Alu Family Members Research Articles

A negative regulatory element with properties similar to those of enhancers is contained within an Alu sequence.

A negative regulatory element has been found within a member of the African green monkey Alu family of interspersed repeated sequences. This "reducer" element decreased transcription in both directions from a cellular simian virus 40-like bidirectional promoter independently of both orientation and position. The reducer was not promoter specific since it also decreased expression from the simian virus 40 early and human beta-globin promoters. In addition, the reducer decreased transcription from a polymerase III promoter. The reducer was contained in 38 base pairs of an Alu family member and interacted specifically with a monkey cell nuclear protein.

Recently amplified Alu family members share a common parental Alu sequence.

Three of the most recently inserted primate Alu family members are exceptionally closely related. Therefore, one, or a few, Alu family members are dominating the amplification process and the vast majority are not actively involved in retroposition. Although individual Alu family members are not under any apparent evolutionary constraint, the sequences of these active members are being moderately conserved.

Existence of at least three distinct Alu subfamilies

Computer-assisted sequence analysis of human Alu family members reveals that Alu repeats belong to one of at least three subfamilies. The insertion of human Alu repeats can be represented by three episodic bursts, each of which was founded by a distinct master sequence.

Recent insertion of an Alu sequence in the beta-globin gene cluster of the gorilla.

We present the nucleotide sequence of a new Alu family member that lies between the delta- and beta-globin genes in gorilla DNA. The sequence exhibits 91% similarity with a consensus sequence of the Alu family. It is flanked by a perfect repetition of a 16-nucleotide target sequence and terminates with 24 adenylic residues. As this sequence is absent at this locus in other primate DNAs, its insertion occurred less than 8 million years ago, thus supporting the idea that Alu sequences are still mobile elements in the hominoid genome.

Cloning of decay-accelerating factor suggests novel use of splicing to generate two proteins

Decay-accelerating factor (DAF), a glycoprotein that is anchored to the cell membrane by phosphatidylinositol, binds activated complement fragments C3b and C4b, thereby inhibiting amplification of the complement cascade on host cell membranes. Here, we report the molecular cloning of human DAF from HeLa cells. Analysis of DAF complementary DNAs revealed two classes of DAF messenger RNA, one apparently derived from the other by a splicing event that causes a coding frameshift near the C terminus. The apparent 'intron' sequence contains an Alu family member and encodes contiguous protein sequence. Two DAF proteins are therefore possible, having divergent C-terminal domains which differ in their hydrophobicity. Both mRNAs are found on polysomes, suggesting that both are translated. We propose that the major (90%) spliced DAF mRNA encodes membrane-bound DAF whereas the minor (10%) unspliced DAF mRNA may encode secreted DAF and we present expression data supporting this. The deduced DAF sequence contains four repeating units homologous to a consensus repeat found in a recently described family of complement proteins.

Clustering and subfamily relationships of the Alu family in the human genome.

Thirteen and 10 sequences of the Alu family of repeated DNA elements found within the human thymidine kinase and beta-tubulin genes, respectively, were compared. These genes have approximately five times the expected density of Alu family members. The consensus sequence that could be drawn from these 23 Alu family members would differ slightly from others drawn from random Alu family sequences but only at very heterogeneous positions. The different Alu family members do show different pairwise percentage identities, with approximately 15% (7 of 48 Alu family members analyzed) of them clearly representing a separate subfamily of sequences. This analysis also confirms the species-specific differences between human and the prosimian Galago crassicaudatus Alu family members. These data are consistent with both the origin of these sequences in primates less than 65-70 Myr ago and amplification since that time to their present 500,000 copies. The data do not show any special relationships among densely clustered Alu family members.

Primate evolution of the α-globin gene cluster and its Alu-like repeats

The arrangement of α-globin genes in Old World and New World monkeys and a prosimian, galago, has been determined by restriction mapping. Recombinant DNAs containing galago and Old World monkey α-globin genes have been isolated and subjected to a partial sequence determination for comparison to α-globin genes in human, chimpanzee and non-primate mammals. The results of this extensive structural analysis are relevant to several topics concerning the evolution of primate α-globin genes and Alu family repeats. All orders of higher primates (i.e. Old and New World monkeys, chimpanzee and human) have the same arrangement of α-globin genes. In contrast, the arrangement and correction of galago α-globin genes differ from those of higher primates, but are similar to those of non-primate mammals. The 5- and 3-flanking regions of the human α1 gene are orthologous to the corresponding region in galago, identifying the human α2 gene as the more recently duplicated gene. The human ψα1 gene is found to be inactivated after divergence of the human and galago lineages but prior to the divergence of human and monkey. Orthologous Alu family members in human and monkey DNAs indicate that the dispersion of some Alu repeats occurred prior to the divergence of these lineages. However, the Alu-like repeats of prosimian and higher primates result from entirely independent events giving rise to different repeat elements inserted at distinct genomic positions.

Removal of the Alu structural domain from signal recognition particle leaves its protein translocation activity intact

Alu-like elements comprise the most abundant family of interspersed repetitive sequences in primates and rodents, and contain many features of processed genes, suggesting that they were initially derived by reverse transcription of processed RNA transcripts. Transcripts containing Alu family members are represented in heterologous nuclear RNAs, cytoplasmic messenger RNAs and small RNAs, although nothing is known about their function. Evolutionary studies strongly suggest that the parent RNA for the Alu-like elements is the highly conserved 7SL RNA, which is an essential component of signal recognition particle (SRP), a small cytoplasmic ribonucleoprotein whose function is the targeting of nascent secretory and membrane proteins to the rough endoplasmic reticulum (for a review see ref. 6). 7SL RNA is composed of both unique and Alu-like sequences. SRP is rod-shaped and, in addition to its RNA, contains four proteins (two monomers composed of a polypeptide of relative molecular mass (Mr) 19,000 (19K) and one of 54K, and two heterodimers, one composed of a 9K and a 14K polypeptide, and the other composed of a 68K and a 72K polypeptide, respectively). The RNA moiety is required for SRP activity, as well as for structural integrity of the particle. To investigate whether the Alu-like segments of 7SL RNA have a specific role in SRP activity, we have now purified and analysed a SRP subparticle that is created upon extensive digestion with micrococcal nuclease and entirely lacks the Alu-like sequences. We find that it contains the 72/68K, 54K and 19K proteins tightly bound, but lacks the 9/14K protein. In vitro activity assays demonstrated that the subparticle could still promote secretory protein translocation across the microsomal membrane, but could no longer trigger an arrest of pre-secretory protein synthesis. Re-addition of the 9/14K protein did not restore the elongation arrest. We conclude that the region of SRP comprised of the Alu-like RNA and the 9/14K protein exists in a distinct structural domain which is not required for the protein translocation promoted by SRP but apparently confers elongation-arresting activity on the particle.

Transcriptional inactivity of Alu repeats in HeLa cells.

The in vivo transcription of human Alu family members has been investigated by a sensitive primer extension method. The selected primers represent various regions of the Alu family consensus sequence, thus assaying the transcriptional activity of the entire family rather than the activity of an individual member sequence. Using this method, a very small number of RNA molecules per HeLa cell is found to have a distribution of 5' ends centered on the in vitro Alu transcription start site. The distribution of these 5' ends suggests that they are more likely the result of hnRNA degradation rather than transcription start sites. Therefore, despite their great numerical abundance, Alu family members are transcriptionally silent in HeLa cells.

Complete nucleotide sequence of the gene for human interleukin 1 alpha.

The chromosomal gene for human interleukin 1 alpha (IL-1 alpha) was isolated from a human genomic DNA library by using as a probe cloned human IL-1 alpha cDNA. Complete nucleotide sequence of about 12 kilobase pairs (kbp) long was determined and the structure and organization of this gene were elucidated. This gene contains seven exons and six introns. The first exon encodes the 5'-untranslated region. Most of the prepeptide portion of the precursor polypeptide is encoded by the next three exons, and the mature form of IL-1 alpha is encoded by the remaining three exons. The last exon also encodes the intronless 3'-untranslated region. The fourth intron as well as both 5'- and 3'-flanking regions contains the sequence of an Alu family member. In the middle part of the last intron, a 46-bp sequence with a unique structure is repeated five times in a head to tail manner. These repeats are flanked by the regions containing alternative purine and pyrimidine tracts. In the 5'-flanking region, immediately upstream of the putative TATA box, the 16-bp sequence highly homologous to the binding site of the adenovirus 2 major late promoter transcription factor is identified. The nucleotide sequence reported here showed only one nucleotide substitution in the 3'-untranslated exon in comparison with the nucleotide sequence of the cDNA from HL-60, a promyelocytic leukemia cell line, previously cloned and sequenced in our laboratories.

A human opal suppressor tRNA gene and pseudogene.

A human DNA library, cloned in bacteriophage lambda, was screened with an opal suppressor tRNA probe. Two genes were isolated, subcloned into pBR322, and sequenced. One is a normal opal suppressor tRNA gene 87 nucleotides in length without intervening sequences. It has a TCA anticodon demonstrating that the mature tRNA reads the termination codon UGA. The 5' internal control region for transcription has two extra nucleotides compared to the consensus sequence for eucaryotic tRNA genes, while the 3' internal control region is normal. This gene differs from a previously sequenced chicken opal suppressor serine tRNA gene (Hatfield, D., Dudock, B., and Eden, F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4940-4944) only at position 11. The second human gene appears to be a pseudogene truncated near the 3' end, since in the cloverleaf form of the mature tRNA there are three noncomplementary bases in the acceptor stem. The two human genes have a high degree of homology and, excluding the truncated 3' terminus of the pseudogene, differ in only two positions. The flanking sequences of the pseudogene are about 90% homologous to the consensus sequence of the human Alu family of repeated sequences. This gene appears to have been inserted between two adjacent Alu family members.

Human satellite I sequences include a male specific 2.47 kb tandemly repeated unit containing one Alu family member per repeat.

A portion of human satellite I DNA is digested by HinfI into three fragments of 775, 875 and 820bp in length which form a tandemly repeated unit 2.47kb in length, specific to male DNA. One Alu family member per repeat is found within the relatively G+C rich 775bp fragment. The 875 and 820bp fragments are highly A+T rich and consist of long stretches of poly dAdT and related sequences.

Transcription in vivo of an Alu family member upstream from the human epsilon-globin gene.

The more distal Alu repeat flanking the epsilon-globin gene is transcribed in K562 cells to generate transcripts 350-400 nucleotides in length. Initiation occurs at the start of the repeat, upstream of a putative PolIII control signal. These transcripts originate from the strand which does not code epsilon-globin and are oriented in the opposite direction from the gene. They are non-polyadenylated, nucleus-confined and are only detectable in association with expression of the epsilon-globin gene.

Molecular evolution of the human adult alpha-globin-like gene region: insertion and deletion of Alu family repeats and non-Alu DNA sequences.

Previous heteroduplex studies have revealed extensive sequence homology between the two human adult alpha-globin-like genes (alpha 2 and alpha 1) and their flanking regions. These homologous regions, which are interrupted by two blocks of nonhomology, each span approximately 4 kilobases [Lauer, J., Shen, C.-K. J. & Maniatis, T. (1980) Cell 20, 119-130]. We have determined 3 kilobases of DNA sequences within and flanking the nonhomologous blocks of these two tandem duplication units. A total of three Alu family repeats has been identified. Two of them are approximately 300 base pairs long and define the 3' ends of the first homology blocks. The third Alu family member is a 600-base-pair-long sequence consisting of two monomeric Alu members arranged in a head-to-tail fashion. It is located in the 3' portion of the first block of nonhomology in alpha 2-gene-containing unit. We present direct evidence that this dimeric Alu sequence was inserted at a staggered break. The second nonhomology block is the result of insertion or deletion of a 224-base-pair sequence. From these data and the calculation of sequence divergence, we propose a history for the evolution of the human adult alpha-globin-like gene region. We also suggest that DNA insertion elements may disrupt gene correction processes in the two duplication units containing alpha 2- and alpha 1-globin genes.

Unusual class of Alu sequences containing a potential Z-DNA segment.

A potential Z-DNA sequence, (dA-dC)9, has been found to replace the customary A-rich region in the middle of an Alu family member in the African green monkey genome. This Alu, bounded by imperfect direct repeats, also contains an unusual 3' end and may be a member of a large subfamily of such sequences.

Unusual class of Alu sequences containing a potential Z-DNA segment.

A potential Z-DNA sequence, (dA-dC)9, has been found to replace the customary A-rich region in the middle of an Alu family member in the African green monkey genome. This Alu, bounded by imperfect direct repeats, also contains an unusual 3' end and may be a member of a large subfamily of such sequences.

Alu family members in the human alpha-like globin-gene cluster.

Alu family members in the human alpha-like globin-gene cluster.

Long internal direct repeat in Epstein-Barr virus DNA.

The nucleotide sequence of the long internal reiteration. IR1, of Epstein-Barr Virus DNA has been determined. The repeat unit is 3,071 base pairs which are 66.8% guanine plus cytosine. There is a CCAAT sequence 39 nucleotides 5' to a TATAA, which could indicate a promotor for transcription. The longest open reading frame is 1,124 base pairs. Also within IR1 is a sequence homologous to the papovavirus origin of DNA replication. The ori-like sequence is within a long palindromic region which is 500 base pairs overall. The palidromic region shares common features with the Alu family members and with eucaryotic transposable elements. The juncture between the short unique region (U1) and IR1 is also sequenced. The transition occurs in BamHI-C at 1,214 base pairs before the BamHI site in the first repeat of IR1. The transition from IR1 to the rightward unique region (U2) has been reported to be at 636 base pairs after the BamHI site in the last repeat of IR1. Thus, relative to the start of IR1 at the juncture with U1, the last copy of IR1 is a partial repeat which contains only the beginning 1,850 base pairs.

Isolation and characterization of human DNA fragments with nucleotide sequence homologies with the simian virus 40 regulatory region.

A recombinant library of human DNA sequences was screened with a segment of simian virus 40 (SV40) DNA that spans the viral origin of replication. One hundred and fifty phage were isolated that hybridized to this probe. Restriction enzyme and hybridization analyses indicated that these sequences were partially homologous to one another. Direct DNA sequencing of two such SV40-hybridizing segments indicated that this was not a highly conserved family of sequences, but rather a set of DNA fragments that contained repetitive regions of high guanine plus cytosine content. These sequences were not members of the previously described Alu family of repeats and hybridized to SV40 DNA more strongly than do Alu family members. Computer analyses showed that the human DNA segments contained multiple homologies with sequences throughout the SV40 origin region, although sequences on the late side of the viral origin contained the strongest cross-hybridizing sequences. Because of the number and complexity of the matches detected, we could not determine unambiguously which of the many possible heteroduplexes between these DNAs was thermodynamically most favored. No hybridization of these human DNA sequences to any other segment of the SV40 genome was detected. In contrast, the human DNA segments isolated cross-hybridized with many sequences within the human genome. We tested for the presence of several functional domains on two of these human DNA fragments. One SV40-hybridizing fragment, SVCR29, contained a sequence which enhanced the efficiency of thymidine kinase transformation in human cells by approximately 20-fold. This effect was seen in an orientation-independent manner when the sequence was present at the 3' end of the chicken thymidine kinase gene. We propose that this segment of DNA contains a sequence analogous to the 72-base-pair repeats of SV40. The existence of such an "activator" element in cellular DNA raises the possibility that families of these sequences may exist in the mammalian genome.

Isolation and characterization of human DNA fragments with nucleotide sequence homologies with the simian virus 40 regulatory region.

A recombinant library of human DNA sequences was screened with a segment of simian virus 40 (SV40) DNA that spans the viral origin of replication. One hundred and fifty phage were isolated that hybridized to this probe. Restriction enzyme and hybridization analyses indicated that these sequences were partially homologous to one another. Direct DNA sequencing of two such SV40-hybridizing segments indicated that this was not a highly conserved family of sequences, but rather a set of DNA fragments that contained repetitive regions of high guanine plus cytosine content. These sequences were not members of the previously described Alu family of repeats and hybridized to SV40 DNA more strongly than do Alu family members. Computer analyses showed that the human DNA segments contained multiple homologies with sequences throughout the SV40 origin region, although sequences on the late side of the viral origin contained the strongest cross-hybridizing sequences. Because of the number and complexity of the matches detected, we could not determine unambiguously which of the many possible heteroduplexes between these DNAs was thermodynamically most favored. No hybridization of these human DNA sequences to any other segment of the SV40 genome was detected. In contrast, the human DNA segments isolated cross-hybridized with many sequences within the human genome. We tested for the presence of several functional domains on two of these human DNA fragments. One SV40-hybridizing fragment, SVCR29, contained a sequence which enhanced the efficiency of thymidine kinase transformation in human cells by approximately 20-fold. This effect was seen in an orientation-independent manner when the sequence was present at the 3' end of the chicken thymidine kinase gene. We propose that this segment of DNA contains a sequence analogous to the 72-base-pair repeats of SV40. The existence of such an "activator" element in cellular DNA raises the possibility that families of these sequences may exist in the mammalian genome.