cilia are in the cytoplasm (non-protruding). This was also observed in situ in the endothelial cells in the umbilical vein. The exact function(s?) of these non-protruding cilia remains unclear.Ultra-structural analysis of cultured HUVECs and the endothelial layer of the human umbilical veins reveal that there are: vesicles inside the ciliary pocket during the early stages of ciliogenesis; tubules/vesicles from the cytoplasm fuse with the ciliary sheath; irregular axoneme patterns, and two round, membranous vesicles inside the basal body.We conclude that cobblestone cultured HUVECs are comparable to the in vivo epithelial lining of the umbilical veins and therefore provide a well defined, relatively simple human model system with a reproducible number of non-protruding primary cilia for studying ciliogenesis.     

The zebrafish as a model for studying neuroblastoma

Neuroblastoma is a tumor arising in the peripheral sympathetic nervous system and is the most common cancer in childhood. Since most of the cellular and molecular mechanisms underlying neuroblastoma onset and progression remain unknown, the generation of new in vivo models might be appropriate to better dissect the peripheral sympathetic nervous system development in both physiological and disease states. This review is focused on the use of zebrafish as a suitable and innovative model to study neuroblastoma development. Here, we briefly summarize the current knowledge about zebrafish peripheral sympathetic nervous system formation, focusing on key genes and cellular pathways that play a crucial role in the differentiation of sympathetic neurons during embryonic development. In addition, we include examples of how genetic changes known to be associated with aggressive neuroblastoma can mimic this malignancy in zebrafish. Thus, we note the value of the zebrafish model in the field of neuroblastoma research, showing how it can improve our current knowledge about genes and biological pathways that contribute to malignant transformation and progression during embryonic life.     

Use of numerical profiles for studying bacterial diversity

A total of 308 bacteria were isolated from oil-storage tanks. Of these 20% were unidentifiable, even at the generic level. A numerical scoring method differentiated between the isolates and was used to estimate the diversity of the bacterial communities in the tanks over a period of 11 months. Although the scoring method suggested a higher diversity than did conventional identification, there was some consistency in the results produced by the two approaches. It is suggested that a scoring method based on only nine tests could be useful for estimating and comparing bacterial diversity in other habitats.     

Muscular dysgenesis in mice: a model system for studying excitation-contraction coupling

Muscular dysgenesis (mdg) is a lethal autosomal, recessive mutation of mice. Skeletal muscle from dysgenic mice is paralyzed due to the failure of excitation-contraction (E-C) coupling. Considerable evidence indicates that this failure results from the absence of a specific gene product, the alpha 1 subunit of the skeletal muscle receptor for dihydropyridine calcium channel modifiers. This dihydropyridine receptor is hypothesized to function in E-C coupling of normal skeletal muscle as the voltage sensor that triggers calcium release from the sarcoplasmic reticulum and thereby causes contraction. The skeletal muscle dihydropyridine receptor is also postulated to function as the ion channel responsible for a slowly activating, dihydropyridine-sensitive calcium current (Islow). Dysgenic skeletal muscle lacks Islow but expresses, at low levels, a distinctly different dihydropyridine-sensitive calcium current (Idys). The channel protein underlying Idys is incapable of serving as a voltage sensor for E-C coupling. Studies using dysgenic skeletal muscle have provided significant insight into the role of dihydropyridine receptors in E-C coupling.     

Flagellar regeneration in Chlamydomonas: a model system for studying organelle assembly

How do the many different components of an organelle assemble into a functional structure at an appropriate place and time? Flagellar regeneration by the biflagellate green alga Chlamydomonas is one experimental system in which genetics, biochemistry and ultrastructural analysis are being combined to investigate the assembly of a microtubule-containing organelle. Recent advances in the molecular biology of this 'green yeast' have made possible several new approaches to the problem of flagellar assembly; insights from these new approaches are the focus of this review.