Structural analysis of type II variants within the mouse intracisternal A-particle sequence family.

Intracisternal A-particle (IAP) elements are present in multiple copies in the mouse and other rodent genomes. The bulk of this sequence family in Mus musculus consists of 7 Kb long elements, but the majority of IAP sequences involved in known transpositions have been deleted forms. The present study describes a subset of deleted IAP sequences (type II IAP) characterized by insertion of a particular short sequence element (AIIins). AIIins are interspersed and the majority occur as part of the type II IAP elements in the mouse genome. AIIins sequences are absent or in low copy number outside Mus musculus. We have isolated clones containing AIIins from a mouse genomic DNA library and have sequenced three isolates of AIIins and their surrounding IAP sequences to define the detailed structure of type II elements. AIIins are 272, 268 and 264 bp long and 90% homologous in sequence. They are bracketed by 9 bp duplications, suggesting they may be inserted elements. A 75 bp region containing a core enhancer sequence is repeated at the 5' end in type II IAP elements. Insertion into the IAP genome, with potential to encode an integrase function, may have played a role in the amplification of AIIins.     

Methylation and rearrangement of mouse intracisternal a particle genes in development, aging, and myeloma.

Sequences of DNA that hybridize on Southern blots with cloned intracisternal A-particle (IAP) sequences have been examined in genomic DNAs of neonatal mice, livers of adult mice (3, 6, 12, 18, 24, and 26 months old), and the solid myeloma tumor MOPC-315. The isoschizomers HpaII (CCGG or mCCGG) and MspI (CCGG or CmCGG) were used to assess methylation. All the DNAs produced a major 0.5-kilobase MspI fragment that hybridizes with IAP probe. Only the myeloma DNA, and to a much lesser degree DNA from senescent mouse liver, produced this fragment in HpaII digest; the other DNAs all had IAP sequences resistant to HpaII digestion. These sequences thus become fully methylated to CmCGG early and remain so in adult life, except in the myeloma cells that are expressing the IAP genes. An increase in MspI-sensitive sites in IAP gene-containing DNA was observed in aging mice. The probe used to assess methylation, a 0.8-kilobase fragment produced by BamHI-HindIII double digestion, is common to several cloned IAP genes and is part of a region of DNA which is conserved in genomes of all mouse tissues. The probe hybridized to 1.5- and 1.4-kilobase doublet bands produced by BamHI, HindIII, and EcoRI triple digestions of neonatal DNA. These two bands were found in neonatal livers of Swiss Webster, BALB/c, and C57BL/6J mouse strains, showed less in adult liver, and were barely detectable in senescent livers from C57BL/6J mice.     

Microarray-based global mapping of integration sites for the retrotransposon, intracisternal A-particle, in the mouse genome

Mammalian genomes contain numerous evolutionary harbored mobile elements, a part of which are still active and may cause genomic instability. Their movement and positional diversity occasionally result in phenotypic changes and variation by causing altered expression or disruption of neighboring host genes. Here, we describe a novel microarray-based method by which dispersed genomic locations of a type of retrotransposon in a mammalian genome can be identified. Using this method, we mapped the DNA elements for a mouse retrotransposon, intracisternal A-particle (IAP), within genomes of C3H/He and C57BL/6J inbred mouse strains; consequently we detected hundreds of probable IAP cDNA–integrated genomic regions, in which a considerable number of strain-specific putative insertions were included. In addition, by comparing genomic DNAs from radiation-induced myeloid leukemia cells and its reference normal tissue, we detected three genomic regions around which an IAP element was integrated. These results demonstrate the first successful genome-wide mapping of a retrotransposon type in a mammalian genome.     

High-Throughput Targeted Repeat Element Bisulfite Sequencing (HT-TREBS): Genome-Wide DNA Methylation Analysis of IAP LTR Retrotransposon

In vertebrates, DNA methylation-mediated repression of retrotransposons is essential for the maintenance of genomic integrity. In the current study, we developed a technique termed HT-TREBS (High-Throughput Targeted Repeat Element Bisulfite Sequencing). This technique is designed to measure the DNA methylation levels of individual loci of any repeat families with next-generation sequencing approaches. To test the feasibility of HT-TREBS, we analyzed the DNA methylation levels of the IAP LTR family using a set of 12 different genomic DNA isolated from the brain, liver and kidney of 4 one-week-old littermates of the mouse strain C57BL/6N. This technique has successfully generated the CpG methylation data of 5,233 loci common in all the samples, representing more than 80% of the individual loci of the five targeted subtypes of the IAP LTR family. According to the results, approximately 5% of the IAP LTR loci have less than 80% CpG methylation levels with no genomic position preference. Further analyses of the IAP LTR loci also revealed the presence of extensive DNA methylation variations between different tissues and individuals. Overall, these data demonstrate the efficiency and robustness of the new technique, HT-TREBS, and also provide new insights regarding the genome-wide DNA methylation patterns of the IAP LTR repeat elements.     

Genome-wide assessments reveal extremely high levels of polymorphism of two active families of mouse endogenous retroviral elements

Endogenous retroviral elements (ERVs) in mice are significant genomic mutagens, causing ~10% of all reported spontaneous germ line mutations in laboratory strains. The majority of these mutations are due to insertions of two high copy ERV families, the IAP and ETn/MusD elements. This significant level of ongoing retrotranspositional activity suggests that inbred mice are highly variable in content of these two ERV groups. However, no comprehensive genome-wide studies have been performed to assess their level of polymorphism. Here we compared three test strains, for which sufficient genomic sequence is available, to each other and to the reference C57BL/6J genome and detected very high levels of insertional polymorphism for both ERV families, with an estimated false discovery rate of only 0.4%. Specifically, we found that at least 60% of IAP and 25% of ETn/MusD elements detected in any strain are absent in one or more of the other three strains. The polymorphic nature of a set of 40 ETn/MusD elements found within gene introns was confirmed using genomic PCR on DNA from a panel of mouse strains. For some cases, we detected gene-splicing abnormalities involving the ERV and obtained additional evidence for decreased gene expression in strains carrying the insertion. In total, we identified nearly 700 polymorphic IAP or ETn/MusD ERVs or solitary LTRs that reside in gene introns, providing potential candidates that may contribute to gene expression differences among strains. These extreme levels of polymorphism suggest that ERV insertions play a significant role in genetic drift of mouse lines.     

Identification of two mariner-like elements in the genome of the mosquito Ochlerotatus atropalpus

Two distinct mariner-like elements, Atmar-1 and Atmar-2, were isolated from the genome of the mosquito Ochlerotatus atropalpus. Full-sized Atmar-1 elements, obtained by screening a genomic library, have a 1293-bp consensus sequence with 27-bp inverted terminal repeats and a 1047-bp open reading frame (ORF) encoding the transposase. The Atmar-2 elements were amplified by polymerase chain reaction from genomic DNA and contain the central part of the transposase ORF. Individual clones of both mariner elements contain deletions, frameshifts, and stop codons. The Atmar-1 elements are present in 370-1200 copies, while the Atmar-2 elements are present in approximately 100-300 copies per haploid genome. One of the Atmar-1 elements, Atmar-1.33, could be mobilized, suggesting the presence of functional Atmar-1 elements elsewhere in the genome. Phylogenetic analysis demonstrated that Atmar-1 elements belong to the irritans subfamily and Atmar-2 elements to the cecropia subfamily of mariner elements.     

DNA topoisomerase VIII: a novel subfamily of type IIB topoisomerases encoded by free or integrated plasmids in Archaea and Bacteria

Type II DNA topoisomerases are divided into two families, IIA and IIB. Types IIA and IIB enzymes share homologous B subunits encompassing the ATP-binding site, but have non-homologous A subunits catalyzing DNA cleavage. Type IIA topoisomerases are ubiquitous in Bacteria and Eukarya, whereas members of the IIB family are mostly present in Archaea and plants. Here, we report the detection of genes encoding type IIB enzymes in which the A and B subunits are fused into a single polypeptide. These proteins are encoded in several bacterial genomes, two bacterial plasmids and one archaeal plasmid. They form a monophyletic group that is very divergent from archaeal and eukaryotic type IIB enzymes (DNA topoisomerase VI). We propose to classify them into a new subfamily, denoted DNA topoisomerase VIII. Bacterial genes encoding a topoisomerase VIII are present within integrated mobile elements, most likely derived from conjugative plasmids. Purified topoisomerase VIII encoded by the plasmid pPPM1a from Paenibacillus polymyxa M1 had ATP-dependent relaxation and decatenation activities. In contrast, the enzyme encoded by mobile elements integrated into the genome of Ammonifex degensii exhibited DNA cleavage activity producing a full-length linear plasmid and that from Microscilla marina exhibited ATP-independent relaxation activity. Topoisomerases VIII, the smallest known type IIB enzymes, could be new promising models for structural and mechanistic studies.     

The sperm-specific proteins of the edible oyster (European flat oyster (Ostrea edulis)) are products of proteolytic processing

Extraction of sperm proteins from the bivalve mollusc Ostrea edulis shows them to contain a normal complement of core histones, together with three sperm-specific proteins, OE1 and OE2, plus the shorter OE3, which shows substantial microheterogeneity. OE1 and OE2 have a very similar amino acid composition, cyanogen bromide (CNBr) cleavage yields products of identical size and possesses a trypsin-resistant core peptide, together indicating that they are closely homologous histone H1-like proteins. Western blotting shows that OE1 and OE2 are closely related to the histone H1-like protein PL-II* of Mytilus trossulus. The amino acid composition of OE3 shows it to be a protamine-like PL-IV type protein. Edman degradation of a CNBr peptide from OE2 gave the sequence (M)KAAFAKGLKSGALVRPKGS-which has 85% identity to a sequence located towards the C-terminal end of the globular domain of the PL-II* protein of M. trossulus. An O. edulis sperm cDNA library yielded a clone of 428 bp. A genomic clone including an open reading frame (ORF) of 750 bp was isolated by PCR amplification from genomic DNA. Hypothetical translation showed the ORF to encode OE1 (or OE2) immediately followed by OE3, separated by a proteolytic processing site. This arrangement (a two-protein ORF) is also found in M. trossulus and Ensis minor.     

A novel homologue of the TIAP/m-survivin gene

The inhibitor of apoptosis (IAP) proteins comprise a highly conserved gene family that prevents cell death in response to a variety of stimuli. TIAP/m-survivin, a murine homologue of human Survivin, is a member of the IAP family. TIAP/m-survivin has one baculovirus IAP repeat (BIR) and lacks a C-terminal RING finger motif. Here we identified the genomic DNA region (TIAP-2) that is homologous to the TIAP/m-survivin gene by a low stringency genomic DNA hybridization. The region is on the chromomsome 9 which is distinct from that (chromosome 11) of the TIAP/m-survivin gene, and contains DNA sequence similar to a part of the BIR and the 3' side of the TIAP/m-survivin gene and the sequence homology between them is 92%. Expression of TIAP-2 mRNA was detected in various murine tissues by RT-PCR. Although expression of TIAP/m-survivin mRNA is upregulated in synchronized cells at S to G2/M phase of the cell cycle, expression of TIAP-2 mRNA was constant in the cell cycle, suggesting the different role of TIAP-2 from that of TIAP/m-survivin.