Structure of circular copies of the 412 transposable element present in Drosophila melanogaster tissue culture cells, and isolation of a free 412 long terminal repeat

We have isolated, from Drosophila melanogaster tissue culture cells, extrachromosomal circular forms of the transposable element 412, and have cloned some of them in bacteriophage lambda. A total of 24 clones have been analysed in detail by restriction and heteroduplex mapping. Seventeen clones are virtually identical, and contain complete 412 elements with one copy of the long terminal direct repeat (LTR). The remaining seven clones are all different and contain various rearrangements. Four have deletions, two have some 412 sequence substituted by other DNA and one has both an inversion and a deletion. The clone containing the inversion has two LTRs in inverted orientation and separated by a few thousand bases of 412 DNA. The base sequences of the two LTRs in this clone, and of the LTR in one of the 17 clones containing complete elements are very similar to that of the 481 base-pair LTR of a genomic 412 element. We have found no evidence, in either cloned or uncloned material, for 412 elements with two LTRs as a tandem direct repeat. We have found that there are several "free" 412 LTRs in genomic DNA from D. melanogaster strains Canton S and Oregon R, and from D. melanogaster tissue culture cells. We have cloned and sequenced one of these free LTRs. It is 475 base-pairs long and is flanked by a direct repeat four base-pairs long. This sequence differs from that of the 481 base-pair repeat at 16 places including a ten base deletion.     

The 1723 element: a long, homogeneous, highly repeated DNA unit interspersed in the genome of Xenopus laevis

We describe a highly repeated DNA element in the Xenopus laevis genome. This sequence, named the 1723 element, was first identified among sequences that are transcribed during embryonic development. The element is present in about 8500 copies per haploid genome, which together accounts for about 2.4% of the genome. Most copies of the element have highly conserved restriction maps, and are interspersed in the genome. The copies range in size from 6000 to 10,000 base-pairs due to an expandable region that contains variable numbers of a tandemly repeating 183 to 204 base-pair unit. The element is framed by an imperfect 18 base-pair inverted sequence, and inverted repeats of 180 to 185 base-pairs are nearby. Sequence analysis of DNA adjacent to three cloned elements shows that the elements are flanked by 8 base-pair direct repeats. These and other properties of 1723 suggest that it may be transposable.     

Plasmid R6K DNA replication. II. Direct nucleotide sequence repeats are required for an active gamma-origin

Insertions of the transposable element Tn5 were obtained at a number of sites within the γ origin region of plasmid R6K. A contiguous region of DNA, 240 base-pairs in length, was identified as the functional γ origin of replication on the basis that Tn5 insertions within this region inactivated the origin. This region contains seven, tandemly arranged, 22 base-pair direct repeats. Spontaneous deletion of a Tn5 sequence that is inserted in one of the repeats was accompanied generally by the loss of nucleotide sequences equivalent to one or more of the repeat sequences. Deletion mutants of the γ origin that lack one or two repeats are still functional but a plasmid containing such a mutant origin is not maintained stably. Deletions of nucleotide sequences equivalent to four or more of the direct repeats result in the inactivation of the R6K γ origin.     

Terminal repeats of the Drosophila transposable element copia: nucleotide sequence and genomic organization

We have determined the nucleotide sequence of the terminal regions of two members of the copia sequence family of D. melanogaster. The first 276 bp at one end of a copia element are repeated in direct orientation at its other end. The direct repeats on a single copia element are identical to each other, but they differ by two nucleotide substitutions between the two elements which were examined; this suggests that during transposition only one direct repeat of the parent element is used as a template for both direct repeats of the transposed element. Each direct repeat itself contain a 17 bp imperfectly matched inveted terminal repetition. The ends of copia show significant sequence homology both to the yeast Ty1 element and to the integrated provirus of avian spleen necrosis virus, two other eucaryotic elements known to insert at many different chromosomal locations. Analysis of the genomic organization of the direct repeat sequence demonstrates that it seldom, if ever, occurs unlinked to an entire copia element.     

The organization of internal telomeric repeats in the polytene chromosomes of the hypotrichous ciliate Stylonychia lemnae.

There exist about 1000-1500 internal telomeric sequences per haploid genome in the polytene chromosomes of the hypotrichous ciliate Stylonychia lemnae. All these telomeric repeats are contained in a very conserved element. This element consists of two 2 kb direct repeats flanking a 2.6 kb sequence. Immediately adjacent to one of the repeats a 18mer C4A4C4A4C2 telomeric sequence is localized. Sequences homologous to macronuclear DNA follow 180 bp downstream of the C4A4-bloc. These macronuclear homologous sequences are flanked by the second direct repeat. The possible origin and function of these telomere containing elements is discussed.     

Subtelomeric regions of yeast chromosomes contain a 36 base-pair tandemly repeated sequence.

We have determined the nucleotide sequence of a region of DNA derived from the end of one chromosome of the yeast, Saccharomyces cerevisiae. Inspection of the sequence reveals the presence of 12 tandem direct repeats, each 36 nucleotides long and having nearly identical sequence. Each 36 base-pair repeat can be further subdivided into three tandem sub-repeats of a similar 12 base-pair sequence. Analysis of total genomic yeast DNA from several strains by Southern hybridization suggests that the number of tandem 36 base-pair repeat units may vary from approximately 8 to 25 among different telomeric regions. Differences in the number of repeats may have arisen by unequal crossing over between them. Furthermore, the finding that the pattern of bases at multiple variable positions within the repeat unit is not random suggests that these regions may undergo gene conversion events that render them homogeneous.     

Rat immunoglobulin E heavy chain locus

A 2100 base-pair long sequence has been established which covers all four constant domains of the rat epsilon-chain. An analysis of messenger RNA from an immunoglobulin E producing rat immunocytoma revealed two separate epsilon-chain mRNA species, 2.3 X 10(3) and 2.8 X 10(3) base-pairs long. The latter mRNA encodes the membrane binding form of the epsilon-chain. The membrane exons which are located approximately 2 X 10(3) base-pairs away from the 3'-side of the CH4 exon were also sequenced. A comparison between the rat and mouse epsilon-chains at the protein sequence level revealed an overall homology of 80% which, as expected, is considerably higher than the homology found between rat and human epsilon-chains. The fourth constant domain together with the two membrane exons exhibited the highest degree of homology, 81 to 89%. Only two differences were found when the epsilon-chains from LOU and Sprague Dawley rats were compared. The most striking difference at the nucleotide sequence level between the rat, mouse, and human epsilon genes was found within the first intron. The mouse genome contains a unique 366 base-pair long sequence in this region. The inserted sequence is repetitive and present in approximately 100 copies in the mouse genome. It is flanked by 22 base-pair long direct repeats and contains also 14 base-pair long inverted repeats, thus having properties in common with transposable elements.     

Molecular and functional organization of yeast plasmid pSR1

The nucleotide sequence of a 6251 base-pair plasmid, pSR1, harbored in an osmophilic haploid yeast, Zygosaccharomyces rouxii (formerly Saccharomyces rouxii), was determined. No homology was detected between the sequences of pSR1 and 2-micron DNA of Saccharomyces cerevisiae. pSR1 has a pair of inverted repeats consisting of completely homologous 959 base-pair sequences, which separate two unique sequences 2654 base-pairs and 1679 base-pairs long. Each inverted repeat has an ARS sequence functional in both Z. rouxii and S. cerevisiae hosts. Short direct repeats or dyad symmetries were observed in the inverted repeats similar to those found close to the replication origin of 2-micron DNA. Three open reading frames, P, S and R, each able to encode a protein of molecular weight larger than 10,000, were found. Insertional inactivation of R gave rise to a defect in the intramolecular recombination at the inverted repeats, and that of S reduced the copy number of pSR1 in the S. cerevisiae host. The maintenance stability of the plasmid was also tested in the heterogeneous S. cerevisiae host, but the results of the insertional inactivation of P, S and R were ambiguous. pSR1 and 2-micron DNA were compatible in S. cerevisiae cells, but the protein factors encoded by these plasmids did not complement each other.     

Mobile dispersed genetic element MDG1 of Drosophila melanogaster: nucleotide sequence of long terminal repeats.

Long terminal repeats (LTRs) of two members of mdg1 family were sequenced. In the both cases, they are represented by perfect direct repeats 442 and 444 bp in length. Sixteen nucleotides in the LTRs of two different mdg1 elements are different. Each LTR contains slightly mismatched 16-nucleotide inverted repeats located at the ends of the LTR. Six base pairs closest to the termini of LTR form perfect inverted repeats. On the gene-distal sides of LTRs, short 4-nucleotide direct repeats are located, probably representing the duplication of a target DNA sequence arising from insertion of mdg. They are different in the two cases analyzed. Just as the other analyzed eukaryotic transposable elements, mdg1 starts with TGT and ends with ACA. Within the both strands of LTR, the sequences similar to Hogness box (a putative signal for RNA initiation, or a selector) and AATAAA blocks (putative polyadenylation signals) are present. The LTR of mdg1 contains many short direct and inverted repetitive sequences. These include a 10-nucleotide sequence forming a perfect direct repeat with the first ten nucleotides of the LTR. A region of LTR about 70 bp long is represented by simple repetitive sequences (TAT).     

Structure-function analysis of the inverted terminal repeats of the sleeping beauty transposon

Translocation of Sleeping Beauty (SB) transposon requires specific binding of SB transposase to inverted terminal repeats (ITRs) of about 230 bp at each end of the transposon, which is followed by a cut-and-paste transfer of the transposon into a target DNA sequence. The ITRs contain two imperfect direct repeats (DRs) of about 32 bp. The outer DRs are at the extreme ends of the transposon whereas the inner DRs are located inside the transposon, 165-166 bp from the outer DRs. Here we investigated the roles of the DR elements in transposition. Although there is a core transposase-binding sequence common to all of the DRs, additional adjacent sequences are required for transposition and these sequences vary in the different DRs. As a result, SB transposase binds less tightly to the outer DRs than to the inner DRs. Two DRs are required in each ITR for transposition but they are not interchangeable for efficient transposition. Each DR appears to have a distinctive role in transposition. The spacing and sequence between the DR elements in an ITR affect transposition rates, suggesting a constrained geometry is involved in the interactions of SB transposase molecules in order to achieve precise mobilization. Transposons are flanked by TA dinucleotide base-pairs that are important for excision; elimination of the TA motif on one side of the transposon significantly reduces transposition while loss of TAs on both flanks of the transposon abolishes transposition. These findings have led to the construction of a more advanced transposon that should be useful in gene transfer and insertional mutagenesis in vertebrates.     

Analysis of putative DNA amplification genes in the element AUD1 of Streptomyces lividans 66

The amplifiable AUD1 element of Streptomyces lividans 66 consists of two copies of a 4.7 kb sequence flanked by three copies of a 1 kb sequence. The DNA sequences of the three 1 kb repeats were determined. Two copies (left and middle repeats) were identical: (1009 by in length) and the right repeat was 1012 bp long and differed at 63 positions. The repeats code for open reading frames (ORFs) with typical Streptomyces codon usage, which would encode proteins of about 36 kD molecular weight. The sequences of these ORFs suggest that they specify DNA-binding proteins and potential palindromic binding sites are found adjacent to the genes. The putative amplification protein encoded by the right repeat was expressed in Escherichia coli.     

Nucleotide sequence analysis and enhancer function of long terminal repeats associated with an endogenous African green monkey retroviral DNA.

The nucleotide sequence and enhancer activity of the long terminal repeats (LTRs) associated with a cloned endogenous African green monkey (AGM) retroviral DNA designated as lambda-AGM-1 was studied. A unique feature of the endogenous AGM proviral LTRs was the presence of multiple copies of two types of directly repeating units in the U3 region: 16 8-base-pair (bp) repeats were present in the 5' LTR and 12 were present in the 3' LTR which were bound by a 6-bp perfect direct repeat; tandem duplication of a 32-bp sequence resulted in 3.5 copies in the 5' LTR and 2.5 copies in the 3' LTR. Nucleotide sequence homology was seen between the 8-bp direct repeats located in the AGM proviral LTRs and a 10-bp repeat unit of the deca-satellite present in AGM cellular DNA. The 32-bp repeats of the AGM proviral LTRs contained sequences which were related to the SV40 21-bp repeats and to the "core" of the SV40 72-bp enhancer element. Furthermore, the AGM provirus was distinct from known infectious retroviruses due to the presence of a primer-binding sequence complementary to the 3' terminus of mammalian tRNAGly. Functional analysis of the 3' LTR present in lambda-AGM-1 DNA by chloramphenicol acetyltransferase assay demonstrated enhancer activity associated with the 32-bp direct repeats. Sequences outside the 32-bp unit were necessary for full activator function, suggesting the presence of multiple enhancer domains in the AGM provirus.     

Sequence, organization, and evolution of the A+T region of Drosophila melanogaster mitochondrial DNA

The long (4.6-kb) A+T region of Drosophila melanogaster mitochondrial DNA has been cloned and sequenced. The A+T region is organized in two large arrays of tandemly repeated DNA sequence elements, with nonrepetitive intervening and flanking sequences comprising only 22% of its length. The first repeat array consists of five repeats of 338-373 bp. The second consists of four intact 464-bp repeats and a fifth partial repeat of 137 bp. Three DNA sequence elements are found to be highly conserved in D. melanogaster and in several Drosophila species with short A+T regions. These include a 300-bp DNA sequence element that overlaps the DNA replication origin and two thymidylate stretches identified on opposite DNA strands. We conclude that the length heterogeneity observed in the A+T regulatory region in mitochondrial DNAs from the genus Drosophila results from the expansion (and contraction) of the number of repeated DNA sequence elements. We also propose that the 300-bp conserved DNA sequence element, in conjunction with another primary sequence determinant, perhaps the adjacent thymidylate stretch, functions in the regulation of mitochondrial DNA replication.      

Interaction between the replication origin and the initiator protein of the filamentous phage f1. Binding occurs in two steps

The replication initiator protein of bacteriophage f1 (gene II protein) binds to the phage origin and forms two complexes that are separable by polyacrylamide gel electrophoresis. Complex I is formed at low gene II protein concentrations, and shows protection from DNase I of about 25 base-pairs (from position +2 to +28 relative to the nicking site) at the center of the minimal origin sequence. Complex II is produced at higher concentrations of the protein, and has about 40 base-pairs (from -7 to +33) protected. On the basis of gel mobility, complex II appears to contain twice the amount of gene II protein as does complex I. The 40 base-pair sequence protected in complex II corresponds to the minimal origin sequence as determined by in-vivo analyses. The central 15 base-pair sequence (from +6 to +20) of the minimal origin consists of two repeats in inverted orientation. This sequence, when cloned into a plasmid, can form complex I, but not complex II. We call this 15 base-pair element the core binding sequence for gene II protein. Methylation interference with the formation of complex I by the wild-type origin indicates that gene II protein contacts six guanine residues located in a symmetric configuration within the core binding sequence. Formation of complex II requires, in addition to the core binding sequence, the adjacent ten base-pair sequence on the right containing a third homologous repeat. A methylation interference experiment performed on complex II indicates that gene II protein interacts homologously with the three repeats. In complex II, gene II protein protects from DNase I digestion not only ten base-pairs on the right but also ten base-pairs on the left of the sequence that is protected in complex I. Footprint analyses of various deletion mutants indicate that the left-most ten base-pairs are protected regardless of their sequence. The site of nicking by gene II protein is located within this region. A model is presented for the binding reaction involving both protein-DNA and protein-protein interactions.     

The transposable element Uhu from Hawaiian Drosophila--member of the widely dispersed class of Tc1-like transposons

We report the complete nucleotide sequence of the transposable element Uhu from the vicinity of the alcohol dehydrogenase (Adh) gene of Drosophila heteroneura (an endemic Hawaiian Drosophila). The complete element is about 1650 base-pairs (bp) long, has 46-50 base-pair inverse imperfect repeats at it's ends, and contains a large open reading frame potentially encoding a 192 amino acid protein. We demonstrate that Uhu belongs to a class of transposable elements which includes Tc1 from Caenorhabditis elegans, Barney from Caenorhabditis briggsae, and HB1 from Drosophila melanogaster. All of these elements share significant sequence similarity, are approximately 1600 base pairs long, have short inverse terminal repeats (ITRs), contain open reading frames (ORFs) with significant sequence identity, and appear to insert specifically at TA sequences generating target site duplications.