A ubiquitous family of repeated DNA sequences in the human genome

Renatured DNA from human and many other eukaryotes is known to contain 300-nucleotide duplex regions formed from renatured repeated sequences. These short repeated DNA sequences are widely believed to be interspersed with single copy DNA sequences. In this work we show that at least half of these 300-nucleotide duplexes share a cleavage site for the restriction enzyme AluI. This site is located 170 nucleotides from one end. This Alu family of repeated sequences makes up at least 3% of the genome and is present in several hundred thousand copies.Inverted repeated sequences are also known to contain a short 300-nucleotide duplex region. We find that at least half of the 300-nucleotide duplex regions in inverted repeated sequences also have an AluI restriction site located 170 nucleotides from one end.By driven renaturation techniques, the Alu family is shown to be distributed over a minimum range of 30% to 60% of the genome. (The breadth of this range reflects the presence of inverted repeated sequences which, in part, include the Alu family.) These findings imply that the interspersion pattern of repeated and single copy sequences in human DNA is largely dominated by one family of repeated sequences.     

Sequence of the cryptic satellite DNA from the plant Sinapis alba

A satellite DNA with a buoyant density equal to that of main band DNA in neutral cesium chloride (‘cryptic satellite’) can be isolated from the DNA of mustard (Sinapis alba) nuclei by Ag⁺/Cs₂SO₄ density gradient centrifugation. This satellite is cleaved into 172 bp repeat units by HinfI, AluI or HaeIII. The HinfI fragments have been further cleaved by AluI, and seven AluI subfragments have been sequenced. As a result two versions of a basic 172 HinfI repeat have been found, one (A + B) with an additional HinfI site. These two sequences (A + B and C) are the most frequent versions of the basic repeat of mustard satellite DNA. The basic 172 bp unit does not contain subrepeats or palindromic sequences. It is not similar (at a criterion of 15 common bases) with any known satellite sequence. It is not unusually highly methylated in the native state.     

Ammonia-Oxidizing Bacteria along Meadow-to-Forest Transects in the Oregon Cascade Mountains

Although nitrification has been well studied in coniferous forests of Western North America, communities of NH₃-oxidizing bacteria in these forests have not been characterized. Studies were conducted along meadow-to-forest transects at two sites (Lookout and Carpenter) in the H. J. Andrews Experimental Forest, located in the Cascade Mountains of Oregon. Soil samples taken at 10- or 20-m intervals along the transects showed that several soil properties, including net nitrogen mineralization and nitrification potential rates changed significantly between vegetation zones. Nonetheless, terminal restriction fragment length polymorphism (T-RFLP) analysis of the PCR-amplified NH₃ monooxygenase subunit A gene (amoA) showed the same DNA fragments (TaqI [283 bp], CfoI [66 bp], and AluI [392 bp]) to dominate ≥45 of 47 soil samples recovered from both sites. Two fragments (491-bp AluI [AluI491] and CfoI135) were found more frequently in meadow and transition zone soil samples than in forest samples at both sites. At the Lookout site the combination AluI491-CfoI135 was found primarily in meadow samples expressing the highest N mineralization rates. Four unique amoA sequences were identified among 15 isolates recovered into pure culture from various transect locations. Six isolates possessed the most common T-RFLP amoA fingerprint of the soil samples (TaqI283-AluI392-CfoI66), and their amoA sequences shared 99.8% similarity with a cultured species, Nitrosospira sp. strain Ka4 (cluster 4). The other three amoA sequences were most similar to sequences of Nitrosospira sp. strain Nsp1 and Nitrosospira briensis (cluster 3). 16S ribosomal DNA sequence analysis confirmed the affiliation of these isolates with Nitrosospira clusters 3 and 4. Two amoA clone sequences matched T-RFLP fingerprints found in soil, but they were not found among the isolates.     

A genetic and functional analysis of the unusually large variable region in the M·Alu I DNA-(cytosine C5 )-methyltransferase

The M·AluI DNA-(cytosine C5)-methyltransferase (5mC methylase) acts on the sequence 5′-AGCT-3′. The amino acid sequences of known 5mC methylases contain ten conserved motifs, with a variable region between Motifs VIII and IX that contains one or more “target-recognizing domains” (TRDs) responsible for DNA sequence specificity. Monospecific 5mC methylases are believed to have only one TRD, while multispecific 5mC methylases have as many as five. M·AluI has the second-largest variable region of all known 5mC methylases, and sequence analysis reveals five candidate TRDs. In testing whether M·AluI is in fact monospecific it was found that AGCT methylation represents only 80–90% of the methylating activity of this enzyme, while control experiments with the enzyme M·HhaI gave no unexplained activity. Because individual TRDs can be deleted from multispecific methylases without general loss of activity, a series of insertion and deletion mutants of the M·AluI variable region were prepared. All deletions that removed more than single amino acids from the variable region caused significant loss of activity; a sensitive in vivo assay for methylase activity based on McrBC restriction suggested that the central portion of the variable region is particularly important. In some cases, multispecific methylases can accommodate a TRD from another multispecific methylase, thereby acquiring an additional specificity. When TRDs were moved from a multispecific methylase into two different locations in the variable region of M·AluI, all hybrid enzymes had greatly reduced activity and no new specificities. M·AluI thus behaves in most respects as a monospecific methylase despite the remarkable size of its variable region.     

Molecular cloning of the murine branched chain α-ketoacid dehydrogenase E2 subunit: presence of 3′ B1 repeat elements

The cDNA sequence encoding the murine E2 subunit (dihydrolipoyl transacylase) of the branched-chain α-ketoacid dehydrogenase (BCKAD) complex was determined. In the region encoding the mature E2 subunit protein, both the nucleotide composition and predicted amino acid sequence are highly conserved between murine, human, and bovine species. In contrast, the 5′ sequence encoding the amino-terminal preprotein sequence and 3′ untranslated region are less well conserved. The 3′-noncoding region contains sequences highly homologous to the rodent B1 repeat elements, which are related to human Alu repeat sequences. This finding is similar to the presence of three Alu repeat sequences in the 3′-noncoding region of human E2 cDNA.     

Alu Repeats in the Human Genome

Highly repetitive DNA sequences account for more than 50% of the human genome. The L1 and Alu families harbor the most common mammalian long and short interspersed elements. An Alu element is a dimer of similar, but not identical, fragments of total size about 300 bp, and originates from the 7SL RNA gene. Each element contains a bipartite promoter for RNA polymerase III, a poly(A) tract located between the monomers, a 3"-terminal poly(A) tract, and numerous CpG islands, and is flanked by short direct repeats. Alu repeats constitute more than 10% of the human genome and are capable of retroposition. Possibly, these elements played an important part in genome evolution. Insertion of an Alu element into a functionally important genome region or other Alu-dependent alterations of gene functions cause various hereditary disorders and are probably associated with carcinogenesis. In total, 14 Alu families differing in diagnostic mutations are known. Some of these, which are present in the human genome, are polymorphic and relatively recently have been inserted into new loci. Alu copies transposed during ethnic divergence of the human population are useful markers for evolutionary genetic studies.     

Is gene deletion in eukaryotes sequence-dependent? A study of nine deletion junctions and nineteen other deletion breakpoints in intron 7 of the human dystrophin gene

Although large deletions comprise 65% of the mutations that underlie most cases of Duchenne and Becker muscular dystrophies, the DNA sequence characteristics of the deletions and the molecular processes leading to their formation are largely unknown. Intron 7 of the human dystrophin gene is unusually large (110 kb) and a substantial number of deletions have been identified with endpoints within this intron. The distribution of 28 deletion endpoints was mapped to local sequence elements by PCR. The break points were distributed among unique sequence, LINE-1, Alu, MIR, MER and microsatellite sequences with frequencies expected from the frequency of those sequences in the intron. Thus, deletions in this intron are not associated primarily with any one of those sequences in the intron. Nine deletion junctions were amplified and sequenced. Eight were deletions between DNA sequences with minimal homology (0–4 bp) and are therefore unlikely to be products of homologous recombination. In the ninth case, a complex rearrangement was found to be consistent with unequal recombinational exchange between two Alu sequences coupled with a duplication. We have hypothesized that a paucity of matrix attachment regions in this very large intron expanded by the insertion of many mobile elements might provoke a chromatin structure that stimulates deletions (McNaughton et al., 1997, Genomics 40, 294–304). The data presented here are consistent with that idea and demonstrate that the deletion sequences are not usually produced by homologous DNA misalignments.     

Study of Alu sequences at the hypoxanthine phosphoribosyltransferase (hprt) encoding region of man

The hypoxanthine phosphoribosyltransferase (hprt) encoding region of man is considered rich in Alu sequences; with 49 sequences present within 57 kilobases. Subfamily classification of the Alu sequences and identification of flanking direct repeats has been carried out to detect past rearrangements associated with their insertion into the region. Members of the Alu-J and three Alu-S subfamilies are present, along with the existence of free left arm sequences. Using available data, a comparison is made of the Alu subfamilies present at different gene regions. The heterogeneity in the number of each subfamily present at different genes shows that no one particular subfamily attained saturation in the genome. Several adjacent insertions of Alu sequences are seen at the hprt region. Furthermore two novel sequences are described, there is an incident where one Alu sequence has inserted into the middle poly(A) tract of an existing sequence at the hprt region; while another resulted from an Alu/Alu cross-over event elsewhere in the genome, before insertion into the hprt region. Once inserted, the Alu sequences are rarely subject to loss or rearrangement.     

Comparative Assessment of 5′ A/T-Rich Overhang Sequences with Optimal and Sub-optimal Primers to Increase PCR Yields and Sensitivity

Efficient PCR amplifications require precisely designed and optimized oligonucleotide primers, components, and cycling conditions. Despite recent software development and reaction improvement, primer design can still be enhanced. The aims of this research are to understand (1) the effect on PCR efficiency and DNA yields of primer thermodynamics parameters, and (2) the incorporation of 5′ A/T-rich overhanging sequences (flaps) during primer design. Two primer sets, one optimal (ΔG = 0) and one sub-optimal (ΔG = 0.9), were designed using web interface software Primer3, BLASTn, and mFold to target a movement protein gene of Tobacco mosaic virus. The optimal primer set amplifies a product of 195 bp and supports higher PCR sensitivity and yields compared to the sub-optimal primer set, which amplifies a product of 192 bp. Greater fluorescence was obtained using optimal primers compared to that with sub-optimal primers. Primers designed with sub-optimal thermodynamics can be substantially improved by adding 5′ flaps. Results indicate that even if the performance of some primers can be improved substantially by 5′ flap addition, not all primers will be similarly improved. Optimal 5′ flap sequences are dependent on the primer sequences, and alter the primer’s T _m value. The manipulation of this feature may enhance primer’s efficiency to increase the PCR sensitivity and DNA yield.     

Opisthorchis viverrini and Haplorchis taichui: Development of a multiplex PCR assay for their detection and differentiation using specific primers derived from HAT-RAPD

Specific primers for the detection of Opisthorchis viverrini and Haplorchis taichui were investigated by using the HAT-RAPD PCR method. Fourteen arbitrary primers (Operon Technologies) were performed for the generation of polymorphic DNA profiles. The results showed that a 319 bp fragment generated from the OPA-04 primer was expected to be O. viverrini-specific while a 256 bp fragment generated from the OPP-11 primer was considered to be H. taichui-specific. Based on each sequence data, two pairs of specific primers were designed and sequences of each primer were as follows; H. taichui; Hapt_F5′-GGCCAACGCAATCGTCATCC-3′and Hapt_R1 5′-CTCTCGACCTCCTCTAGAAT-3′ which yielded a 170 bp PCR product. For O. viverrini, OpV-1F: 5′-AATCGGGCTGCATATTGACCGAT-3′ and OpV-1R: 5′-CGGTGTTGCTTATTTTGCAGACAA-3′ which generated a 319 bp PCR product. These specific primers were tested for efficacy and specific detection for all parasites DNA samples. The results showed that 170 and 319 bp specific PCR products were generated as equivalent to positive result in H. taichui and O. viverrini, respectively by having no cross-reaction with any parasites tested. PCR conditions are recommended at 68 °C annealing temperature and with 0.5 mM magnesium chloride (Mg Cl₂). Additionally, specific primers developed in this study were effective to determine the presence of both parasites in fish and snail intermediate hosts, which the DNA of O. viverrini was artificially spiked since it is rarely found in northern Thailand.The H. taichui and O. viverrini-specific primers successfully developed in this study can be use for epidemiological monitoring, preventing management and control programs.     

The orotidine-5′-phosphate decarboxylase gene (URA3) of a methylotrophic yeast,Candida boidinii: Nucleotide sequence and its expression in Escherichia coli

Previously, we cloned a DNA fragment from a genomic library of a methylotrophic yeast, Candida boidinii. This 3.5-kb SalI fragment was capable of complementing the pyrF mutation in Escherichia coli. In this report, we identify this fragment as that harboring an orotidine-5′-phosphate decarboxylase (ODCase) gene (C. boidinii URA3); we have also determined the complete DNA sequence of the C. boidinii URA3 gene. The deduced amino acid sequence of the gene showed homology to ODCase genes from other sources, and it could complement the ura3 mutation of Saccharomyces cerevisiae. The DNA fragment, which harbored the C. boidinii URA3 gene, was able to express ODCase activity in the E. coli pyrF mutant strain without an exogenous E. coli promoter. From nested-deletion analysis, both the 5′-(136 bp) and 3′-(58 bp) flanking regions were shown to be required for pyrF-complementation of the E. coli mutant. The 5′-flanking region had sequences homologous to E. coli promoter consensus sequences (−35 and −10 regions) which may function in the expression of the C. boidinii URA3 gene in E. coli.     

A consensus Alu repeat probe for physical mapping

Physical mappinf of the human genome involves a variety of complex hybridization-based procedures, some of which rely upon the ability to seperate human clones derived from human-rodent hybrid cell lines from those that contain background rodent-derived DNA sequences. The ability to block the repititive element (Alu repeat) portion of inter-Alu PCR products derived from a variety of complex sources is also crucial for the isolation of unique DNA sequences. Here we report the construction and characterization of a new consensus Alu repeat probe (pPD39) designed for these purposes.     

A specific endonuclease from Arthrobacter luteus.

A new restriction-like endonuclease, AluI, has been partially purified from Arthrobacter luteus. This enzyme cleaves bacteriophage λ DNA, adenovirus-2 DNA and simian virus 40 DNA at many sites including all sites cleaved by the endonuclease HindIII from Haemophilus influenzae serotype d. Radioactive oligonucleotides in pancreatic DNAase digests of (5′-³²P)-labelled fragments of phage λ DNA released by the action of AluI had the 5′ terminal sequence pC-T-N-. The enzyme recognises the tetranucleotide sequence

and cleaves it at the position marked by the arrows.     

Transposition of Tnr1 in rice genomes to 5′-PuTAPy-3′ sites,duplicating the TA sequence

Tnr1 is a repetitive sequence in rice with several features characteristic of a transposable DNA element. Its copy number was estimated to be about 3500 per haploid genome by slot-blot hybridization. We have isolated six members of Tnr1 located at different loci by PCR (polymerase chain reaction) and determined their nucleotide sequences. The Tnr1 elements were similar in size and highly homologous (about 85%) to the Tnr1 sequence identified first in the Waxy gene in Oryza glaberrima. A consensus sequence of 235 by could be derived from the nucleotide sequences of all the Tnr1 members. The consensus sequence showed that base substitutions occurred frequently in Tnr1 by transition, and that Tnr1 has terminal inverted repeat sequences of 75 bp. Almost all the chromosomal sequences that flank the Tnr1 members were 5′-PuTA-3′ and 5′-TAPy-3′, indicating that Tnr1 transposed to 5′-PuTAPy-3′ sites, duplicating the TA sequence. PCR-amplified fragments from some rice species did not contain the Tnr1 members at corresponding loci. Comparison of nucleotide sequences of the fragments with or without a Tnr1 member confirmed preferential transposition of Tnr1 to 5′-PuTAPy-3′ sites, duplicating the TA sequence. One amplified sequence suggested that imprecise excision had occurred to remove a DNA segment containing a Tnr1 member and its neighboring sequences at the Waxy locus of rice species with genome types other than AA. We also present data that may suggest that Tnr1 is a defective form of an autonomous transposable element.     

Recombinations between Alu repeat sequences that result in partial deletions within the C1 inhibitor gene

Genomic DNA sequence analysis was used to define the extent of deletions within the C1 inhibitor gene in two families with type I hereditary angioneurotic edema. Southern blot analysis initially indicated the presence of the partial deletions. One deletion was approximately 2 kb and included exon VII, whereas the other was approximately 8.5 kb and included exons IV–VI. Genomic libraries from an affected member of each family were constructed and clones containing the deletions were analyzed. Sequence analysis of the deletion joints of the mutants and corresponding regions of the normal gene in the two families demonstrated that both deletion joints resulted from recombination of two Alu repetitive DNA elements. Alu repeat sequences from introns VI and VII combined to make a novel Alu in family A, and Alu sequences in introns III and VI were spliced to make a new Alu in family B. The splice sites in the Alu sequences of both mutants were located in the left arm of the Alu element, and both recombination joints overlapped one of the RNA polymerase III promoter sequences. Because the involved Alu sequences, in both instances, were oriented in the same direction, unequal crossingover is the most likely mechanism to account for these mutations.     

Simplified plasmid rescue of host sequences adjacent to integrated proviruses

We have previously described a Moloney murine leukemia retroviral (MoMLV) vector useful for the generation of anchored long-range maps of complex mammalian genomes. We now report the development of a modified vector carrying the ColE1 origin of replication and the chloramphenicol-resistance (Cm^R) gene to facilitate the recovery of genomic sequences adjacent to integrated proviruses. We demonstrate the utility of this new vector for the rescue in plasmid form of a 6-kb human fragment containing portions of an Alu element adjacent to the proviral 3′-LTR from an infected human primary fibroblast clone. We generated a sequence-tagged site (STS) from the derived sequence outside the Alu element, and used a somatic cell hybrid mapping panel to assign this STS to human chromosome 10 by a polymerase chain reaction-based assay. We suggest that this new Cm^R vector will be useful for recovering sequences of genes interrupted by proviral insertion, to generate directional maps with respect to the inserted provirus using the single cleavage sites within the vector, and to generate site-specific STS for defining and mapping the integration site.