Duplicated region of the mouse genome containing a cytoplasmic gamma-actin processed pseudogene associated with long interspersed repetitive elements

The structures of two cloned recombinants of bacteriophage lambda and mouse genomic DNA (lambda mA14 and lambda mA36) were compared by electron microscopic analysis of various heteroduplex DNAs, restriction endonuclease mapping and nucleotide sequence determination. Each clone was shown to be derived from a distinct region of the mouse genome, but the two exhibited structural similarity over a region of at least 11,000 bases which included a cytoskeletal gamma-actin processed pseudogene of approximately 1800 bases. It is concluded that the two genomic regions were derived from a common ancestral region by duplication or amplification. The homologous regions of the two clones contained members of the long interspersed repetitive L1Md (long interspersed repeated sequence 1 of Mus domesticus) family lying in opposite orientation to one another, so that single-stranded DNA from the clones could form intra-molecular heteroduplexes. The complete nucleotide sequences of three L1Md members in lambda mA14 were determined. The longest of these (L1Md-14LH) had inserted into the gamma-actin processed pseudogene and, although it contained internal deletions, appeared to possess intact 5' and 3' ends. A second L1Md member (L1Md-14RH1) also appeared to have an intact 5' end but had lost most of its 3' portion, and a third member (L1Md-14RH2) was an internal fragment. The repeated sequence at the 5' ends of L1Md-14LH and L1Md-14RH1 showed these to be members of the L1Md-A family.     

GEM, a cluster of repetitive sequences in the Drosophila subobscura genome

GEM is a new family of repetitive sequences detected in the D. subobscura genome. Two of the four described GEM elements encompass a heterogeneous central module, with no detectable ORF, flanked by two long inverted repeats. These elements are composed of a set of repetitive modules, which are inverted repeat (IR), direct repeat (DR), palindromic sequence (PS), long sequence (LS) and short sequence (SS). These five modules can be found either clustered or dispersed as single modules in the D. subobscura genome, in euchromatic and heterochromatic regions. In addition to the 3' region of Adh retrosequences, single IR and LS blocks were found associated with the promoter region of different genes, in particular, LS-like blocks have also been found associated with functional genes in D. melanogaster and D. virilis. Conversely, the DR block is highly similar to satellite DNAs from some other species of the obscura group. In addition, GEM elements share some structural features with IS elements described in different Drosophila species. It is likely that both GEM and IS sequences would be vestiges of an ancestral transposable element.     

Palindromic repetitive elements in the mitochondrial genome of Volvox

Group I introns were found in the cob and cox I genes of Volvox carteri. These introns contain tandem arrays of short palindromic sequences that are related to each other. Inspection of other regions in the mtDNA revealed that similar palindromic repetitive sequences are dispersed in the non-protein coding regions of the mitochondrial genome. Analysis of the group I intron in the cob gene of another member of Volvocaceae, Volvox aureus, has shown that its sequence is highly homologous to its counterpart in V. carteri with the exception of a cluster of palindromic sequences not found in V. carteri. This indicates that the palindromic clusters were inserted into the introns after divergence of the two species, presumably due to frequent insertions of the palindromic elements during evolution of the Volvocaceae. Possible involvement of the palindromic repetitive elements in the molecular evolution of functional RNAs is discussed.     

Microsatellites and associated repetitive elements in the sheep genome

To determine the frequency and clustering of a variety of simple di-and trinucleotide repeats, an Artiodactyl short interspersed element (SINE), an ovine satellite repeat, and a human Alu 1 repeat were used to screen a random selection of cosmids containing inserts of ovine genomic DNA. In total, 197 individual cosmids were digested with EcoRI and the fragments separated on 0.7% agarose gels. Southern blots of these gels were then sequentially probed with (AC)₇, (CT)₉, and (CAC)₆ oligonucleotides, and the repeats described above. The frequency at which (AC)₁, (CT)_n, and (CAC)_n repeats were found in the cosmids indicated that they occurred at average intervals of 65 kb, 367 kb, and 213 kb respectively within the ovine genome. The Artiodactyl SINE was the most common, occurring at an average interval of 20 kb. No human Alu 1 sequences were detected. There was a significant positive association between the (AC)_n and the Artiodactyl SINE. This association is quite strong as there was significant clustering of the two repeats both within cosmids and also within the EcoRI fragments of the digested genomic fragments. With the exception of the sheep satellite sequence, which occurs in tandem arrays, none of the other repeats showed significant clustering within the 41-kb (average size) cosmid inserts. The first 25 ovine microsatellites we characterized had an average polymorphic information content (PIC) of 0.65. The different microsatellite types, containing either perfect, imperfect, or compound repeats, had similar average PICs of 0.64, 0.65, and 0.66 respectively. There was a weak regression relationship (R²(adj)%=21.9) between the length of the longest uninterrupted dinucleotide repeat in the largest allele and the PIC of the microsatellite.     

A new family of interspersed repetitive DNA sequences in the mouse genome

Repetitive DNA sequences near immunoglobulin genes in the mouse genome (Steinmetz et al., 1980a,b) were characterized by restriction mapping and hybridization. Six sequences were determined that turned out to belong to a new family of dispersed repetitive DNA. From the sequences, which are called R1 to R6, a 475 base-pair consensus sequence was derived. The R family is clearly distinct from the mouse B1 family (Krayev et al., 1980). According to saturation hybridization experiments, there are about 100,000 R sequences per haploid genome, and they are probably distributed throughout the genome. The individual R sequences have an average divergence from the consensus sequence of 12.5%, which is largely due to point mutations and, among those, to transitions. Some R sequences are severly truncated. The R sequences extend into A-rich sequences and are flanked by short direct repeats. Also, two large insertions in the R2 sequence are flanked by direct repeats. In the neighbourhood of and within R sequences, stretches of DNA have been identified that are homologous to parts of small nuclear RNA sequences. Mouse satellite DNA-like sequences and members of the B1 family were also found in close proximity to the R sequences. The dispersion of R sequences within the mouse genome may be a consequence of transposition events. The possible role of the R sequences in recombination and/or gene conversion processes is discussed.     

Integration of a vector containing rodent repetitive elements in the rat genome.

We have previously shown that integration of a polyoma vector containing rodent repetitive elements into rat cellular DNA is non-random (Wallenburg et al. J. Virol. 50: 678-683). Junctions between the polyoma vector and the host DNA occur in the repetitive sequences of the vector about ten times more frequently than would be expected if sequences from the vector were used randomly for integration. In this paper we looked at the host sequences involved in these junctions. Our analysis did not reveal any repetitive or specific sequences and we presume therefore that the repetitive sequences of the vector acted as hot spots for illegitimate recombination. We also analysed the integration mechanism and found that: First, even though the polyoma vector was transfected in the presence of carrier DNA, integration did not involve the formation of a transgenome. Second, in at least one of the clones analysed, integration resulted in deletion of host DNA sequences. Third, the host DNA displaced at the integration site was considerably longer than the integrated segment.     

Organization of a family of highly repetitive sequences within the human genome

Recombinant clones containing the highly repetitive human DNA sequence approximately 340 base-pairs in length obtained after EcoRI digestion (αRI-DNA) were cloned in plasmid pAT153. Two clones contained a single copy of the αRI-DNA sequence, and the third had an insert with two copies of the sequence in tandem. When radioactive recombinant DNA was hybridized to total human DNA partially digested with EcoRI, a series of multiple bands was obtained up to 22 repeats in length, demonstrating that the αRI-DNA sequences occur in tandem arrays in the genomic DNA. A reassociation analysis using isolated insert DNA from one of the recombinant clones showed that the family of sequences is repeated 22,000 times in the human genome. Clones containing the αRI-DNA sequence were also isolated from a library of human genomic DNA in bacteriophage λ. Using these clones it was shown that, in at least some cases, the repetitive element is bounded by DNA less abundant than the αRI sequence.     

A tandemly-oriented late gene cluster within the vaccinia virus genome.

The nucleotide sequence of a 5.1 kilobase-pair fragment from the central portion of the vaccinia virus genome has been determined. Within this region, five complete and two incomplete open reading frames (orfs) are tightly-clustered, tandemly-oriented, and read in the leftward direction. Late mRNA start sites for the five complete orfs and one incomplete orf were determined by S1 nuclease mapping. The two leftmost complete orfs correlated with late polypeptides of 65,000 and 32,000 molecular weight previously mapped to this region. When compared with each other and with sequences present in protein data banks, the five complete orfs showed no significant homology matches amongst themselves or any previously reported sequence. The six putative promoters were aligned with three previously sequenced late gene promoters. While all of the nine are A-T rich, the only apparent consensus sequence is TAA immediately preceeding the initiator ATG. Identification of this tandemly-oriented late gene cluster suggests local organization of the viral genome.     

Molecular evolution of a repetitive region within the per gene of Drosophila

The clock gene period (per) controls a number of biological rhythms in Drosophila. In D. melanogaster, per has a repetitive region that encodes a number of alternating threonine-glycine residues. We sequenced and compared this region from several different Drosophila species belonging to various groups within the Drosophila and Sophophora subgenera. This part of per shows a great variability in both DNA sequence and length. Furthermore, analysis of the data suggests that changes in the length of this variable region might be associated with amino acid replacements in the more conserved flanking sequences.      

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.     

Insertions of transposable elements in the promoter proximal region of the gene cluster for Escherichia coli H+-ATPase: 8 base pair repeat generated by insertion of IS1

Summary A plasmid pKY159 (Yamaguchi and Yamaguchi 1983) carrying a promoter proximal portion of the gene cluster of the proton-translocating ATPase (H⁺-ATPase) of Escherichia coli causes growth inhibition of wild-type cells. Insertion of a transposable element in this plasmid released this inhibitory effect. In analyzing this inhibitory effect, we determined the insertion points at the nucleotidesequence level of transposable elements on 30 independent derivatives of pKY159. Insertions of IS1, IS5, and were found between the promoter and the gene for a possible component of 14,000 daltons of the H⁺-ATPase. Of 31 insertions, 26 were of IS1 and were located at the same site, indicating that this site is a hotspot for IS1 insertion and that IS1 insertion is much more frequent than that of IS5 of in this region. Four different sites for IS1 insertion were found; in two of these an 8 base pair (bp) duplicate of the target sequence (AAAAACGT and AAACGTTG) was generated, while in the other two a 9 bp duplicate was found. In all cases in this study the nucleotide sequence of IS1 was the same as that of IS1-K. In the two cases with an 8 bp duplicate in different sites, a common 6 bp sequence (AAACGT) was found. These results suggested that generation of the 8 bp duplicate is related to the common sequence rather than a mutation in IS1 suggested by Iida et al. (1981) and also suggested that the essential length of the duplicate is 8 bp or less than 8 bp. A 6 bp sequence (GTGATG) homologous to the end portion of IS1 was found at the hotspot, but not at other sites, suggesting that this homology contributed to the high frequency of IS1 insertion. The direction of IS1 insertion at the hotspot was the same in 25 of 26 instances, suggesting that the direction of IS1 insertion is determined by the structure of the target and/or its nearby sequence.Abbreviations bp base pairs - 14 K protein a possible component of the H⁺-ATPase with molecular weight of 14,000 (see Kanazawa and Futai 1982 for details) - EDTA ethylenediaminetetraacetate     

Immunochemical detection of multiple conformations within a 36 base pair oligonucleotide.

A 36 base pair chimeric oligonucleotide containing a central core of DNA duplex flanked by RNA/DNA hybrid at each end was synthesized. These distinct regions of the oligonucleotide adopt different conformations which were detected with antibody probes. Enzyme linked immunosorbent assays (ELISA) and a gel electrophoresis retardation assay were used to demonstrate the binding of antibodies which recognize B-DNA, Z-DNA and RNA/DNA hybrid. The DNA duplex core of this oligonucleotide adopts the B-conformation in 0.14 M NaCl. In high salt solution (4 M NaCl) the DNA core adopts the Z-conformation. The RNA/DNA hybrid at the ends of the oligomer adopt a conformation which is distinct from both B-DNA and A-RNA.     

Foreign DNA introduced by calcium phosphate is integrated into repetitive DNA elements of the mouse L cell genome.

We investigated the sites of integration of exogenous DNA fragments introduced by DNA-mediated gene transfer. Mouse Ltk- cells were transformed with the herpes simplex virus thymidine kinase gene and pBR322 DNA by the calcium phosphate precipitation method. Some of the integrated exogenous DNA sequences were recovered from the stable tk+ transformants in the form of plasmids that were capable of propagation in bacteria. Four plasmids derived from two cloned cell lines were analyzed in detail by nucleotide sequencing and hybridization techniques. These plasmids contained a total of seven cellular-exogenous DNA junctions. In all cases, there was no sequence homology between the exogenous and cellular DNA sequences adjacent to the joining sites, and no specific exogenous or cellular sequences occurred at the junctions. Rearrangement or deletion of Ltk- DNA was always associated with the integration of exogenous DNA. All of the assignable cellular sequences at the junctions were repetitive sequences. Two of these sequences were from the MIF-1 repetitive sequence family, and a third consisted of a 40-base pair simple copolymer of alternating deoxyadenosine-deoxythymidine. Our results suggest that repetitive sequences are relatively favorable sites for the integration of exogenous DNA.     

Neighboring base composition is strongly correlated with base substitution bias in a region of the chloroplast genome

Nucleotide sequence from a region of the chloroplast genome is presented for 12 species spanning four subfamilies of the grass family. The region contains the coding sequence for the rbcL gene and the intergenic spacer between the gene coding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase rbcL and the photosystem I gene psal. This intergenic spacer contains a pseudogene for rpl23 as well as two noncoding segments with different A+T contents. Using the sequence of rbcL a chloroplast phylogeny of this family was constructed by parsimony. Variable sites of the two noncoding segments were traced onto the phylogeny to study the dynamics of base substitution. This was also performed for the fourfold-degenerate sites of the rbcL gene. A wide variation in transversion/transition is observed between the two noncoding segments and between the noncoding DNA and the fourfold-degenerate sites of rbcL This variation is correlated with regional A+T content. As regional A+T content decreases, the ratio of transversions to transitions also decreases. Substitutions were then scored in relation to neighboring base composition. The composition of the two bases immediately flanking each substitution is highly correlated with the transversion/transition bias. When both the 5 and 3 flanking bases are an A or a T, transversions are observed 2.2 times as frequently as transitions. When either or both neighbors are a C or a G, the opposite trend is found; transitions are observed 1.5 times more frequently than transversions. Correspondence to: Brian R. Morton     

Genomic organization and nucleotide sequence of a long mosaic repetitive DNA in the mouse genome

A long mosaic repetitive sequence (LMRS) was isolated from a mouse liver genome library using a mouse repetitive DNA as a probe. LMRS exhibits the following features: (1) it is almost 15 kb in length; (2) it is partly organized in tandem array and frequently interrupted by other repeated sequences; and (3) it is located predominantly on the A3 band of the mouse X Chromosome (Chr). One fragment of LMRS (B6) shows restriction fragment length polymorphism (RFLP) between different mouse strains, and is thus potentially useful for mapping studies. The nucleotide sequence confirms a mosaic organization of LMRS which includes three repeats in the 5 part, showing similarity with the 5 end of L1Md-A2, and seven long A+T rich segments in the central part of the element. Our findings suggest that this sequence may have arisen from the duplication of an ancestral motif and has expanded by successive waves of amplification and invasion by foreign sequences.The nucleotide sequence data reported in this paper have been submitted to EMBL and have been assigned the accession number X55036.