Evolution of Conserved Non-Coding Sequences Within the Vertebrate Hox Clusters Through the Two-Round Whole Genome Duplications Revealed by Phylogenetic Footprinting Analysis

As a result of two-round whole genome duplications, four or more paralogous Hox clusters exist in vertebrate genomes. The paralogous genes in the Hox clusters show similar expression patterns, implying shared regulatory mechanisms for expression of these genes. Previous studies partly revealed the expression mechanisms of Hox genes. However, cis-regulatory elements that control these paralogous gene expression are still poorly understood. Toward solving this problem, the authors searched conserved non-coding sequences (CNSs), which are candidates of cis-regulatory elements. When comparing orthologous Hox clusters of 19 vertebrate species, 208 intergenic conserved regions were found. The authors then searched for CNSs that were conserved not only between orthologous clusters but also among the four paralogous Hox clusters. The authors found three regions that are conserved among all the four clusters and eight regions that are conserved between intergenic regions of two paralogous Hox clusters. In total, 28 CNSs were identified in the paralogous Hox clusters, and nine of them were newly found in this study. One of these novel regions bears a RARE motif. These CNSs are candidates for gene expression regulatory regions among paralogous Hox clusters. The authors also compared vertebrate CNSs with amphioxus CNSs within the Hox cluster, and found that two CNSs in the HoxA and HoxB clusters retain homology with amphioxus CNSs through the two-round whole genome duplications.     

Cytochemical and electron microscopic demonstration of organelle translocation and the inhibition of such translocation by colchicine during the mitosis of rat hepatocytes

Hepatocyte lysosomes, mitochondria, and peroxisomes show a dramatic translocation during mitosis induced by partial hepatectomy. During prophase, all three organelles move to the perinuclear cytoplasm. In metaphase, they become concentrated in the polar regions. During telophase, these organelles form clusters in the juxtanuclear regions. This organelle translocation is inhibited by the administration of a low concentration of colchicine, suggesting an involvement of microtubules in their movement.     

Chromosome distribution of intracisternal A-particle sequences in the Syrian hamster and mouse

Metaphase chromosomes of Syrian hamster and BALB/c mice were hybridized in situ with radiolabeled probes derived from cloned intracisternal A-particle (IAP) genes of the corresponding species. The DNAs of these species are known to contain about 900 and 1,000 copies, respectively, of the retrovirus-like IAP sequence elements per haploid genome. Multiple IAP sequences were found on all chromosomes of both hamster and mouse. In the hamster, more than half of the IAP sequences were located in regions of non-centromeric constitutive heterochromatin, at an average concentration per unit chromosome length 5 times greater than in the euchromatic regions. The other dispersed sequences showed marked local variations in concentration along the chromosome lengths; both discrete foci and large grain clusters were observed as well as regions apparently lacking IAP sequences. Within the resolution of the techniques, IAP sequences appeared to be more evenly distributed over the mouse chromosomes; however, some prominent variations in concentration were seen. The number of potentially active IAP genes in the Syrian hamster, and by extension in the mouse, may be restricted by the preferential location of IAP sequences in genetically inert regions of the genome.     

Acetylcholine receptor clusters are associated with nuclei in rat myotubes

Clustered and diffuse acetylcholine receptors are present in cultured myotubes. These clustered AChRs represent regions of myotube membrane containing high receptor density. We have studied the distribution of the AChR clusters and nuclei to determine whether there is an association in the distribution of nuclei beneath AChR clusters. AChR clusters were visualized with alpha-bungarotoxin conjugated to tetramethylrhodamine (alpha BTX-TMR) and the nuclei were stained with bisbenzimide which binds specifically to DNA. This double label procedure, and the computerized analysis of the data allowed us to determine the distribution of nuclei and AChR clusters in the same myotube. During early stages of myotube development the nuclei formed aggregates which were comprised of 4 to 10 nuclei in close apposition to one another. This association of AChR clusters with nuclear aggregates was greatest at Day 4 after plating. As the number of nuclear aggregates associated with clusters decreased the number of nuclei in the aggregates also decreased and the AChR clusters decreased in size as well as number. At all time points examined, the concentration of myotube nuclei in the cells was 3 to 12 times higher beneath areas of AChR clusters than away from clusters. Our computerized analysis shows that there is an association of the AChR clusters with the nuclear region during myotube development.     

Two plus two does not equal three: statistical tests for multiple genome comparison

Gene clusters that span three or more chromosomal regions are of increasing importance, yet statistical tests to validate such clusters are in their infancy. Current approaches either conduct several pairwise comparisons or consider only the number of genes that occur in all of the regions. In this paper, we provide statistical tests for clusters spanning exactly three regions based on genome models of typical comparative genomics problems, including analysis of conserved linkage within multiple species and identification of large-scale duplications. Our tests are the first to combine evidence from genes shared among all three regions and genes shared between pairs of regions. We show that our tests of clusters spanning three regions are more sensitive than existing approaches, and can thus be used to identify more diverged homologous regions.     

Brain extract causes acetylcholine receptor redistribution which mimics some early events at developing neuromuscular junctions

We studied the effect of rat brain extract on rat muscle cells in vitro by light and electron microscope (EM) autoradiography after labeling acetylcholine receptors (AChR's) with 125I-alpha-bungarotoxin. We found that: (a) In the absence of brain extract, peak site densities within AChR clusters usually do not exceed 4,000 sites/micrometer2. (b) Within hours after exposure to brain extract, AChR's redistribute to form clusters in which the peak site densities are greater than 10,000 sites/micrometer2. Receptor concentration within extract-induced clusters is thus within a factor of 2 of that at the neuromuscular junction (nmj). (c) In the absence of extract, the AChR's and AChR clusters are predominantly on the bottom surface of the myotubes (facing the tissue culture dish). After extract treatment, they are predominantly at the top surface. (d) Plasma membrane in regions of high-density AChR clusters is enriched in membrane with enhanced electron density and surface basal lamina whether or not cells are treated with extract. Extract causes an increase in both these specializations on the top surface of the myotubes. (e) Brain extract does not produce an overall increase in AChR site density or a marked change in degradation rate of receptors in either clustered or nonclustered regions. By producing AChR clusters with junctional site densities and enhanced surface specialization, and by causing an overall shift in AChR's distribution, brain extract mimics early events reported at developing neuromuscular junctions.     

Photobleaching. A novel fluorescence method for diffusion studies in lipid system.

(1) The fluorescent molecular 12(9-anthroyloxy)-stearic acid dimerises on irradiation with light of 366 nm wavelength. (2) The dimer is nonfluorescent and can be reconverted to the parent compound by irradiation at 254 nm. (3) Kinetic analysis suggests that the dimerisation proceeds by a diffusion-limited second order mechanism in many solvents. (4) Anomalously high rates seen in other systems can be attributed to localised high concentration regions (clusters) of the fluorescent molecule. (5) The analysis has been extended to oriented lipid bilayers and the results suggest that below the gel-liquid crystalline transition temperature the 12(9-anthroyloxy)-stearic acid is excluded by the lipid matrix and forms regions of localised high concentration. (6) In fluid lipid the results suggest an isotropic distribution of the probe. Calculated diffusion coefficients correspond to those found by other techniques.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

Identification of unique transcripts from a mouse full-length, subtracted inner ear cDNA library

A small-scale full-length library construction approach was developed to facilitate production of a mouse full-length cDNA encyclopedia representing approximately 250 enriched, normalized, and/or subtracted cDNA libraries. One library produced using this approach was a subtracted adult mouse inner ear cDNA library (sIEa). The average size of the inserts was approximately 2.5 kb, with the majority ranging from 0.5 to 7.0 kb. From this library 22,574 sequence reads were obtained from 15,958 independent clones. Sequencing and chromosomal localization established 5240 clusters, with 1302 clusters being unique and 359 representing new ESTs. Our sIEa library contributed 56.1% of the 7773 nonredundant Unigene clusters associated with the four mouse inner ear libraries in the NCBI dbEST. Based on homologous chromosomal regions between human and mouse, we identified 1018 UniGene clusters associated with the deafness locus critical regions. Of these, 59 clusters were found only in our sIEa library and represented approximately 50% of the identified critical regions.     

Monitoring expression and clustering of the ionotropic 5HT3 receptor in plasma membranes of live biological cells

The ionotropic 5HT(3) receptor was expressed in transiently transfected mammalian cells, yielding an unprecedented high concentration of up to 12 million receptors per cell. Receptor traffic in the plasma membrane of live cells was observed continuously over 24 h by fluorescence scanning confocal microscopy. This was possible by using 5HT(3) receptor-specific fluorescent ligands with high binding affinity and low off-rate to pulse label receptors at any time after appearance on the cell surface, and label subsequently those receptors expressed later by another, spectrally distinguishable, high-affinity fluorescent ligand. Having reached a critical cell surface concentration of approximately 3000 receptors/microm(2), the receptors started to aggregate in patches with a 4-fold increased surface concentration. The clusters were constantly delivered from a pool of freshly expressed receptors isotropically distributed within the basolateral region of the cell membrane. From there, they migrated to and accumulated on the apical cell surface approximately 9 h after transfection. Individual clusters grew until they reached a critical size of 1-2 microm when they merged to form with 3-5 microm large macroclusters. Clustered receptors were immobile on the minute time scale but always coexisted with monomeric receptors in the regions surrounding the clusters as revealed by fluorescence correlation spectroscopy. Because the receptor density of 12 000 receptors/microm(2) in the patches is as high as that found in two-dimensional crystals of certain membrane proteins, such patches might be a proper source for direct crystallization of membrane proteins without prior purification.     

Probabilistic Clustering of the Human Connectome Identifies Communities and Hubs

A fundamental assumption in neuroscience is that brain function is constrained by its structural properties. This motivates the idea that the brain can be parcellated into functionally coherent regions based on anatomical connectivity patterns that capture how different areas are interconnected. Several studies have successfully implemented this idea in humans using diffusion weighted MRI, allowing parcellation to be conducted in vivo. Two distinct approaches to connectivity-based parcellation can be identified. The first uses the connection profiles of brain regions as a feature vector, and groups brain regions with similar connection profiles together. Alternatively, one may adopt a network perspective that aims to identify clusters of brain regions that show dense within-cluster and sparse between-cluster connectivity. In this paper, we introduce a probabilistic model for connectivity-based parcellation that unifies both approaches. Using the model we are able to obtain a parcellation of the human brain whose clusters may adhere to either interpretation. We find that parts of the connectome consistently cluster as densely connected components, while other parts consistently result in clusters with similar connections. Interestingly, the densely connected components consist predominantly of major cortical areas, while the clusters with similar connection profiles consist of regions that have previously been identified as the ‘rich club’; regions known for their integrative role in connectivity. Furthermore, the probabilistic model allows quantification of the uncertainty in cluster assignments. We show that, while most clusters are clearly delineated, some regions are more difficult to assign. These results indicate that care should be taken when interpreting connectivity-based parcellations obtained using alternative deterministic procedures.     

Site-specific maturation of enveloped viruses in L cells treated with cytochalasin B

Treatment of infected L cells with 10 micrograms/ml cytochalasin B (CB) was found to promote a rapid relocalization of viral glycoproteins on the cell surface. Whereas the vesicular stomatitis virus G protein and the influenza virus hemagglutinin were uniformly distributed on the surface of untreated cells, in CB-treated cells, they were strikingly concentrated at cell extremities in the regions of clustered blebs. Glycoprotein concentration at cell extremities was accompanied by preferential maturation of virus particles from the same sites; both vesicular stomatitis and influenza viruses budded predominantly from the vicinity of clustered blebs. This effect of CB was completely reversible. Removal of CB from the cell growth medium resulted in a return of viral glycoproteins to the uniform distribution characteristic of untreated cells and to uniform virus budding. The results of this study are interpreted in terms of a model that suggests that preferential budding of viruses from the regions of bleb clusters is due to the concentration of viral glycoproteins at these sites.     

A morphometric study of the spatial patterns of TDP-43 immunoreactive neuronal inclusions in frontotemporal lobar degeneration (FTLD) with progranulin (GRN) mutation

Mutations of the progranulin (GRN) gene are a major cause of familial frontotemporal lobar degeneration with transactive response (TAR) DNA-binding protein of 43 kDa (TDP-43) proteinopathy (FTLD-TDP). We studied the spatial patterns of TDP-43 immunoreactive neuronal cytoplasmic inclusions (NCI) and neuronal intranuclear inclusions (NII) in histological sections of the frontal and temporal lobe in eight cases of FTLD-TDP with GRN mutation using morphometric methods and spatial pattern analysis. In neocortical regions, the NCI were clustered and the clusters were regularly distributed parallel to the pia mater; 58% of regions analysed exhibiting this pattern. The NII were present in regularly distributed clusters in 35% of regions but also randomly distributed in many areas. In neocortical regions, the sizes of the regular clusters of NCI and NII were 400-800 μm, approximating to the size of the modular columns of the cortico-cortical projections, in 31% and 36% of regions respectively. The NCI and NII also exhibited regularly spaced clustering in sectors CA1/2 of the hippocampus and in the dentate gyrus. The clusters of NCI and NII were not spatially correlated. The data suggest degeneration of the cortico-cortical and cortico-hippocampal pathways in FTLD-TDP with GRN mutation, the NCI and NII affecting different clusters of neurons.     

Clusters of intramembrane particles associated with binding sites for alpha-bungarotoxin in cultured chick myotubes

Developing chick myotubes in tissue culture were freeze-fractured to yield complementary replicas of large areas of membrane. Regions of muscle fibers with high concentrations of acetylcholine receptors were identified by binding of fluorescent-labeled alpha-bungarotoxin. Membranes in such regions contained clusters of large (100 A Diam) angular particles, similar in appearance to particles found in postsynaptic membranes of cholinergic synapses. Particles appeared in apposing areas of cytoplasmic and external leaflets but were more prevalent in the cytoplasmic leaflet. The areas of high particle concentration were coextensive with the fluorescence due to bound toxin. Treatment of cultures with tetrodotoxin increased the size of fluorescent spots and areas of high concentration of particles relative to those found in control cultures. In muscle cultures grown in the presence of spinal cord explants, some neurites contacted and innervated nearby myotubes. Intense fluorescence due to binding or alpha-bungarotoxin was present at portions of such neurite-myotube contacts. At these same portions, a high concentration of large angular particles was present in the sarcolemma adjacent to the neurite. In addition, an ordered arrangement of large particles was seen in the cytoplasmic leaflet of the neuronal plasmalemma directly apposing the muscle. The possible significance of these arrangements is discussed. Clusters on myotubes tended to be larger (contain more particles) when they occurred in groups, defined as three or more clusters with an intercluster distance of less than 0.5 micrometers. Clusters were also larger in myotubes treated with tetrodotoxin and in myotubes adjacent to some neurites in nerve-muscle cocultures. Several depressions containing particles similar to those in the clusters were found in the sarcolemma. The implications of these depressions are discussed in light of current theories of incorporation of proteins into cell membranes.     

Identification and High-Density Mapping of Gene-Rich Regions in Chromosome Group 5 of Wheat

The distribution of genes and recombination in the wheat genome was studied by comparing physical maps with the genetic linkage maps. The physical maps were generated by mapping 80 DNA and two phenotypic markers on an array of 65 deletion lines for homoeologous group 5 chromosomes. The genetic maps were constructed for chromosome 5B in wheat and 5D in Triticum tauschii. No marker mapped in the proximal 20% chromosome region surrounding the centromere. More than 60% of the long arm markers were present in three major clusters that physically encompassed <18% of the arm. Because 48% of the markers were cDNA clones and the distributions of the cDNA and genomic clones were similar, the marker distribution may represent the distribution of genes. The gene clusters were identified and allocated to very small chromosome regions because of a higher number of deletions in their surrounding regions. The recombination was suppressed in the centromeric regions and mainly occurred in the gene-rich regions. The bp/cM estimates varied from 118 kb for gene-rich regions to 22 Mb for gene-poor regions. The wheat genes present in these clusters are, therefore, amenable to molecular manipulations parallel to the plants with smaller genomes like rice.     

Intercalary heterochromatin in the genome of Drosophila

The modern concept of intercalary heterochromatin as polytene chromosome regions exhibiting a number of specific characteristics is formulated. DNA constituting these regions is replicated late in the S period; therefore, some strands of polytene chromosomes are underrepresented; i.e., they are underreplicated. Late-replicating regions account for about 7% of the genome; genes are located there in clusters of as many as 40. In general, the gene density in the clusters is substantially lower than in the main part of the genome. Late-replicating regions have an inactivating capacity: genes incorporated into these regions as parts of transposons are inactivated with a higher probability. These regions contain a specific protein SUUR affecting the rate of replication completion.     

Backbone cluster identification in proteins by a graph theoretical method

A graph theoretical algorithm has been developed to identify backbone clusters of residues in proteins. The identified clusters show protein sites with the highest degree of interactions. An adjacency matrix is constructed from the non-bonded connectivity information in proteins. The diagonalization of such a matrix yields eigenvalues and eigenvectors, which contain the information on clusters. In graph theory, distinct clusters can be obtained from the second lowest eigenvector components of the matrix. However, in an interconnected graph, all the points appear as one single cluster. We have developed a method of identifying highly interacting centers (clusters) in proteins by truncating the vector components of high eigenvalues. This paper presents in detail the method adopted for identifying backbone clusters and the application of the algorithm to families of proteins like RNase-A and globin. The objective of this study was to show the efficiency of the algorithm as well as to detect conserved or similar backbone packing regions in a particular protein family. Three clusters in topologically similar regions in the case of the RNase-A family and three clusters around the porphyrin ring in the globin family were observed. The predicted clusters are consistent with the features of the family of proteins such as the topology and packing density. The method can be applied to problems such as identification of domains and recognition of structural similarities in proteins.     

GM1-containing lipid rafts are depleted within clathrin-coated pits

Recent studies show that markers for lipid rafts are among the plasma membrane components most likely to be internalized independently of clathrin-coated pits, and there is evidence to suggest that lipid rafts may play a functional role in endocytic trafficking [1-5]. However, lipid rafts themselves are commonly defined purely in biochemical terms, by resistance to detergent extraction. The existence of rafts in live-cell membranes remains controversial [6-8], and their distribution relative to endocytic machinery has not been investigated. This study employs fluorescence resonance energy transfer (FRET) to show that in the plasma membrane (PM) of living cells the glycosphingolipid GM1, labeled with cholera toxin B subunit (CTB) [9,10], is found at least in part within clusters that also include GPI-linked proteins. These clusters are cholesterol-dependent and exclude non-raft proteins such as transferrin receptor and so possess predicted properties of lipid rafts. This type of lipid raft is largely excluded from clathrin-positive regions of the PM. They are found within Caveolin-positive regions at the same concentration as at the rest of the cell surface. The data provide evidence for a model in which lipid rafts are distributed uniformly across most of the PM of nonpolarized cells but are prevented from entering clathrin-coated pits.     

Association of beta-cytoplasmic actin with high concentrations of acetylcholine receptor (AChR) in normal and anti-AChR-treated primary rat muscle cultures

Previous immunocytochemical studies in which an antibody specific for mammalian cytoplasmic actin was used showed that a high concentration of cytoplasmic actin exists at neuromuscular junctions of rat muscle fibers such that the distribution of actin corresponded exactly to that of the acetylcholine receptors. Although clusters of acetylcholine receptors also are present in noninnervated rat and chick muscle cells grown in vitro, neither the mechanism for the formation and maintenance of these clusters nor the relationship of these clusters to the high density of acetylcholine receptors at the neuromuscular junction in vivo are known. In the present study, a relationship between beta-cytoplasmic actin and acetylcholine receptors in vitro has been demonstrated immunocytochemically using an antibody specific for the beta-form of cytoplasmic actin. Networks of cytoplasmic actin-containing filaments were found in discrete regions of the myotube membrane that also contained high concentrations of acetylcholine receptors; such high concentrations of acetylcholine receptors have been described in regions of membrane-substrate contact. Moreover, when primary rat myotubes were exposed to human myasthenic serum, gross morphological changes, accompanied by an apparent rearrangement of the cytoplasmic actin-containing cytoskeleton, were produced. Although whether the distribution of cytoplasmic actin-containing structures was influenced by the organization of acetylcholine receptor or vice versa cannot be determined from these studies, these findings suggest that in primary rat muscle cells grown in vitro, acetylcholine receptors and beta-cytoplasmic actin-containing structures may be somehow connected.