Characterization of microsatellites and repetitive flanking sequences (ReFS) from the topmouth culter (Culter alburnus Basilewsky)

The topmouth culter (Culter alburnus) is an economically important freshwater fish in China. We obtained 159 microsatellite containing sequences (MCSs) from genomic DNA in this species enriched by (CAA)₈ and (GAA) ₈ probes. Careful examination of these sequences revealed the existence of cryptic repeated elements on presumed unique flanking regions. These cryptic elements can be grouped into three families, with the MCSs of the each family sharing regions of similarity ranging between 40 and 130 bp in length, with 96% sequence similarity. Repbase scans revealed that a large proportion of the cryptic repetitive DNA was identified as transposable elements (TEs). Complex patterns were apparent among these sequences. In most (89.2%), a single TE was identified in an MCS, in three instances, the same TE was observed twice in the same MCS. Some MCS have two or even four different TEs. We isolated nine polymorphic microsatellite loci from sequences with no matches to TEs. In a sample of 30 cultured C. alburnus, we found that the average allele number was 8.1 per locus (range = 4–17), with polymorphism informative content ranging from 0.364 to 0.898. These microsatellites can be used to study the population genetic diversity of this species.     

Selective inhibition of Alu retrotransposition by APOBEC3G

The non-LTR retrotransposon LINE-1 (L1) comprises  17% of the human genome, and the L1-encoded proteins can function in trans to mediate the retrotransposition of non-autonomous retrotransposons (i.e., Alu and probably SVA elements) and cellular mRNAs to generate processed pseudogenes. Here, we have examined the effect of APOBEC3G and APOBEC3F, cytidine deaminases that inhibit Vif-deficient HIV-1 replication, on Alu retrotransposition and other L1-mediated retrotransposition processes. We demonstrate that APOBEC3G selectively inhibits Alu retrotransposition in an ORF1p-independent manner. An active cytidine deaminase site is not required for the inhibition of Alu retrotransposition and the resultant integration events lack G to A or C to T hypermutation. These data demonstrate a differential restriction of L1 and Alu retrotransposition by APOBEC3G, and suggest that the Alu ribonucleoprotein complex may be targeted by APOBEC3G.     

Isolation, analysis and marker utility of novel miniature inverted repeat transposable elements from the barley genome

Four previously undescribed families of miniature inverted repeat transposable elements (MITEs) were isolated by searching barley genomic DNA using structure-based criteria. Putative MITEs were confirmed by PCR to determine their insertional polymorphism in a panel of diverse barley germplasm. Copy numbers for all these familes are somewhat low (less than 1,000 copies per family per haploid genome). In contrast to previous studies, a higher proportion of insertions of the new MITEs are found within known transposable elements (27%) than are associated with genes (15%). Preliminary studies were conducted on two of the new MITE families to test their utility as molecular markers. Insertional polymorphism levels for both the families are high and diversity trees produced by both the families are similar and congruent with known relationships among the germplasm studied, suggesting that both the MITE families are useful markers of barley genetic diversity.     

Ty1-copia group retrotransposon sequences in amphibia and reptilia

We have isolated sequences belonging to Tyl-copia group retrotransposons from the genomes of an amphibian (Pyxicephalus adspersa) and three reptiles (Conolophus subscristatus, Amblyrynchus cristatus and Pytas mucosus). Two different seqences were found in the amphibian (Tpa1 and Tpa2). Each is present in several copies per genome and absent from the genomes of two other amphibian species. The C. subcristatus sequence Tcs1 is present in multiple copies in both its host genome (Galapagos land iguana) and the genome of the related Galapagos marine iguana (A. cristatus). There is little or no polymorphism in Tcs1 insertions between different individual animals, suggesting that this sequence is not transposing rapidly in either iguana genome. The P. mucosus sequence Tpm1 shows a discontinuous distribution in snake species, suggesting that it has either been lost from many lineages during vertical germline transmission or has been transferred horizontally in some snake species. Phylogenetic comparisons of all these sequences with each other and with other members of this retrotransposon group from other animals and plants show that sequences within a particular vertebrate species are most closely related to each other, consistent with a vertical transmission model for their evolution.     

Isolation and characterization of Tnd-1, a retrotransposon marker linked to black root rot resistance in tobacco

 In Nicotiana debneyi, resistance to a wide range of black root rot (Chalara elegans) isolates is conferred by a single dominant gene. This gene has been transferred to cultivated tobacco (Nicotiana tabacum) and was recently discovered to be linked in coupling to a 1050-bp random amplified polymorphic DNA (RAPD) marker generated with the UBC 418 primer. We have cloned and sequenced the UBC418₁₀₅₀ marker and found it to be part of a retrotransposon. This retrotransposon is a remnant of the N. debneyi genome and is the first to be isolated from this species. Transposon N. debneyi (Tnd)-1 is present in the tobacco genome as two directly repeated copies, and in multiple copies in the donor species N. debneyi and in a number of related Nicotiana species. The retrotransposon appears to have been introduced into the Nicotiana genome after the development of the Suavolentes progenitors. The gene associated with black root rot resistance co-segregates with the retrotransposon in tobacco and is thought to be contained within the introgressed fragment marked by Tnd-1. The retrotransposon will therefore be a useful species-specific landmark that can be used for future cloning of the resistance gene. Received: 3 March 1998 / Accepted: 18 August 1998     

Updating of transposable element annotations from large wheat genomic sequences reveals diverse activities and gene associations

Triticeae species (including wheat, barley and rye) have huge and complex genomes due to polyploidization and a high content of transposable elements (TEs). TEs are known to play a major role in the structure and evolutionary dynamics of Triticeae genomes. During the last 5 years, substantial stretches of contiguous genomic sequence from various species of Triticeae have been generated, making it necessary to update and standardize TE annotations and nomenclature. In this study we propose standard procedures for these tasks, based on structure, nucleic acid and protein sequence homologies. We report statistical analyses of TE composition and distribution in large blocks of genomic sequences from wheat and barley. Altogether, 3.8 Mb of wheat sequence available in the databases was analyzed or re-analyzed, and compared with 1.3 Mb of re-annotated genomic sequences from barley. The wheat sequences were relatively gene-rich (one gene per 23.9 kb), although wheat gene-derived sequences represented only 7.8% (159 elements) of the total, while the remainder mainly comprised coding sequences found in TEs (54.7%, 751 elements). Class I elements [mainly long terminal repeat (LTR) retrotransposons] accounted for the major proportion of TEs, in terms of sequence length as well as element number (83.6% and 498, respectively). In addition, we show that the gene-rich sequences of wheat genome A seem to have a higher TE content than those of genomes B and D, or of barley gene-rich sequences. Moreover, among the various TE groups, MITEs were most often associated with genes: 43.1% of MITEs fell into this category. Finally, the TRIM and copia elements were shown to be the most active TEs in the wheat genome. The implications of these results for the evolution of diploid and polyploid wheat species are discussed. Electronic Supplementary Material Supplementary material is available for this article at     

skippy, a retrotransposon from the fungal plant pathogen Fusarium oxysporum

A retrotransposon from the fungal plant pathogen Fusarium oxysporum f. sp. lycopersici has been isolated and characterized. The element, designated skippy (skp) is 7846 by in length, flanked by identical long terminal repeats (LTR) of 429 by showing structural features characteristic of retroviral and retrotransposon LTRs. Target-site duplications of 5 bp were found. Two long overlapping open reading frames (ORF) were identified. The first ORF, 2562 by in length, shows homology to retroviral gag genes. The second ORF, 3888 bp in length, has homology to the protease, reverse transciptase. RNase H and integrase domains of retroelement pol genes in that order. Sequence comparisons and the order of the predicted proteins from skippy indicate that the element is closely related to the gypsy family of LTR-retrotransposons. The element is present in similar copy numbers in the two races investigated, although RFLP analysis showed differences in banding patterns. The number of LTR sequences present in the genome is higher than the number of copies of complete elements, indicating excision by homologous recombination between LTR sequences.     

Development of a Purification Procedure for the Isolation of Nucleosides from Urine Prior to Mass Spectrometric Analysis

Abstract

A chromatographic separation of nucleosides from urine has been developed in order to facilitate their mass spectrometric analysis for clinical diagnosis. A number of chromatographic resins were studied in order to develop an effective and efficient purification procedure. The optimized sequential protocol comprises a centrifugation, acidification and neutralization step, followed by application of an affinity chromatographic column and finally further separation on an acidic cation exchange column and a basic anion exchanger. This scheme shows effective clean-up of a standard radiolabelled nucleoside with a recovery of 92.5%, and recovery of nucleosides added to urine samples before extraction showed recoveries of 72 – 82%.     

In silico mapping of 1758 new SSR markers developed from public genomic sequences for sorghum

In order to develop simple sequence repeat (SSR) markers for sorghum, a total of 2,113 primer pairs were designed from 81,342 public genomic sequence contigs of sorghum [methyl-filtered sequences downloaded from SAMI (Sorghum Assembled Genomic Island)]. After screening eight sorghum lines, 1,758 primers amplifying polymorphisms were identified. Of these 1,710 SSR markers were predominantly polymorphic in the eight lines and could detect from two to eight alleles. In addition, 1,692 of the markers were mapped on to the ten sorghum chromosomes by in silico mapping. The number of markers per chromosome ranged from 107 to 285. These SSR markers will provide an ideal marker system to help with gene targeting, gene cloning, quantitative trait locus mapping, and marker-assisted selection in sorghum species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.