Novel dendritic kinesin sorting identified by different process targeting of two related kinesins: KIF21A and KIF21B

Neurons use kinesin and dynein microtubule-dependent motor proteins to transport essential cellular components along axonal and dendritic microtubules. In a search for new kinesin-like proteins, we identified two neuronally enriched mouse kinesins that provide insight into a unique intracellular kinesin targeting mechanism in neurons. KIF21A and KIF21B share colinear amino acid similarity to each other, but not to any previously identified kinesins outside of the motor domain. Each protein also contains a domain of seven WD-40 repeats, which may be involved in binding to cargoes. Despite the amino acid sequence similarity between KIF21A and KIF21B, these proteins localize differently to dendrites and axons. KIF21A protein is localized throughout neurons, while KIF21B protein is highly enriched in dendrites. The plus end-directed motor activity of KIF21B and its enrichment in dendrites indicate that models suggesting that minus end-directed motor activity is sufficient for dendrite specific motor localization are inadequate. We suggest that a novel kinesin sorting mechanism is used by neurons to localize KIF21B protein to dendrites since its mRNA is restricted to the cell body.     

Analysis of the genusZea (Poaceae) using polymorphic chloroplast simple sequence repeats

We have used polymorphic chloroplast simple sequence repeats (cpSSRs) to analyse levels of diversity and relationships within the genusZea. Between two and nine alleles were found at 15 polymorphic loci and combining the data from these loci gave 32 haplotypes in the 37 accessions studied. Genetic differentiation between the two sections within the genus was calculated using theST statistic which showed that 70% of the total variation was found to exist between the sections. A phylogenetic analysis based on the ² distance metric showed a large split between the two sections and suggested multiple origins of modern cultivated maizeZea mays subsp.mays. The agreement of the phylogenetic tree with other molecular, morphological and karyological studies suggests that cpSSRs may have value in phylogenetic studies in plants.     

α1β1-Integrin is an essential signal for neurite outgrowth induced by thrombospondin type 1 repeats of SCO-spondin

In the central and peripheral nervous systems a heterogeneous group of proteins constituting the thrombospondin superfamily provides a cue for axonal pathfinding. They either contain or are devoid of the tripeptide RGD, and the sequence(s) and mechanism(s) which trigger in vitro their neurite-promoting activity have remained unclear. In this study, we reconsider the problem of whether sequences present in the thrombospondin type 1 repeats (TSRs), and independent of the well-known RGD-binding site, may activate integrins and account for their neurite-promoting activity. SCO-spondin is a newly identified member of the thrombospondin superfamily, which shows a multidomain organization with a great number of TSR motifs but no RGD sequence. Previous research has implicated oligopeptides derived from SCO-spondin TSRs in in-vitro development of various neuronal cell types. In this study, we investigate whether function-blocking antibodies directed against integrin subunits can block these effects in cell line B104, cloned from a neuroblastoma of the rat central nervous system. By two different approaches: flow cytometry revealing short-term effects and cell cultures revealing long-term effects, we show that: (a) activation of cell metabolism, (b) changes in cell size and structure, and (c) neurite-promoting activity induced by TSR oligopeptides are inhibited by function-blocking antibodies to 1-subunit. Using a panel of function-blocking antibodies directed against various integrin -subunits we show that the 1-subunit might be the partner of the 1-subunit in B104 cells. Thus, we demonstrate that an original sequence within a TSR motif from SCO-spondin promotes neurite outgrowth through an intracellular signal driven by integrins, independently of an RGD-binding site.     

Retrotransposon-gene associations are widespread among D. melanogaster populations

We have surveyed 18 natural populations of Drosophila melanogaster for the presence of 23 retrotransposon-gene-association alleles (i.e., the presence of an LTR retrotransposon sequence in or within 1,000 bp of a gene) recently identified in the sequenced D. melanogaster genome. The identified associations were detected only in the D. melanogaster populations. The majority (61%) of the identified retrotransposon-gene associations were present only in the sequenced strain in which they were first identified. Thirty percent of the associations were detected in at least one of the natural populations, and 9% of the associations were detected in all of the D. melanogaster populations surveyed. Sequence analysis of an association allele present in all populations indicates that selection is a significant factor in the spread and/or maintenance of at least some of retroelement-gene associations in D. melanogaster.     

Inter-simple sequence repeat markers and flow cytometry for the characterization of closely related Citrus limon germplasms

The genetic relationship among commercial cultivars of Citrus limon (lemon) was analysed by inter-simple sequence repeats (ISSR) and flow cytometry techniques. Two cultivars with a close germplasm were distinguished by screening 10 SSR primers and by measuring DNA content of prestained nuclei.     

Structural analysis of DNA-PKcs: modelling of the repeat units and insights into the detailed molecular architecture

DNA-dependent protein kinase (DNA-PK) is part of the eukaryotic DNA double strand break repair pathway and as such is crucial for maintenance of genomic stability, as well as for V(D)J (variable-diversity-joining) recombination. The catalytic subunit of DNA-PK (DNA-PKcs) belongs to the phosphatidylinositol-3 (PI-3) kinase-like kinase (PIKK) superfamily and is comprised of approximately 4100 amino acids. We have used a novel repeat detection method to analyse this enormous protein and have identified two different types of helical repeat motifs in the N-terminal region of the sequence, as well as other previously unreported features in this repeat region. A comparison with the ATMs, ATRs, and TORs show that the features identified are likely to be conserved throughout the PIKK superfamily. Homology modelling of parts of the DNA-PKcs sequence has been undertaken and we have been able to fit the models to previously obtained electron microscopy data. This work provides an insight into the overall architecture of the DNA-PKcs protein and identifies regions of interest for further experimental studies.     

Unpaired structures in SCA10 (ATTCT)n.(AGAAT)n repeats

A number of human hereditary diseases have been associated with the instability of DNA repeats in the genome. Recently, spinocerebellar ataxia type 10 has been associated with expansion of the pentanucleotide repeat (ATTCT)(n).(AGAAT)(n) from a normal range of ten to 22 to as many as 4500 copies. The structural properties of this repeat cloned in circular plasmids were studied by a variety of methods. Two-dimensional gel electrophoresis and atomic force microscopy detected local DNA unpairing in supercoiled plasmids. Chemical probing analysis indicated that, at moderate superhelical densities, the (ATTCT)(n).(AGAAT)(n) repeat forms an unpaired region, which further extends into adjacent A+T-rich flanking sequences at higher superhelical densities. The superhelical energy required to initiate duplex unpairing is essentially length-independent from eight to 46 repeats. In plasmids containing five repeats, minimal unpairing of (ATTCT)(5).(AGAAT)(5) occurred while 2D gel analysis and chemical probing indicate greater unpairing in A+T-rich sequences in other regions of the plasmid. The observed experimental results are consistent with a statistical mechanical, computational analysis of these supercoiled plasmids. For plasmids containing 29 repeats, which is just above the normal human size range, flanked by an A+T-rich sequence, atomic force microscopy detected the formation of a locally condensed structure at high superhelical densities. However, even at high superhelical densities, DNA strands within the presumably compact A+T-rich region were accessible to small chemicals and oligonucleotide hybridization. Thus, DNA strands in this "collapsed structure" remain unpaired and accessible for interaction with other molecules. The unpaired DNA structure functioned as an aberrant replication origin, in that it supported complete plasmid replication in a HeLa cell extract. A model is proposed in which unscheduled or aberrant DNA replication is a critical step in the expansion mutation.     

Reticulate Evolution of Parthenospecies of the Lacertidae Rock Lizards: Inheritance of CLsat Tandem Repeats and Anonymous RAPD Markers

The genetic relatedness of several bisexual and of four unisexual Lacerta saxicola complex lizards was studied, using monomer sequences of the complex-specific CLsat tandem repeats and anonymous RAPD markers. Genomes of parthenospecies were shown to include different satellite monomers. The structure of each such monomer is specific for a certain pair of bisexual species. This fact might be interpreted in favor of co-dominant inheritance of these markers in bisexual species hybridogenesis. This idea is supported by the results obtained with RAPD markers; i.e., unisexual species genomes include only the loci characteristic of certain bisexual species. At the same time, in neither case parthenospecies possess specific, autoapomorphic loci that were not present in this or that bisexual species.