The Alu family of dispersed repetitive sequences.

A family of related sequences that includes approximately 500,000 members is the most prominent short dispersed repeat family in primate and rodent DNA's. The primate sequence is approximately 300 base pairs in length and is composed of two imperfectly repeated monomer units, whereas the rodent repeat consists of only a single monomer. Properties of this repeat sequence, its flanking sequences in chromosomal DNA, and RNA's transcribed from it suggest that it may be a mobile DNA element inserted at hundreds of thousands of different chromosomal locations.

Thermal stability of human DNA and chimpanzee DNA heteroduplexes.

The base pairing fidelity of heteroduplexes formed from human DNA and chimpanzee DNA has been studied by the criterion of thermal stability to test the evolutionary conservation of repeated DNA base sequences.

A gradient of sequence divergence in the human adult alpha-globin duplication units.

The nucleotide sequences of the two 5'-homology blocks of human alpha-globin gene duplication units were determined. The sequence difference between the two blocks is essentially zero in the 5' portions, and increases gradually toward the 3' ends until it reaches a value of 18 percent. This gradient of sequence divergence is similar to the distribution of the frequencies of gene conversion along several loci in Ascobolus and yeast. Hot spots for initiation of gene correction processes appear to exist near the 5' ends of the human alpha-globin duplication units. The data provide the physical evidence for polar gene correction process in a mammalian genome.

Sequence-specific packaging of DNA in human sperm chromatin.

The DNA in human sperm chromatin is packaged into nucleoprotamine (approximately 85%) and nucleohistone (approximately 15%). Whether these two chromatin fractions are sequence-specific subsets of the spermatozoon genome is the question addressed in this report. Sequence-specific packaging would suggest distinct structural and functional roles for the nucleohistone and nucleoprotamine in late spermatogenesis or early development or both. After removal of histones with 0.65M NaCl, exposed DNA was cleaved with Bam HI restriction endonuclease and separated by centrifugation from insoluble nucleoprotamine. The DNA sequence distribution of nucleohistone DNA in the supernatant and nucleoprotamine DNA in the pellet was compared by cloning size-selected single-copy sequences and by using the derived clones as probes of nucleohistone DNA and nucleoprotamine DNA. Two clones derived from nucleohistone DNA preferentially hybridized to nucleohistone DNA, and two clones derived from nucleoprotamine DNA preferentially hybridized to nucleoprotamine DNA, which demonstrated the existence of sequence-specific nucleohistone and nucleoprotamine components within the human spermatozoon.

Structure and evolution of the U2 small nuclear RNA multigene family in primates: gene amplification under natural selection?

The organization of U2 genes was compared in apes, Old World monkeys, and the prosimian galago. In humans and all apes (gibbon, orangutan, gorilla, and chimpanzee), the U2 genes were organized as a tandem repeat of a 6-kb element; however, the restriction maps of the 6-kb elements in these divergent species differed slightly, demonstrating that mechanisms must exist for maintaining sequence homogeneity within this tandem array. In Old World monkeys, the U2 genes were organized as a tandem repeat of an 11-kb element; the restriction maps of the 11-kb elements in baboon and two closely related macaques, bonnet and rhesus monkeys, also differed slightly, confirming that efficient sequence homogenization is an intrinsic property of the U2 tandem array. Interestingly, the 11-kb monkey repeat unit differed from the 6-kb hominid repeat unit by a 5-kb block of monkey-specific sequence. Finally, we found that the U2 genes of the prosimian galago were dispersed rather than tandemly repeated, suggesting that the hominid and Old World monkey U2 tandem arrays resulted from independent amplifications of a common ancestral U2 gene. Alternatively, the 5-kb monkey-specific sequence could have been inserted into the 6-kb array or deleted from the 11-kb array soon after divergence of the hominid and Old World monkey lineages.

A transpositionally and transcriptionally competent Alu subfamily.

DNA base sequence comparisons indicate that a subfamily of recently transposed human Alu repeats are distinguished from most Alu repeats by diagnostic sequence differences. Using an oligonucleotide hybridization probe that incorporates these sequence features, we found that there was an expansion of this Alu subfamily following the divergence of humans and African apes. This oligonucleotide was used to select human genomic clones containing representatives of this subfamily. One representative member of this subfamily was evidently absent from the corresponding chimpanzee locus and was associated with a restriction fragment length polymorphism in the human genome. This apparently polymorphic member had all the diagnostic sequence features that initially predicted the existence of a newly expanding Alu subfamily. A transpositionally active sequence variant should also be transcriptionally active in at least some cell types or tissues. Northern (RNA) blot hybridization, primer extension, and RNA sequence analysis demonstrated the existence of different-length polyadenylated and nonpolyadenylated transcripts corresponding to this subfamily. Evidence for 3' processing and subcellular localization of these transcripts is discussed. Most of the nearly one million human Alu repeats are pseudogenes with respect to coding for either an RNA product or new family members; a select and identifiable subset of Alu repeats serve as transcriptionally and transpositionally competent source genes.

Developmental differences in methylation of human Alu repeats.

Alu repeats are especially rich in CpG dinucleotides, the principal target sites for DNA methylation in eukaryotes. The methylation state of Alus in different human tissues is investigated by simple, direct genomic blot analysis exploiting recent theoretical and practical advances concerning Alu sequence evolution. Whereas Alus are almost completely methylated in somatic tissues such as spleen, they are hypomethylated in the male germ line and tissues which depend on the differential expression of the paternal genome complement for development. In particular, we have identified a subset enriched in young Alus whose CpGs appear to be almost completely unmethylated in sperm DNA. The existence of this subset potentially explains the conservation of CpG dinucleotides in active Alu source genes. These profound, sequence-specific developmental changes in the methylation state of Alu repeats suggest a function for Alu sequences at the DNA level, such as a role in genomic imprinting.

p53 inhibits RNA polymerase III-directed transcription in a promoter-dependent manner.

Wild-type p53 represses Alu template activity in vitro and in vivo. However, upstream activating sequence elements from both the 7SL RNA gene and an Alu source gene relieve p53-mediated repression. p53 also represses the template activity of the U6 RNA gene both in vitro and in vivo but has no effect on in vitro transcription of genes encoding 5S RNA, 7SL RNA, adenovirus VAI RNA, and tRNA. The N-terminal activation domain of p53, which binds TATA-binding protein (TBP), is sufficient for repressing Alu transcription in vitro, and mutation of positions 22 and 23 in this region impairs p53-mediated repression of an Alu template both in vitro and in vivo. p53's N-terminal domain binds TFIIIB, presumably through its known interaction with TBP, and mutation of positions 22 and 23 interferes with TFIIIB binding. These results extend p53's transcriptional role to RNA polymerase III-directed templates and identify an additional level of Alu transcriptional regulation.

Potential Alu function: regulation of the activity of double-stranded RNA-activated kinase PKR.

Cell stress, viral infection, and translational inhibition increase the abundance of human Alu RNA, suggesting that the level of these transcripts is sensitive to the translational state of the cell. To determine whether Alu RNA functions in translational homeostasis, we investigated its role in the regulation of double-stranded RNA-activated kinase PKR. We found that overexpression of Alu RNA by cotransient transfection increased the expression of a reporter construct, which is consistent with an inhibitory effect on PKR. Alu RNA formed stable, discrete complexes with PKR in vitro, bound PKR in vivo, and antagonized PKR activation both in vitro and in vivo. Alu RNAs produced by either overexpression or exposure of cells to heat shock bound PKR, whereas transiently overexpressed Alu RNA antagonized virus-induced activation of PKR in vivo. Cycloheximide treatment of cells decreased PKR activity, coincident with an increase in Alu RNA. These observations suggest that the increased levels of Alu RNAs caused by cellular exposure to different stresses regulate protein synthesis by antagonizing PKR activation. This provides a functional role for mammalian short interspersed elements, prototypical junk DNA.

Silk worm Bm1 SINE RNA increases following cellular insults.

The effect of cell stresses upon the expression of the Bm1 short interspersed element (SINE) family in cultured silk worm cells is examined. Primer extension analysis shows that Bm1 repeats are transcribed by RNA polymerase III (Pol III) into cytoplasmic RNAs. Five consecutive T residues, which would normally terminate Pol III transcription, occur within the Bm1 consensus and are included within cDNA sequences representing these transcripts. In analogy to mammalian SINEs, the level of the Bm1 transcripts increases in response to either heat shock, inhibiting protein synthesis by cycloheximide or viral infection. The lifetime of Bm1 RNA increases following cell insults so that post-transcriptional events partially account for stress induced increases in its abundance. In the case of heat shock, the increase in Bm1 RNA follows the transient increase in hsp70 mRNA indicating that this response is temporally regulated to occur later in heat shock recovery. These results support the proposal that SINE RNAs serve a role in the cell stress response that predates the divergence of insects and mammals implying that SINEs are essentially a class of cell stress genes.

K562 cells implicate increased chromatin accessibility in Alu transcriptional activation.

Alu repeats in K562 cells are unusually hypomethylated and far more actively transcribed than those in other human cell lines and somatic tissues. Also, the level of Alu RNA in K562 cells is relatively insensitive to cell stresses, namely heat shock, adenovirus infection and treatment with cycloheximide, which increase the abundance of Alu RNA in HeLa and 293 cells. Recent advances in understanding the interactions between DNA methylation, transcriptional activation and chromatin conformation reveal reasons for the constitutively high level of Alu expression in K562 cells. Methylation represses transcription of transiently transfected Alu templates in all cell lines tested but cell stresses do not relieve this repression suggesting that they activate Alu transcription through another pathway. A relatively large fraction of the Alus within K562 chromatin is accessible to restriction enzyme cleavage and cell stresses increase the chromatin accessibility of Alus in HeLa and 293 cells. Cell stress evidently activates Alu transcription by rapidly remodeling chromatin to recruit additional templates.

Related single copy sequences in the human genome.

Human single copy DNA renatured under relatively non-stringent conditions (50 degrees C, 0.36 M Na+) forms two components with different thermal stabilities: a high temperature component melting at a temperature (81 degrees C, 0.18 M Na+) expected for well-paired duplex and a low temperature component melting at 55 degrees C (0.18 M Na+). The production of the high and low temperature components has been examined as a function of C0t (product of concentration and time in units of mol/s per l) value and renaturation temperature. The duplex formed has been characterized by its resistance to S1 endonuclease and its length has been determined by gel electrophoresis. As a result of these experiments and related controls we conclude that the low temperature component is not an artifact of DNA fragment length, nonspecific base pairing or hydroxyapatite chromatography conditions. We attribute the low thermal stability of this component to the renaturation of distantly related single copy sequences to form a highly mismatched duplex DNAs with an average length of about 100 base pairs.

The effect of silver ion binding and pH on the buoyant density of DNA and its use in fractionating heterogeneous DNA.

1. The effect of pH on the buoyant density of the complexes of Ag+ with DNA has been studied using 3H-labeled human DNA and several bacterial DNAs to determine the conditions necessary for the maximum resolution of compositional heterogeneity. In neutral CS2SO4 density gradients, Ag+ complexes with (G - C)-rich components are always denser than those with (A - T)-rich components, since (G - C)rich DNAs have a larger affinity for Ag+ than (A - T)-rich DNAs and their complexes are denser than (A - T)-rich complexes. In alkaline (pH greater than 9) CS2SO4 gradients, the buoyant density of the Ag+ - DNA complex is not a simple function of base composition. The Ag+ affinity of (A - T)-rich DNA is larger than that of (G - C)-rich DNA but the density of a (G - C)-rich complex is larger. Thus the ordering of the buoyant density changes depends on the amount of added Ag+. 2. The problem of resolving the density heterogeneity within a tracer DNA, and minor components of DNA, is explored and useful fractionation techniques are developed.

Fractionation of the sequence organization of human DNA by preparative HgCl2/Cs2SO4 density gradients.

Human DNA has been fractionated on preparative HgCl2/Cs2SO4 density gradients. Repetitious sequences are found in DNA components of all base compositions but the (G + C)-rich sequences are enriched for repetitive sequences in (A + T)-rich components are tandemly arranged; those in (G + C)-rich components tend to be interspersed with single copy sequences. It is concluded that the interspersed repetitious sequences have high (G + C) compositions.

A phase diagram of the binding of mismatched duplex DNAs to hydroxyapatite.

The hydroxyapatite binding properties of imperfectly base-paired DNA have been investigated and summarized in a phase diagram. This diagram defines the conditions at which thermal elution results in both denaturation and desorption of mismatched duplex DNA from hydroxyapatite.

Unusual sequences of two old, inactive human Alu repeats.

Two human Alu repeats terminating in an oligo(T) run rather than the usual A-rich 3' tail were isolated by library screening. Base sequence comparisons reveal that these unusual Alus are also exceptionally divergent from other Alu family members implying that they are evolutionarily old. Unlike other members of the family, they are not transcribed in vitro by RNA polymerase III (Pol III) suggesting a partial explanation for how Alu source genes might become inactive with age.

Fractionation of renatured repetitive human DNA according to thermal stability, sequence length, and renaturation rate.

Renatured repetitive human DNA sequences have been fractionated according to three criteria: length, thermal stability, and rate of renaturation. Satellite sequences are found in the long repetitive sequence fractions and renatured satellite sequences are well base paired by the criterion of thermal stability. No satellites are observed in short repeated sequences. Short repeated sequences are interspersed with single copy sequences and are poorly base paired. This implies satellite components are relatively homogeneous in base sequence whereas short interspersed repeated sequences are inexact copies of each other. A method for isolating satellite DNAs based on their high thermal stabilities is discussed.

A critical examination of possible fractionations of human DNA according to base composition.

Human DNA has been fractionated according to base composition by sedimentation equilibrium in an HgCl2/Cs2SO4 density gradient, followed by sedimentation equilibrium in an actinomycin/cesium formate density gradient. The fractions of different base composition resulting from this procedure were subsequently analyzed by sedimentation equilibrium in CsCl, DNA renaturation kinetics, and electron microscopy. All fractions contain similar kinetic classes of repeated DNA sequences as judged by renaturation studies. Short (300 nucleotides) interspersed repeated sequences are found in all fractions with no noticeable enrichment for these sequences in any fraction. Repeated sequences from fractions of different base composition are partially able to cross-hybridize, demonstrating that nearly identical repeated sequences occur in molecules of different base composition. These findings are critically compared to reports of successful density gradient fractionations of different human DNA sequence classes.

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

The arrangement of alpha-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 alpha-globin genes have been isolated and subjected to a partial sequence determination for comparison to alpha-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 alpha-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 alpha-globin genes. In contrast, the arrangement and correction of galago alpha-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 alpha 1 gene are orthologous to the corresponding region in galago, identifying the human alpha 2 gene as the more recently duplicated gene. The human psi alpha 1 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.

Cloned extrachromosomal circular DNA copies of the human transposable element THE-1 are related predominantly to a single type of family member.

The 2300 base-pair transposon-like human element, THE-1, has been identified in the extrachromosomal circular DNA of the established human cell line HeLa as a relatively homogeneous population of covalently closed 1900 base-pair molecules. THE-1, which has been classified tentatively as a retroviral-like transposable element (a retrotransposon), is present in the extrachromosomal circular DNA of African green monkey (BSC-1) and human lymphoblastoid (Jurkat) cell lines. The 1900 base-pair extrachromosomal elements isolated and cloned from HeLa cells (1) appear to contain only THE-1-specific nucleotide sequences, (2) are circularized versions of the linear chromosomal sequence, and (3) are related predominantly to a single, or single type of, family member.