Dense Alu clustering and a potential new member of the NF kappa B family within a 90 kilobase HLA class III segment.

We have conducted a detailed structural analysis of 90 kilobases (kb) of the HLA Class III region from the Bat2 gene at the centromeric end to 23 kb beyond TNF. A single contig of 80 kb was sequenced entirely with a group of four smaller contigs covering 10 kb being only partly sequenced. This region contains four known genes and a novel telomeric potential coding region. The genes are bracketed by long, dense clusters of Alu repeats belonging to all the major families. At least six new families of MER repeats and one pseudogene are intercalated within and between the Alu clusters. The most telomeric 3.8 kb contains three potential exons, one of which bears strong homology to the ankyrin domain of the DNA binding factors NF kappa B and I kappa B.

Repeats in genomic DNA: mining and meaning.

For hundreds of millions of years, perhaps from the very beginning of their evolutionary history, eukaryotic cells have been habitats and junkyards for countless generations of transposable elements, preserved in repetitive DNA sequences. Analysis of these sequences, combined with experimental research, reveals a history of complex 'intracellular ecosystems' of transposable elements that are inseparably associated with genomic evolution.

Clustering, duplication and chromosomal distribution of mouse SINE retrotransposons.

We analyzed potential mechanisms determining chromosomal distributions of the mouse B1 and B2 non-LTR retrotransposons, also known as SINE elements. We report that young B1 and B2 SINEs are underrepresented on chromosome X relative to autosomes, which is consistent with their integration in male germ lines. As the age of the SINE elements progresses, their densities on chromosome X increase relative to autosomal densities, possibly due to differences in ectopic recombination rates between chromosome X and autosomes. Furthermore, unlike young human Alus that tend to be integrated outside Alu-dense regions, young B1 and B2 elements are found mostly in SINE-rich clusters. The B1- or B2-rich clusters are more likely to contain duplicated elements than B1- or B2-poor chromosomal regions. We also present evidence indicating potential association of B1 and B2 elements with intra-chromosomal segmental duplications. No such association was found with inter-chromosomal duplications. We propose that the accumulation of mouse SINE elements observed in GC-rich regions may be due to the excess of DNA duplications over deletions in gene-rich regions that tend to be GC rich.

Repbase Update, a database of eukaryotic repetitive elements.

Repbase Update is a comprehensive database of repetitive elements from diverse eukaryotic organisms. Currently, it contains over 3600 annotated sequences representing different families and subfamilies of repeats, many of which are unreported anywhere else. Each sequence is accompanied by a short description and references to the original contributors. Repbase Update includes Repbase Reports, an electronic journal publishing newly discovered transposable elements, and the Transposon Pub, a web-based browser of selected chromosomal maps of transposable elements. Sequences from Repbase Update are used to screen and annotate repetitive elements using programs such as Censor and RepeatMasker. Repbase Update is available on the worldwide web at http://www.girinst.org/Repbase_Update.html.

Ancestral, mammalian-wide subfamilies of LINE-1 repetitive sequences.

Analysis of the 3'-ends of approximately 900 separate human LINE-1 (L1) elements from primates revealed 47 contiguous but distinct subfamilies with the L1 family. Eight previously described medium reiteration frequency sequences (MERs) were found to be parts of ancient L1 untranslated 3'-regions which show little or no sequence similarity to the presently active L1 3'-end. Some of the major changes in 3'-end sequence can be explained by recombination events between different L1 repeats as well as between L1 and unrelated repetitive sequences. One of these sequences, MER42, is reported in this paper. With the set of consensus sequences for different subfamilies and their diagnostic features, it is possible to estimate the age of individual LINE-1 elements. Contrary to earlier suggestions, the majority of L1 copies in the human genome is very old; more than half of the identifiable elements were inserted into the genome before the mammalian radiation, as evidenced by elements at orthologous sites in human and other mammalian genomes. Multiple distinct L1 source genes seem to have been active simultaneously over long periods of time.

Birth of a gene: locus of neuronal BC200 snmRNA in three prosimians and human BC200 pseudogenes as archives of change in the Anthropoidea lineage.

The gene encoding brain-specific dendritic BC200 small non-messenger RNA is limited to the primate order and arose from a monomeric Alu element. It is present and neuronally expressed in all Anthropoidea examined. By comparing the human sequence of about 13.2 kb with each of the prosimian (lemur 14.6 kb, galago 12 kb, and tarsier 13.8 kb) orthologous loci, we could establish that the BC200 RNA gene is absent from the prosimian lineages. In Strepsirhini (lemurs and lorises), a dimeric AluJ-like element integrated very close to the BC200 insertion point, while the corresponding tarsier region is devoid of any repetitive element. Consequently, insertion of the Alu monomer that gave rise to the BC200 RNA gene must have occurred after the anthropoid lineage diverged from the prosimian lineage(s). Shared insertions of other repetitive elements favor proximity of simians and tarsiers in support of their grouping into Haplorhini and the omomyid hypothesis. On the other hand, the nucleotide sequences in the segment that is available for comparison in all four species reveal less exchanges between Strepsirhini (lemur and galago) and human than between tarsier and human. Our data imply that the early activity of dimeric Alu sequences must have been concurrent with the activity of monomeric Alu elements that persisted longer than is usually thought. As BC200 RNA gave rise to more than 200 pseudogenes, we used their consensus sequence variations as a molecular archive recording the BC200 RNA sequence changes in the anthropoid lineage leading to Homo sapiens and timed these alterations over the past 35-55 million years.

Repetitive DNA in and around translocation breakpoints of the Philadelphia chromosome.

This paper describes systematic sequence studies of repetitive DNA in and around translocation breakpoints on chromosomes 9 and 22, which are involved in the formation of the Philadelphia chromosome in acute leukemias. In addition to Alu repeats described in previous studies, the breakpoint regions appear to contain many other repetitive elements, including a member of a new repetitive family (MER34) reported in this paper. Identification of these repeats broadens current studies on the possible involvement of repetitive DNA in this intensely studied chromosomal translocation.

Comparison of repetitive elements in the third intron of human and rodent mitochondrial benzodiazepine receptor-encoding genes.

The third intron of the mitochondrial benzodiazepine receptor (MBR)-encoding gene was sequenced from hamster, mouse and human. The rodent species were found to include an Alu-like sequence, as was first discovered in the rat gene. Differences with the rat intron were evident by an insertion of an additional B1 element in the hamster and the introduction of a complete and two partial B2 sequences in the mouse intron. The human intron contained a cluster of four Alu sequences; however, all of these repetitive elements were found to be in the opposite orientation relative to the Alu-like sequence present in the rodent genes. These findings support the possibility that the rodent Alu-like sequence is a remnant of a retropositional insertion in this gene prior to the divergence of rodent species. Because the human intron does not contain the same Alu remnant, it cannot be concluded that the rodent sequence represents an insertion of a primordial Alu element prior to the divergence of rodent and primate lineages.

Stimulatory effect of hnRNA on template activity of isolated rat liver chromatin.

Transcription of chromatin isolated from rat liver with E. coli RNA polymerase was stimulated up to five-fold with heterogeneous nuclear RNA (hnRNA) from rat liver. The transcription product had features of DNA-primed RNA. Neither cytoplasmic poly(A)-containing RNA nor high or low molecular weight cytoplasmic poly(A)-lacking RNA from rat liver exhibited stimulation of transcription. The stimulatory effect seems to be confined to middle repetitive sequences of hnRNA. With purified DNA templates, addition of hnRNA resulted in complete inhibition of transcription. The stimulatory effect of hnRNA on chromatin transcription was also observed with endogenous RNA polymerase. This effect was dependent ionic strength and was most pronounced under conditions optimal for RNA-polymerase B activity.

Mammalian retroposons integrate at kinkable DNA sites.

Integration of retroposed RNA in mammals occurs at staggered breaks resulting from an enzyme-generated pair of nicks at opposite DNA strands, preferably within 15-16 bp. Although consensus sequences associated with the two nicks appear somewhat different from one another, both nicking sites are rich in TA, CA and TG dinucleotide steps which are known as specific DNA sites where kinks may occur under bending constraints. This suggests that during interaction with the endonucleolytic enzyme, or enzymes, DNA undergoes bending at the integration sites and kinks are formed, as initial steps in generating the nicks. Nicking at kinkable sites, particularly at TA steps, may also play a role in integration of other insertion elements.

Additional Watson-Crick interactions suggest a structural core in large subunit ribosomal RNA.

Two new Watson-Crick type interactions in 23S-like ribosomal RNA have been identified by comparative sequence analysis. These interactions, A1269/U2011 and C1270/G2010 (E. coli numbering) along with the previously proposed A1262/U2017 suggest an anti-parallel helical arrangement characteristic of secondary structure in the 1265/2015 region of 23S rRNA. Nested within these three interactions are three universal juxtapositions which in principle allow the formation of an irregular helix containing two additional A-G interactions and a universal A-U pair. Whether or not this extended helix is biologically significant is uncertain. The proponderance of interactions in the 1265/2015 region and its location relative to the known structural domains of 23S rRNA suggest that this region may be part of a central structural core similar to that already known in 16S rRNA.

Characteristic archaebacterial 16S rRNA oligonucleotides.

A method of analyzing 16S rRNA catalog data has been developed in which groupings at various taxonomic levels can be characterized in terms of specific "signature" oligonucleotides. This approach provides an alternative means for evaluating higher order branching possibilities and can be used to assess the phylogenetic position of isolates that are poorly placed by the usual clustering procedures. This signature approach has been applied to forty archaebacterial catalogs and every oligonucleotide with significant signature value has been identified. Sets of specific oligonucleotides were identified for every major group on a dendrogram produced by cluster analysis procedures. Signatures that would establish between group relationships were also sought and found. In the case of the Methanobacteriaceae the clustering methods suggest a specific relationship to the Methanococcaceae. This inclusion is in fact supported by six strong signature oligonucleotides. However there are also significant numbers of signature oligonucleotides supporting a specific relationship of the Methanobacteriaceae to either the Halobacteriaceae or the Methanomicrobiaceae. Thus the placement of the Methanobacteriaceae is less certain than the usual dendrograms imply. The signature approach also was used to assess the phylogenetic position of Thermoplasma acidophilum which is found to be more closely related to the methanogen/halophile Division than to the sulfur dependent Division of the archaebacteria. This does not imply however that Thermoplasma acidophilum is properly regarded as being in the methanogen/halophile Division.

MER53, a non-autonomous DNA transposon associated with a variety of functionally related defense genes in the human genome.

We report a new medium reiteration frequency repeat MER53 present in human and mammalian genomes. A 189 bp MER53 consensus sequence has been reconstructed based on the computer analysis of GenBank sequences. TA target site duplication and terminal inverted repeats indicate that the MER53 repeat is a non-autonomous DNA transposon related to the mariner family. Two MER53 repeats were found integrated within different mobile elements. We have found that most of the genes harboring the MER53 repeat are involved in the host defense system. The reasons for this non-random distribution of the repeat are discussed.

Genome sequence, comparative analysis, and population genetics of the domestic horse.

We report a high-quality draft sequence of the genome of the horse (Equus caballus). The genome is relatively repetitive but has little segmental duplication. Chromosomes appear to have undergone few historical rearrangements: 53% of equine chromosomes show conserved synteny to a single human chromosome. Equine chromosome 11 is shown to have an evolutionary new centromere devoid of centromeric satellite DNA, suggesting that centromeric function may arise before satellite repeat accumulation. Linkage disequilibrium, showing the influences of early domestication of large herds of female horses, is intermediate in length between dog and human, and there is long-range haplotype sharing among breeds.

Conserved eukaryotic transposable elements and the evolution of gene regulation.

Multiple remnants of transposable elements preserved in cis-regulatory modules may represent a record of mutations that were critical to the evolution of gene regulation and speciation.

Subfamily structure and evolution of the human L1 family of repetitive sequences.

Comparative analysis of the available 3'-portions of the human L1 (LINE-1) family of repeated sequences indicates that all the sequences can be classified in two major subfamilies. The division is based on patterns of diagnostic bases shared within L1 subfamilies of sequences but differing between them. The overall ratio of replacement to synonymous positions, occupied by the diagnostic bases in the large open reading frame of the L1 sequence, is 1.15. This indicates that both subfamilies were obtained from genes coding for functional proteins. The L1 subfamilies appear to be of different ages and may represent a "fossil record" of the same active gene at different times in the history of primates. The younger subfamily can be split further into at least two closely related branches of sequences. The above facts combined with the recent data for the Alu subfamily structure show that LINE and SINE families of interspersed repeats share discontinuous patterns in their evolution. These data are consistent with the model that both Alu and L1 families, as well as other pseudogene families, contain active genes producing discrete layers of pseudogenes throughout the history of primates. Models of evolutionary processes that could generate these discontinuities are discussed together with the possible biological role of Alu and L1 genes.

Novel families of interspersed repetitive elements from the human genome.

Six novel families of interspersed repetitive elements have been detected in the available human DNA sequences using computer-assisted analyses. The estimated total number of elements in the reported six families is over 17,000. Sequences representative for each family range from approximately 150 to 650 base pairs (bp) in length and are predominantly (A + T)-rich. Sequences from four families contain stretches of patchy complementarity up to 45 bp long. Member of one of the families is likely be directly involved in a multigene deletion on chromosome 14. Two of the six sequence families are homologous to 'low reiteration frequency sequences' from monkey cells, detected first in defective variants of simian virus 40. Like Alu and L1 families, the newly discovered families are probably composed of pseudogenes derived from functional genes.

Sequence patterns indicate an enzymatic involvement in integration of mammalian retroposons.

It is commonly accepted that the reverse-transcribed cellular RNA molecules, called retroposons, integrate at staggered breaks in mammalian chromosomes. However, unlike what was previously thought, most of the staggered breaks are not generated by random nicking. One of the two nicks involved is primarily associated with the 5'-TTAAAA hexanucleotide and its variants derived by a single base substitution, particularly A --> G and T --> C. It is probably generated in the antisense strand between the consensus bases 3'-AA and TTTT complementary to 5'-TTAAAA. The sense strand is nicked at variable distances from the TTAAAA consensus site toward the 3' end, preferably within 15-16 base pairs. The base composition near the second nicking site is also nonrandom at positions preceding the nick. On the basis of the observed sequence patterns it is proposed that integration of mammalian retroposons is mediated by an enzyme with endonucleolytic activity. The best candidate for such enzyme may be the reverse transcriptase encoded by the L1 non-long-terminal-repeat retrotransposon, which contains a freshly reported domain homologous to the apurinic/apyrimidinic (AP) endonuclease family [Martin, F., Olivares, M., Lopez, M. C. & Alonso, C. (1996) Trends Biochem. Sci. 21, 283-285; Feng, Q., Moran, J. V., Kazazian, H. H. & Boeke, J. D. (1996) Cell 87, 905-916] and shows nicking in vitro with preference for targets similar to 5'-TTAAAA/3'-AATTTT consensus sequence [Feng, Q., Moran, J. V., Kazazian, H. H. & Boeke, J. D. (1996) Cell 87, 905-916]. A model for integration of mammalian retroposons based on the presented data is discussed.