Family Of Repetitive Elements Research Articles

A complex population of RNAs exists in human ejaculate spermatozoa: implications for understanding molecular aspects of spermiogenesis.

The presence of mRNAs in human ejaculate spermatozoa is well established, yet little is known of the representation or function of these transcripts. To address these issues, the complexity of spermatozoal RNA was examined. As expected, testis-expressed mRNAs were detected by RT-PCR in mature human spermatozoa. Interestingly, when a testis cDNA library was probed with total spermatozoal RNA, less than 2% of plaques gave a strong hybridization signal, suggesting a rather unique sperm-derived population. To further define the sequence distribution, 18 strongly hybridizing clones were selected at random for end-sequence analysis. Twelve matched unique sequences in the EST, STS and NR databases, whereas five showed no similarity to any of the sequences in the databases. In addition, one clone belonged to the SINE repetitive element family. As demonstrated by sequencing randomly primed cloned inserts, short (SINE/MER) or long (LINE/ORF2) interspersed repeat-like sequences are also contained as part of the spermatozoal RNA fraction. It is now evident that human spermatozoa contain a rich repertoire of both known and unknown protein-encoding and non-coding RNAs. This provides a unique opportunity to identify and investigate the many genes responsible for the structure and function/dysfunction of the male gamete using spermatozoal RNA as the template.

A ribosomal orphon sequence from Xenopus laevis flanked by novel low copy number repetitive elements.

We have used a differential cloning approach to isolate ribosomal/non-ribosomal frontier sequences from Xenopus laevis. A ribosomal intergenic spacer sequence (IGS) was cloned and shown not to be physically linked with the ribosomal locus. This ribosomal orphon contained the IGS sequences found immediately downstream of the 28S gene and included an array of enhancer repetitions and a non-functional spacer promoter. The orphon sequence was flanked by a member of the novel 'Frt' low copy repetitive element family. Three individual Frt repeats were sequenced and all members of this family were shown to lie clustered at two chromosomal sites, one of which contained the ribosomal orphon. One of the Frt elements contained an insertion of 297 bp that showed extensive homology to sequences within at least three other Xenopus genes. Each homology region was flanked by members of the T2 family of short interspersed repetitive elements, (SINEs), and by its target insertion sequence, suggesting multiple translocation events. The data are discussed in terms of the evolution of the ribosomal gene locus.

Structural, genomic, and phylogenetic analysis of Lian, a novel family of non-LTR retrotransposons in the yellow fever mosquito, Aedes aegypti.

A retrotransposon named Lian-Aa1 was discovered in an intron of an AaHR3-1 gene of the yellow fever mosquito, Aedes aegypti. This retrotransposon contained a long open reading frame with 1,219 amino acids that included endonuclease, reverse transcriptase, and RNase H domains. It was shown that in the Rock strain of Ae. aegypti, there were up to 1,380 copies of Lian elements, equivalent to 0.8% of the entire genome. Five additional copies of Lian elements were isolated, mapped by restriction digestion, and partially sequenced. The 5' and 3' ends of the Lian family were determined by comparing the terminal sequences of the six copies and were subsequently confirmed by the identification of putative target duplications flanking Lian-Aa1 and Lian-Aa2. The Lian family is likely a novel family of non-long-terminal-repeat (non-LTR) retrotransposons that terminate in a repeat of (CTGA-TAC)2. On average, the six copies of Lian elements showed only 0.6% sequence divergence at the nucleotide level in both a 735-bp region at the 5' end and a 1,124-bp coding region. Genomic Southern blots also revealed a very high degree of similarity among hundreds of Lian elements, suggesting very recent activity of Lian. Furthermore, all six analyzed Lian elements were closely associated with one or more different families of repetitive elements. It is possible that these associations could reflect the complex relationship between Lian elements and the rest of the Ae. aegypti genome. Phylogenetic analyses based on the reverse transcriptase, domains of 36 non-LTR retrotransposons including Lian-Aa1 identified five major subgroups that were supported by bootstrap replications. In contrast to the majority of non-LTR retrotransposons, Lian-Aa1 has an RNase H domain that is similar to a few other non-LTR retrotransposons and some retroviruses, which is consistent with the previously proposed independent assortment of different domains during the evolution of retroelements.

Macromolecular organization and genetic mapping of a rapidly evolving chromosome-specific tandem repeat family (B77) in cotton (Gossypium).

Isolation and characterization of the most prominent repetitive element families in the genome of tetraploid cotton (Gossypium barbadense L; [39]) revealed a small subset of families that showed very different properties in tetraploids than in their diploid progenitors, separated by 1-2 million years. One element, B77, was characterized in detail, and compared to the well-conserved 5S and 45S rRNA genes. The 572 bp B77 repeat was found to be concentrated in several discontinuous tandem arrays confined to a single 550 kb SalI fragment in tetraploid cotton. Genetic mapping based on the absence of the pentameric 'rung' in the G. barbadense 'ladder' showed that B77 maps to a D-subgenome chromosome. In situ hybridization supports the contention that the array is confined largely to a single chromosomal site in the D-subgenome. The B77 repeat has undergone a substantial increase in copy number since formation of tetraploid cotton from its diploid relatives. RFLPs observed among tetraploid cotton species suggest that amplification and/or rearrangement of the repeat may have continued after divergence of the five tetraploid cotton species. B77 contains many short direct repeats and shares significant DNA sequence homology with a Nicotiana alata retrotransposon Tna1-2 integrase motif. The recent amplification of B77 on linkage group D04 suggests that the D-subgenome of tetraploid cotton may be subject to different evolutionary constraints than the D-genome diploid chromosomes, which exhibit few genome-specific elements. Further, the abundance of B77 in G. gossypioides supports independent evidence that it may be the closest extant relative of the D-genome ancestor of cotton.

Molecular Characterization of a Mouse Genomic Element Mobilized by Advanced Glycation Endproduct Modified-DNA (AGE-DNA)

DNA modified by advanced glycation endproducts (AGEs) undergoes a high frequency of insertional mutagenesis. In mouse lymphoid cells, these mutations are due in part to the transposition of host genomic elements that contain a DNA region homologous to the Alu family of repetitive elements. One particular 853 bp insertion, designated INS-1, was identified previously as a DNA element common to plasmids recovered from multiple, independent lymphoid cell transfections. To characterize the genomic origin of this element, we used a 281-bp region of non-Alu-containing INS-1 sequence, designated. CORE, as a probe in Southern hybridization and for screening a bacteriophage mouse genomic DNA library. The resultant clones were sequenced and localized within the mouse genome. Two distinct genomic clones of 15 kB and 17 kB in size were isolated. A 522-bp unique region common to INS-1 and corresponding to the CORE sequence was identified in each clone. In both cases, CORE was found to be surrounded by repetitive DNA sequences: a 339-bp MT repeat at the 5' end, and a 150-bp B1 repeat at the 3' end. The CORE sequence was localized to mouse chromosome 1. These studies revealed that the CORE region of INS is present in low copy number but is associated with known repetitive DNA elements. The presence of these repetitive elements may facilitate the transposition of CORE by recombination or other, more complex rearrangement events, and explain in part the origin of AGE-induced insertional mutations.

Enrichment of middle repetitive element Bm-1 transcripts in translationally active RNA fractions of the silkmoth, Bombyx mori.

The Bm-1 repetitive element family represents a group of transcribed repetitive sequences in the genome of the silkmoth Bombyx mori. In the Bm-5 and BmN permanent cell lines studied here, alpha-amanitin inhibition and nuclear 'run-on' experiments demonstrated that approximately 80% of the Bm-1 transcripts are produced by RNA polymerase II. Bm-1 transcripts are dramatically enriched in poly A+ and polysomal RNA fractions compared to total RNA in these two cell lines. In the Bm-5 cell line, from total to poly A+ and polysomal RNA fractions, Bm-1 transcripts are enriched approximately 4 and 2 times, respectively, while in the BmN cell line these same fractions are enriched about 2 and 19 times compared to total RNA. This suggests that the Bm-1 transcripts may be involved in post-transcriptional processes or control of translation. Our data also revealed less size heterogeneity of Bm-1 transcripts in polysomal as compared to nuclear fractions. In the Bm-5 and BmN cell lines, the size of most transcripts containing Bm-1 sequences increases from approximately 1700 nt in the nucleus to 3000 nt in the polysomal fraction, both fractions with RNA much larger than the Bm-1 consensus sequence (250 bp). This raises the possibility that some Bm-1 elements are transcribed as part of larger transcripts containing mRNA by way of 'read-through', and may be involved in post-transcriptional regulation of gene expression as cis and/or trans acting elements.

T-DNA integrations in a new family of repetitive elements of Nicotiana tabacum

A number of T-DNA insertions in the genome of Nicotiana tabacum were characterized. One class of integrations was found to have occurred in a new family of highly repetitive sequences. Three genomic regions (ecoA, ecoB, and ecoC) were isolated, all of which contain basic units of 180 bp, organized in direct tandem repeats. Several of the 180-bp elements contain an EcoRI recognition site within the repeating unit and are therefore named "eco repeats." All members of this family are weakly homologous in sequence to a previously described class of repeat elements which contained a BamHI site (HRS60 repeat family), which suggests that both groups of sequences are of common evolutionary origin. The allotetraploid genome of N. tabacum is presumed to originate from the hybridization of two diploid genomes. The HRS60 elements previously described have been found exclusively in the genome of one of the ancestors, N. sylvestris, and in N. tabacum itself. Our DNA hybridization data suggest that the eco elements originate from the genome of the other ancestor, N. tomentosiformis. Whereas the HRS60 elements are transcriptionally silent, at least some eco elements appear to be transcribed.

Fast Identification of Repetitive Elements in Biological Sequences

We have developed a fast filtering method for searching repetitive sequences in databases that allows the simultaneous identification of different families of repetitive elements during the same scanning. It discriminates between repetitive elements and non-related sequences by comparing the frequencies of k-words found in both groups of sequences. The distance used to sort out the sequences is based on a weighting of the k-words, which is obtained by performing a correspondence analysis on learning sets of correctly chosen sequences.The identification of Alu elements in human sequences is given as an illustration of the method. The Alu sequences are divided in four distinct groups of elements: the left and right monomers located on the direct and on the complementary strands. The results obtained on the test sets show that a very good discrimination is achieved with a word length of 6 b.p. Indeed, only 0·5% of the non-Alu sequences were incorrectly predicted as Alu elements for a threshold value allowing the identification of all Alu monomers. The misclassification of the different Alu monomers (1·4%) in the four groups of examples occurs only when the left and the right monomers are in the same orientation. Moreover, during the scanning of 63 GenBank sequences longer than 10 Kb, all the Alu elements were correctly identified (616 elements) and only a few non-Alu sequences were wrongly predicted as Alu elements (22 fragments).There is a real need for this kind of method since most of the repetitive elements are not annotated in the database entries. This method can then be used for a systematic screening of new sequences before their insertion in databases. It can also allow the creation of specific databases devoted to repetitive elements, which is a required step for any further analysis of those elements.

Characterization and genetic organization of full-length copies of a LINE retroposon family dispersed in the genome of Culex pipiens mosquitoes

Many full-length copies of a long interspersed repetitive element family, designated Juan-C, are reiterated in the genome of Culex pipiens mosquitoes. The complete Juan-C elements have a length of 4.48 kb. They are terminated at one end with an adenosine-rich sequence preceded with an AATAAA polyadenylation signal, lack terminal repeats and cause duplication of the host DNA at the site of their integration. Full-length Juan-C copies display two long open reading frames potentially encoding two proteins. The first one includes a domain typical of nucleic-acid-binding proteins, while the second resembles reverse transcriptases. Therefore, Juan-C elements are similar to LINE retroposons in their overall genetic organization and can probably be transposed by reverse transcription of an RNA intermediate. Juan-C elements are most similar in their sequence and coding potential to the Juan-A elements which are reiterated in mosquito species belonging to the genus Aedes. They also display homologies with some Drosophila LINEs such as Jockey, suggesting that all these elements have arisen from a common precursor. Nearly identical full-length Juan-C copies are amplified in C. pipiens strains from different contiments. This finding that Juan-C retroposons reiterated in different strains form an homogeneous family is interpreted to indicate that these elements have spread recently in the C. pipiens species.

Human chromosome 5 sequence primer amplifies Alu polymorphisms on chromosomes 2 and 17

Members of the Alu family of repetitive elements occur frequently in the human genome and are often polymorphic. Techniques involving Alu element mediated polymerase chain reactions (Alu PCR) allow the isolation of region-specific human DNA fragments from mixed DNA sources. Such fragments are a source of region-specific Alu elements useful for the detection of Alu-related polymorphisms. A clone from human chromosome 5, corresponding to locus D5F40S1, was isolated using Alu PCR differential hybridization. Alu elements within this clone were investigated for the presence of potentially polymorphic 3' polyA tails. Primers were devised to amplify the 3' polyA tail of an Alu element present within the clone. One primer, D5F40S1-T, was specific to the DNA flanking the 3' end of the Alu element, and the other primer was homologous to sequences within the element. When these primers were used in PCR reactions, products from chromosomes 2 and 17 (loci D2F40S2 and D17F40S3) were amplified in addition to the expected product from chromosome 5. The most likely explanation for this nonspecific amplification is that the D5F40S1-T primer is located within a low-copy repetitive element that is 3' of the Alu element. This phenomenon presents a potential problem for the identification of region-specific Alu polymorphisms.

Structural organization of the 5′ flanking region of the human corticotropin releasing hormone gene

We have determined the nucleotide sequence of the proximal 3625 nucleotides 5' flanking the major mRNA start site of the human corticotropin releasing hormone gene (hCRH) and identified several putative regulatory elements. Interestingly, we did not detect any glucocorticoid responsive elements; we did however find five interspersed perfect half palindromic estrogen responsive elements, which might confer estrogen regulatability to the hCRH gene. We have identified a segment spanning from -2835 to -2972, which has about 72% homology to the 3' terminal half of the human Alu I family of highly repetitive elements, and another one, which spans from -2213 to -2580 and has greater than 80% homology to members of human type O family of repetitive elements. These elements may confer DNA fragility, since the loci for hCRH and the human fragile site FRA8F colocalize in human chromosome 8. The structural information reported represents a first step in the study of regulation of the hCRH gene at the molecular level.

Prototypic sequences for human repetitive DNA.

We report a collection of 53 prototypic sequences representing known families of repetitive elements from the human genome. The prototypic sequences are either consensus sequences or selected examples of repetitive sequences. The collection includes: prototypes for high and medium reiteration frequency interspersed repeats, long terminal repeats of endogenous retroviruses, alphoid repeats, telomere-associated repeats, and some miscellaneous repeats. The collection is annotated and available electronically.

Characterization of the IS895 family of insertion sequences from the cyanobacterium Anabaena sp. strain PCC 7120.

A family of repetitive elements from the cyanobacterium Anabaena sp. strain PCC 7120 was identified through the proximity of one element to the psbAI gene. Four members of this seven-member family were isolated and shown to have structures characteristic of bacterial insertion sequences. Each element is approximately 1,200 bp in length, is delimited by a 30-bp inverted repeat, and contains two open reading frames in tandem on the same DNA strand. The four copies differ from each other by small insertions or deletions, some of which alter the open reading frames. By using a system designed to trap insertion elements, one of the elements, denoted IS895, was shown to be mobile. The target site was not duplicated upon insertion of the element. Two other filamentous cyanobacterial strains were also found to contain sequences homologous to IS895.

Diverse Gene Sequences Are Overexpressed in Werner Syndrome Fibroblasts Undergoing Premature Replicative Senescence

Genes that play a role in the senescent arrest of cellular replication are likely to be overexpressed in human diploid fibroblasts (HDF) derived from subjects with Werner syndrome (WS) because these cells have a severely curtailed replicative life span. To identify some of these genes, a cDNA library was constructed from WS HDF after they had been serum depleted and repleted (5 days in medium containing 1% serum followed by 24 h in medium containing 20% serum). Differential screening of 7,500 colonies revealed 102 clones that hybridized preferentially with [32P]cDNA derived from RNA of WS cells compared with [32P]cDNA derived from normal HDF. Cross-hybridization and partial DNA sequence determination identified 18 independent gene sequences, 9 of them known and 9 unknown. The known genes included alpha 1(I) procollagen, alpha 2(I) procollagen, fibronectin, ferritin heavy chain, insulinlike growth factor-binding protein-3 (IGFBP-3), osteonectin, human tissue plasminogen activator inhibitor type I, thrombospondin, and alpha B-crystallin. The nine unknown clones included two novel gene sequences and seven additional sequences that contained both novel segments and the Alu class of repetitive short interspersed nuclear elements; five of these seven Alu+ clones also contained the long interpersed nuclear element I (KpnI) family of repetitive elements. Northern (RNA) analysis, using the 18 sequences as probes, showed higher levels of these mRNAs in WS HDF than in normal HDF. Five selected mRNAs studied in greater detail [alpha 1(I) procollagen, fibronectin, insulinlike growth factor-binding protein-3, WS3-10, and WS9-14] showed higher mRNA levels in both WS and late-passage normal HDF than in early-passage normal HDF at various intervals following serum depletion/repletion and after subculture and growth from sparse to high-density confluent arrest. These results indicate that senescence of both WS and normal HDF is accompanied by overexpression of similar sets of diverse genes which may play a role in the senescent arrest of cellular replication and in the genesis of WS, normal biological aging, and attendant diseases.

Diverse gene sequences are overexpressed in werner syndrome fibroblasts undergoing premature replicative senescence.

Genes that play a role in the senescent arrest of cellular replication are likely to be overexpressed in human diploid fibroblasts (HDF) derived from subjects with Werner syndrome (WS) because these cells have a severely curtailed replicative life span. To identify some of these genes, a cDNA library was constructed from WS HDF after they had been serum depleted and repleted (5 days in medium containing 1% serum followed by 24 h in medium containing 20% serum). Differential screening of 7,500 colonies revealed 102 clones that hybridized preferentially with [32P]cDNA derived from RNA of WS cells compared with [32P]cDNA derived from normal HDF. Cross-hybridization and partial DNA sequence determination identified 18 independent gene sequences, 9 of them known and 9 unknown. The known genes included alpha 1(I) procollagen, alpha 2(I) procollagen, fibronectin, ferritin heavy chain, insulinlike growth factor-binding protein-3 (IGFBP-3), osteonectin, human tissue plasminogen activator inhibitor type I, thrombospondin, and alpha B-crystallin. The nine unknown clones included two novel gene sequences and seven additional sequences that contained both novel segments and the Alu class of repetitive short interspersed nuclear elements; five of these seven Alu+ clones also contained the long interpersed nuclear element I (KpnI) family of repetitive elements. Northern (RNA) analysis, using the 18 sequences as probes, showed higher levels of these mRNAs in WS HDF than in normal HDF. Five selected mRNAs studied in greater detail [alpha 1(I) procollagen, fibronectin, insulinlike growth factor-binding protein-3, WS3-10, and WS9-14] showed higher mRNA levels in both WS and late-passage normal HDF than in early-passage normal HDF at various intervals following serum depletion/repletion and after subculture and growth from sparse to high-density confluent arrest. These results indicate that senescence of both WS and normal HDF is accompanied by overexpression of similar sets of diverse genes which may play a role in the senescent arrest of cellular replication and in the genesis of WS, normal biological aging, and attendant diseases.

Evidence for a relationship between the Bombyx mori middle repetitive Bm1 sequence family and U1 snRNA

Several genomic library equivalents of Bombyx mori were constructed in the EMBL-4 lambda derivative. The genomic bank was screened with purified Bombyx mori U1 RNA and twenty positive clones for the U1 gene were isolated. Three U1-related sequences were subcloned and sequenced. Two of the sequences are U1 pseudogenes while a third sequence represents a member of the Bm 1 family of repetitive elements of B.mori with significant sequence similarity to U1 small nuclear RNA. The U1-related Bm1 element exhibits 82% sequence similarity with the Bm1 consensus sequence and, under less stringent computer comparison parameters, 60% similarity with a composite B.mori/Drosophila melanogaster U1 gene. The Bm1 family consensus sequence exhibits 53% sequence similarity with the composite U1 gene. The two pseudogenes possess highly conserved sequences with the B.mori U1 gene only for the first 101 nucleotides. These findings are indicative of at least two different categories of U1-related sequences in B.mori, one with a possible evolutionary relationship to the Bm1 family of repetitive elements and the other representing characteristic processed pseudogenes with retroposon mode of dispersion and target selection for the TTTA hotspot. In addition, the U1-related Bm1 element may demonstrate for the first time that a family of retroposons is ultimately derived from a U snRNA.

ERIC sequences: a novel family of repetitive elements in the genomes of Escherichia coli, Salmonella typhimurium and other enterobacteria

We describe a family of highly conserved, Enterobacterial Repetitive Intergenic Consensus (ERIC) sequences, 14 of which have been identified in Escherichia coli and Salmonella typhimurium and a further three in other enterobacterial species (Yersinia pseudotuberculosis, Klebsiella pneumoniae and Vibrio cholerae). ERIC sequences are 126 bp long and appear to be restricted to transcribed regions of the genome, either in intergenic regions of polycistronic operons or in untranslated regions upstream or downstream of open reading frames. ERIC sequences are highly conserved at the nucleotide sequence level but their chromosomal locations differ between species. Several features of ERIC sequences resemble those of REP sequences (Stern et al., 1984) although the nucleotide sequence is entirely different. The question of whether ERICs have a specific function, or represent a form of 'selfish' DNA, is discussed.

Isolation and sequence analysis ofCaenorhabditis briggsae repetitive elements related to theCaenorhabditis elegans transposon Tc1

We have identified two repetitive element families in the genome of the nematode Caenorhabditis briggsae with extensive sequence identity to the Caenorhabditis elegans transposable element Tc1. Five members each of the TCb1 (previously known as Barney) and TCb2 families were isolated by hybridization to a Tc1 probe. Tc1-hybridizing repetitive elements were grouped into either the TCb1 or TCb2 family based on cross-hybridization intensities among the C. briggsae elements. The genomic copy number of the TCb1 family is 15 and the TCb2 family copy number is 33 in the C. briggsae strain G16. The two transposable element families show numerous genomic hybridization pattern differences between two C. briggsae strains, suggestive of transpositional activity. Two members of the TCb1 family, TCb1#5 and TCb1#10, were sequenced. Each of these two elements had suffered an independent single large deletion. TCb1#5 had a 627-bp internal deletion and TCb1#10 had lost 316 bp of one end. The two sequenced TCb1 elements were highly conserved over the sequences they shared. A 1616-bp composite TCb1 element was constructed from TCb1#5 and TCb1#10. The composite TCb1 element has 80-bp terminal inverted repeats with three nucleotide mismatches and two open reading frames (ORFs) on opposite strands. TCb1 and the 1610-bp Tc1 share 58% overall nucleotide sequence identity, and the greatest similarity occurs in their ORF1 and inverted repeat termini.

The polydeoxyadenylate tract of Alu repetitive elements is polymorphic in the human genome.

To identify DNA polymorphisms that are abundant in the human genome and are detectable by polymerase chain reaction amplification of genomic DNA, we tested the hypothesis that the polydeoxyadenylate tract of the Alu family of repetitive elements is polymorphic among human chromosomes. We analyzed the 3' ends of three specific Alu sequences and found that two (in the adenosine deaminase gene and the beta-globin pseudogene) were polymorphic. This novel class of polymorphisms, termed AluVpA [Alu variable poly(A)] may represent one of the most useful and informative group of DNA markers in the human genome.

Molecular genetics of the Caenorhabditis elegans heterochronic gene lin-14.

We describe a general strategy for the genetic mapping in parallel of multiple restriction fragment length polymorphism (RFLP) loci. This approach allows the systematic identification for cloning of physical genetic loci within about 100 kb of any gene in Caenorhabditis elegans. We have used this strategy of parallel RFLP mapping to clone the heterochronic gene lin-14, which controls the timing and sequence of many C. elegans postembryonic developmental events. We found that of about 400 polymorphic loci in the C. elegans genome associated with the Tc1 family of repetitive elements, six are within 0.3 map unit of lin-14. The three closest lin-14-linked Tc1-containing restriction fragments were cloned and used to identify by hybridization an 830-kb region of contiguous cloned DNA fragments assembled from cosmid and yeast artificial chromosome libraries. A lin-14 intragenic recombinant that separated a previously cryptic lin-14 semidominant mutation from a cis-acting lin-14 suppressor mutation was used to map the location of the lin-14 gene to a 25-kb region of this 830-kb contig. DNA probes from this region detected lin-14 allele-specific DNA alterations and a lin-14 mRNA. Two lin-14 semi-dominant alleles, which cause temporally inappropriate lin-14 gene activity and lead to the reiterated expression of specific early developmental events, were shown to delete sequences from the lin-14 gene and mRNA. These deletions may define cis-acting sequences responsible for the temporal regulation of lin-14.