Effective Isolation of Retrotransposons and Repetitive DNA Families from the Wheat Genome

New classes of repetitive DNA elements were effectively identified by isolating small fragments of the elements from the wheat genome. A wheat A genome library was constructed from Triticum monococcum by degenerate cleavage with EcoO109I, the recognition sites of which consisted of 5'-PuGGNCCPy-3'multi-sequences. Three novel repetitive sequences pTm6, pTm69 and pTm58 derived from the A genome were screened and tested for high copy number using a blotting approach. pTm6 showed identity with integrase domains of the barley Ty1-Copia-retrotransposon BARE-1 and pTm58 showed similarity to the barley Ty3-gypsy-like retrotransposon Romani. pTm69, however, constituted a tandem array with useful genomic specificities, but did not share any identity with known repetitive elements. This study also sought to isolate wheat D-genome-specific repetitive elements regardless of the level of methylation, by genomic subtraction. Total genomic DNA of Aegilops tauschii was cleaved into short fragments with a methylation-insensitive 4 bp cutter, Mbol, and then common DNA sequences between Ae. tauschii and Triticum turgidum were subtracted by annealing with excess T. turgidum genomic DNA. The D genome repetitive sequence pAt1 was isolated and used to identify an additional novel repetitive sequence family from wheat bacterial artificial chromosomes with a size range of 1 395-1 850 bp. The methods successfully led pathfinding of two unique repetitive families.     

Expression of cab Genes in Douglas-Fir Is Not Strongly Regulated by Light

Dark-grown Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) seedlings had approximately 30% of the major polypeptide of the light-harvesting chlorophyll a/b binding protein, 30% of cab mRNA, 54% of psbA mRNA, and 14% of total chlorophyll, in comparison with amounts in light-grown seedlings. Seedlings entrained under a 24-hour photoperiod of light and dark showed small diurnal fluctuations in cab and psbA mRNA levels and, when transferred to continuous conditions, no circadian rhythms in mRNA levels were apparent. These results suggest that regulation of cab gene expression in Douglas-fir differs from regulation in angiosperms, because in the latter, both light and circadian factors strongly influence the expression of cab genes.     

Allelic Skewing of DNA Methylation Is Widespread across the Genome

DNA methylation is assumed to be complementary on both alleles across the genome, although there are exceptions, notably in regions subject to genomic imprinting. We present a genome-wide survey of the degree of allelic skewing of DNA methylation with the aim of identifying previously unreported differentially methylated regions (DMRs) associated primarily with genomic imprinting or DNA sequence variation acting in cis. We used SNP microarrays to quantitatively assess allele-specific DNA methylation (ASM) in amplicons covering 7.6% of the human genome following cleavage with a cocktail of methylation-sensitive restriction enzymes (MSREs). Selected findings were verified using bisulfite-mapping and gene-expression analyses, subsequently tested in a second tissue from the same individuals, and replicated in DNA obtained from 30 parent-child trios. Our approach detected clear examples of ASM in the vicinity of known imprinted loci, highlighting the validity of the method. In total, 2,704 (1.5%) of our 183,605 informative and stringently filtered SNPs demonstrate an average relative allele score (RAS) change ≥0.10 following MSRE digestion. In agreement with previous reports, the majority of ASM (∼90%) appears to be cis in nature, and several examples of tissue-specific ASM were identified. Our data show that ASM is a widespread phenomenon, with >35,000 such sites potentially occurring across the genome, and that a spectrum of ASM is likely, with heterogeneity between individuals and across tissues. These findings impact our understanding about the origin of individual phenotypic differences and have implications for genetic studies of complex disease.     

Functional Interactions between Unlinked Muscle Genes within Haploinsufficient Regions of the Drosophila Genome

Mutations in 13 genes affecting muscle development in Drosophila have been examined in pairwise combinations for evidence of genetic interactions. Heterozygous combinations of mutations in five genes, including the gene coding for myosin heavy chain, result in more severe phenotypes than respective single heterozygous mutant controls. The various mutant interactions include examples showing allele-specific intergenic interactions, gene specific interactions, and allele-specific intragenic complementations, suggesting that some interactions result from the manner in which mutant gene products associate. Interactions that result from alterations in ``+' gene copy number were also uncovered, suggesting that normal myofibril development requires that the relative amounts of respective gene products produced be tightly regulated. The importance of the latter parameter is substantiated by the finding that all five interacting loci map to disperse haploinsufficient or haplolethal regions of the genome. The implications of the present findings are discussed in relation to pursuing the phenomena involving genetic interactions to identify new genes encoding interacting myofibrillar proteins, to examine the nature of intermolecular interactions in mutant and normal development and to decipher the quantitative and temporal regulation of a large family of functionally related gene products.     

Repetitive DNA, Genome and Species Relationships in Avena and Arrhenatherum(Poaceae)

Repetitive sequences have been widely used for examining genomeand species relationships by in situ and Southern hybridization.In the present study, double-stranded DNA sequences, from denaturedDNA reannealed to Cot = 1, from Avena strigosa(2 n = 2x = 14;A genome; referred to as CotA) and Avena sativa(2n = 6 x =42; ACD genome; referred to as CotACD) were isolated with ahydroxyapatite column, and were used for in situ hybridizationon hexaploid A. sativa chromosomes. Probe CotACD labelled allchromosomes evenly throughout their length at the same intensity.Probe CotA labelled the 28 A and D genome chromosomes stronglyand the 14 C genome chromosomes weakly. Three cloned repetitivesequences, pAvKB9 (126 bp), pAvKB26 (223 bp) and pAvKB32 (721bp) were characterized in the A, B, C and D Avena genomes andthe genus Arrhenatherum using molecular and cytological methods.Clones pAvKB9 and pAvKB26 were absent from the Avena C genome,while both could identify the presence of the D genome by Southernhybridization. In situ hybridization to diploid and tetraploidAvena species revealed that the probes showed a dispersed genomicorganization and that they are present on both arms of all chromosomes.These sequences were excluded from areas where tandem repeats,such as rRNA genes and telomeres, are present. These resultsindicate the close relationship between A and D genomes andthe presence of common DNA sequences between A and C Avena genomes.All three clones hybridized to Southern blots containingArrhenatherumdigested genomic DNA, indicating Arrhenatherum’s closeaffinity to A, B and D Avena genomes. Copyright 2000 Annalsof Botany Company Cereals, DNA, hydroxyapatite, in situ hybridization, oats, reassociation kinetics, repetitive DNA     

Host Species-specific Repetitive DNA Sequence in the Genome of Magnaporthe grisea,the Rice Blast Fungus

We cloned a repetitive sequence to show RFLPs in the genome of Magnaporthe grisea, a fungal pathogen responsible for rice blast. As the sequence was 0.8 kb in length and dispersed in the genome, it was named MGSR1 (for Magnaporthe grisea short repeat 1). MGSR1 was conserved highly in the genome of rice pathogens, but poorly in the genome of pathogens of grasses other than rice. And the RFLPs, displayed with the sequence, could distinguish between clonal lineages in rice-pathogenic isolates. The nucleotide sequence showed the presence of an internal promoter of RNA polymerase III, a 3?-poly(T), and an 8-bp direct repeat in it.     

The Repetitive DNA Elements Called CRISPRs and Their Associated Genes: Evidence of Horizontal Transfer Among Prokaryotes

We have found direct DNA repeats 21–47 bp in length interspersed with nonrepetitive sequences of similar length, or clustered regularly interspaced short palindromic repeats (CRISPRs) in a wide range of diverse prokaryotes, including many Archaeal and Eubacterial species. A number of cas, CRISPR-associated genes have also been characterized in many of the same organisms. Phylogenetic analysis of these cas genes suggests that the CRISPR loci have been propagated via HGT, horizontal gene transfer. We suggest a mechanism by which this HGT has occurred, namely, that the CRISPR loci can be carried between cells on megaplasmids ≥40 kb in length. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Stuart Newfeld]     

水稻基因组中的tRNA和rRNA基因

在最近完成测序的水稻籼稻和粳稻两个亚种基因组中,各找到564和519个较为可靠的tRNA基因,进一步证实了于2002年发表的基于基因组序列草图的分析结果。修正的摆动假设,即至少需要46种tRNA基因才能译出61种可能的反密码子,在这两个亚种中均准确成立。在这46种tRNA中,有些在籼稻和粳稻中的序列均全同。有18种水稻tRNA与拟南芥中的相应序列全同。在籼稻基因组序列中还发现了384个5S rRNA基因,一批17S和5．8S rRNA基因以及一个25S rRNA基因。这些rRNA基因的不完备是由于它们通常以串接阵列形式存在于异染色质区域,而后者在全基因组霰弹法测序中不易完整测出。在tRNA和rRNA基因序列之间发现了多处互补片段,这将有助于研究它们的进化和相互作用。     

Identification of Imprinted Genes and Their Differentially Methylated Regions in Porcine

Russian Journal of Genetics - Genomic imprinting is an epigenetic mechanism that leads to parent-specific gene expression. A number of imprinted genes have been identified in humans and...     

Connexin Genes in the Mouse and Human Genome

Gap junctions serve for direct intercellular communication by docking of two hemichannels in adjacent cells thereby forming conduits between the cytoplasmic compartments of adjacent cells. Connexin genes code for subunit proteins of gap junction channels and are members of large gene families in mammals. So far, 17 connexin (Cx) genes have been described and characterized in the murine genome. For most of them, orthologues in the human genome have been found (see White and Paul 1999; Manthey et al. 1999; Teubner et al. 2001; Söhl et al. 2001). We have recently performed searches for connexin genes in murine and human gene libraries available at EMBL/Heidelberg, NCBI and the Celera company that have increased the number of identified connexins to 19 in mouse and 20 in humans. For one mouse connexin gene and two human connexin genes we did not find orthologues in the other genome. Here we present a short overview on distinct connexin genes which we found in the mouse and human genome and which may include all members of this gene family, if no further connexin gene will be discovered in the remaining non-sequenced parts (about 1-5%) of the genomes.     

Loss of the Mitochondrial Nucleoid Protein, Abf2p, Destabilizes Repetitive DNA in the Yeast Mitochondrial Genome

Loss of Abf2p, an abundant mitochondrial nucleoid-associated protein, results in increased mitochondrial frameshifts and direct-repeat mediated deletions but has no effect on the rate of mitochondrial point mutations. The instability of repeated sequences in this strain may be linked to the loss of mitochondrial DNA in abf2-Δ strains.     

An Alphoid-Like Satellite DNA Sequence Is Present in the Genome of a Lacertid Lizard

A PstI DNA family was isolated from the genome of a lacertid, Lacerta graeca. The 185-bp monomeric unit (pGPS) was cloned and hybridized to DNAs and chromosomes of several lacertid species. The data showed that pGPS hybridizes to the (1) centromeric or pericentromeric heterochromatin of almost all the chromosomes of L. graeca and (2) genomic DNA of species phylogenetically related and unrelated to L. graeca. The presence of pGPS even in species immunologically apart more than 30 million years suggests that this repeated family might be either very ancient or have been conserved during evolution due to its functional role. The latter hypothesis might be supported by the results of sequence analysis which showed some homology with both several alphoid sequences of primates and the CDEIII centromeric sequence of yeast. Segments of the satellite sequence are similar to the mammalian CENP-B box. These observations suggest that pGPS might have a role in determining the centromeric function in lacertid lizards. Received: 6 February 1997 / Accepted: 14 May 1997     

MeCP2 Represses the Rate of Transcriptional Initiation of Highly Methylated Long Genes

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Run-Off Replication of Host-Adaptability Genes Is Associated with Gene Transfer Agents in the Genome of Mouse-Infecting Bartonella grahamii

The genus Bartonella comprises facultative intracellular bacteria adapted to mammals, including previously recognized and emerging human pathogens. We report the 2,341,328 bp genome sequence of Bartonella grahamii, one of the most prevalent Bartonella species in wild rodents. Comparative genomics revealed that rodent-associated Bartonella species have higher copy numbers of genes for putative host-adaptability factors than the related human-specific pathogens. Many of these gene clusters are located in a highly dynamic region of 461 kb. Using hybridization to a microarray designed for the B. grahamii genome, we observed a massive, putatively phage-derived run-off replication of this region. We also identified a novel gene transfer agent, which packages the bacterial genome, with an over-representation of the amplified DNA, in 14 kb pieces. This is the first observation associating the products of run-off replication with a gene transfer agent. Because of the high concentration of gene clusters for host-adaptation proteins in the amplified region, and since the genes encoding the gene transfer agent and the phage origin are well conserved in Bartonella, we hypothesize that these systems are driven by selection. We propose that the coupling of run-off replication with gene transfer agents promotes diversification and rapid spread of host-adaptability factors, facilitating host shifts in Bartonella.