Studies on HSAG, a middle repetitive family of genetic elements which elicit a leukemia-related cellular surface antigen.

HSAG is a family of genetic elements capable of eliciting, in transfected cells, a cellular surface antigen which is correlated with human chronic lymphocytic leukemia (CLL). Its prototype member, HSAG-1, was cloned as a 3.4 kb insert and contains numerous Alu-related elements, including its left hand 1.4 kb antigen-eliciting end. These elements are present in mammalian cells with copy numbers varying from 7,000 to 200,000 per haploid genome, depending on how closely their sequence conforms to the Alu consensus sequence. They are present in the configuration found in HSAG-1, a 3.4 kb EcoRI fragment which is part of a larger unit of at least 12.7 kb, at a frequency of 20-50 per haploid genome, and dispersed around the genome. A second family member, HSAG-2, isolated using a functional assay, was cloned as a 9.5 kb insert and contained a 1.5 kb antigen-eliciting left hand end. As in HSAG-1, the antigen-eliciting portion of the insert also contained Alu-like elements, unlike most of the remainder of the insert. A number of HSAG family members were cloned from a library of human CLL genomic DNA by sequence homology with the antigen-eliciting portion of HSAG-1. Most of these members were also shown to be capable of eliciting antigen. Their only sequence similarity with HSAG-1 appeared to be in their content of numerous Alu-like elements. The evidence thus supports the view that the HSAG functional family consists of clusters of Alu-like elements.     

Specific binding of Alu sequences by HeLa nuclear extracts

Plasmid Blur 8 which contains the 300bp human Alu consensus sequence and plasmid pBR322 were digested with restriction enzymes and the fragments obtained end labelled with 32P-gamma-ATP. The end labelled fragments were incubated with HeLa nuclear extracts and the incubation mixtures passed through a nitrocellulose filter. The 300bp alu consensus sequence was preferentially retained on the filter. The HeLa nuclear extract did not preferentially bind any fragments generated from pBR322 and histones which bind nonspecifically all DNA fragments did not preferentially bind the alu sequence. We conclude that the HeLa nuclear extract contains components which specifically bind the human alu sequence.     

Birth of a gene: locus of neuronal BC200 snmRNA in three prosimians and human BC200 pseudogenes as archives of change in the Anthropoidea lineage

The gene encoding brain-specific dendritic BC200 small non-messenger RNA is limited to the primate order and arose from a monomeric Alu element. It is present and neuronally expressed in all Anthropoidea examined. By comparing the human sequence of about 13.2 kb with each of the prosimian (lemur 14.6 kb, galago 12 kb, and tarsier 13.8 kb) orthologous loci, we could establish that the BC200 RNA gene is absent from the prosimian lineages. In Strepsirhini (lemurs and lorises), a dimeric AluJ-like element integrated very close to the BC200 insertion point, while the corresponding tarsier region is devoid of any repetitive element. Consequently, insertion of the Alu monomer that gave rise to the BC200 RNA gene must have occurred after the anthropoid lineage diverged from the prosimian lineage(s). Shared insertions of other repetitive elements favor proximity of simians and tarsiers in support of their grouping into Haplorhini and the omomyid hypothesis. On the other hand, the nucleotide sequences in the segment that is available for comparison in all four species reveal less exchanges between Strepsirhini (lemur and galago) and human than between tarsier and human. Our data imply that the early activity of dimeric Alu sequences must have been concurrent with the activity of monomeric Alu elements that persisted longer than is usually thought. As BC200 RNA gave rise to more than 200 pseudogenes, we used their consensus sequence variations as a molecular archive recording the BC200 RNA sequence changes in the anthropoid lineage leading to Homo sapiens and timed these alterations over the past 35-55 million years.     

Human 7SL RNA consists of a 140 nucleotide middle-repetitive sequence inserted in an alu sequence

We have cloned and sequenced a cDNA copy of in vitro-polyadenylated 7SL RNA of HeLa cells. The cloned fragment is 303 bp long and has a composite structure. A central block of 140 bp is homologous to a new set of human middle-repetitive sequences. This block appears to be inserted in an Alu consensus sequence, 100 bp from the 5' end and 40 bp from the 3' end of the Alu monomer. Two 6 bp direct repeats are found at the junction between the Alu flanking sequences and the central element. The analysis of several clones shows the existence of sequence microheterogeneity in the 5' portion of the molecule. The 7L DNA probably represents a subset of the Alu family of DNA, highly conserved in evolution.     

A dimorphic Alu Sb-like insertion in COL3A1 is ethnic-specific

Alu elements are a class of repetitive DNA sequences found throughout the human genome that are thought to be duplicated via an RNA intermediate in a process termed retroposition. Recently inserted Alu elements are closely related, suggesting that they are derived from a single source gene or closely related source genes. Analysis of the type III collagen gene (COL3A1) revealed a polymorphic Alu insertion in intron 8 of the gene. The Alu insertion in the COL3A1 gene had a high degree of nucleotide identity to the Sb family of Alu elements, a family of older Alu elements. The Alu sequence was less similar to the consensus sequence for the PV or Sb2 subfamilies, subfamilies of recently inserted Alu elements. These data support the observations that at least three source genes are active in the human genome, one of which is distinct from the PV and Sb2 subfamilies and predates either of these two subfamilies. Appearance of the Alu insertion in different ethnic populations suggests that the insertion may have occurred in the last 100,000 years. This Alu insert should be a useful marker for population studies and for marking COL3A1 alleles.     

The beta globin gene cluster of the prosimian primate Galago crassicaudatus: nucleotide sequence determination of the 41-kb cluster and comparative sequence analyses.

The nucleotide sequence of the beta globin gene cluster of the prosimian Galago crassicaudatus has been determined. A total sequence spanning 41,101 bp contains and links together previously published sequences of the five galago beta-like globin genes (5'-epsilon-gamma-psi eta-delta-beta-3'). A computer-aided search for middle interspersed repetitive sequences identified 10 LINE (L1) elements, including a 5' truncated repeat that is orthologous to the full-length L1 element found in the human epsilon-gamma intergenic region. SINE elements that were identified included one Alu type I repeat, four Alu type II repeats, and two methionine tRNA-derived Monomer (type III) elements. Alu type II and Monomer sequences are unique to the galago genome. Structural analyses of the cluster sequence reveals that it is relatively A+T rich (about 62%) and regions with high G+C content are associated primarily with globin coding regions. Comparative analyses with the beta globin cluster sequences of human, rabbit, and mouse reveal extensive sequence homologies in their genic regions, but only human, galago, and rabbit sequences share extensive intergenic sequence homologies. Divergence analyses of aligned intergenic and flanking sequences from orthologous human, galago, and rabbit sequences show a gradation in the rate of nucleotide sequence evolution along the cluster where sequences 5' of the epsilon globin gene region show the least sequence divergence and sequences just 5' of the beta globin gene region show the greatest sequence divergence.     

Sequence of an expressed human beta-tubulin gene containing ten Alu family members

The complete sequence of a functionally expressed human beta-tubulin gene (5 beta) is presented. The amino acid sequence encoded by this gene constitutes a distinct isotype, differing from a previously described human beta-tubulin sequence at 21 positions throughout the polypeptide chain. The beta-tubulin coding sequence in 5 beta is interrupted by three intervening sequences of 1014, 117 and 4826 nucleotides. The largest of these contains ten members of the Alu family of middle repetitive sequences. Together, these regions account for sixty percent of this intervening sequence. Two of the Alu elements are juxtaposed head to tail, and share the same flanking direct repeat. The ten Alu sequences are substantially divergent, both from each other and from an Alu consensus sequence, and several contain deletions of up to half the entire sequence.     

Analysis of the regions flanking the human insulin gene and sequence of an Alu family member.

The regions around the human insulin gene have been studied by heteroduplex, hybridization and sequence analysis. These studies indicated that there is a region of heterogeneous length located approximately 700 bp before the 5' end of the gene; and that the 19 kb of cloned DNA which includes the 1430 bp insulin gene as well as 5650 bp before and 11,500 bp after the gene is single copy sequence except for 500 bp located 6000 bp from the 3' end of the gene. This 500 bp segment contains a member of the Alu family of dispersed middle repetitive sequences as well as another less highly repeated homopolymeric segment. The sequence of this region was determined. This Alu repeat is bordered by 19 bp direct repeats and also contains an 83 bp sequence which is present twice. The regions flanking the human and rat I insulin genes were compared by heteroduplex analysis to localize homologous sequences in the flanking regions which could be involved in the regulation of insulin biosynthesis. The homology between the two genes is restricted to the region encoding preproinsulin and a short region of approximately 60 bp flanking the 5' side of the genes.     

AluElements: Repetitive DNA as Facilitators of Chromosomal Rearrangement

Alu repeats are the most common type of repetitive DNA sequences dispersed throughout the human genome. Technical advances in the field of cytogenetics and molecular biology have facilitated the analysis of epithelial tumors and hematologic malignancies which has led to the observation of Alu elements in and near sites often involved in chromosomal rearrangements. Repair mechanisms of double strand breaks (DSB) such as homol-ogous recombination (HR) may rely on the sequence homology of Alu repeats, potentially leading to chromosomal rearrange-ments. Databases have confirmed the strong association between Alu repeats, specifically the 26 bp consensus sequence and chro-mosomal regions involved in deletions and translocations. Although the Alu repetitive sequence is a potential "hotspot" during homologous recombination, there are other cellular mech-anisms that may play a more prominent role in the initiation of chromosomal rearrangements.     

A human dihydrofolate reductase intronless pseudogene with an Alu repetitive sequence: multiple DNA insertions at a single chromosomal site

A dihydrofolate reductase (DHFR) pseudogene, hDHFR-psi 3 has been isolated from a human genomic DNA fragment library. Sequence analysis of this gene revealed a lack of introns and the presence of a tract of nine adenines, 90 bp downstream from the end of the coding sequence. These features suggest that hDHFR-psi 3 was derived from a processed RNA molecule that has been converted into DNA and inserted into a chromosome, analogous to the origin of three intronless human DHFR genes previously described. An interesting feature of hDHFR-psi 3 is the presence of a member of the Alu moderately repetitive DNA sequence family within the DHFR coding region. This Alu element is flanked by a 16 bp directly repeated DNA segment derived from DHFR coding sequences. The Alu element apparently has been inserted into the intronless DHFR pseudogene and thus, there have been two insertions at a single chromosomal locus. The hDHFR-psi 3 contains only the 3' half of the DHFR coding sequence. Immediately upstream from the directly repeated sequence before the Alu element is an adenine-rich tract. The DNA farther upstream is moderately repetitive and is related to neither DHFR nor Alu DNA sequence. Therefore, it seems possible that a third insertion has occurred at the same site further disrupting the hDHFR coding sequences.     

Structure of the human blood platelet membrane glycoprotein Ib alpha gene

The gene for human platelet glycoprotein Ib alpha-chain has been cloned from a genomic cosmid library using a partial cDNA clone as probe. 3530 bp were sequenced including the entire transcribed part, as well as additional 5' and 3' regions. A single intron was found 6 bp upstream of the ATG initiation codon. An exceptionally long exon was identical to the recently published cDNA sequence (1). The 5' upstream promoter region is atypical for eukaryotic genes with only a weak homology to the characteristic promoter consensus sequences. The 3' region contains two repetitive Alu elements, belonging to distinct subfamilies, connected by an oligo(dA) linker.     

Revision of consensus sequence of human Alu repeats--a review

Nucleotide sequences of 50 human Alu repeats and their flanking regions are presented together with the consensus sequence based on the literature and our findings. The results indicate the need for some revisions of the Alu consensus sequence published by Deininger et al. (1981). Most nucleotide substitutions among the Alu members are transitions, rather than transversions. The Alu sequence seems to consist of 'conserved' regions and 'variable' regions. The conserved regions consist of a 25-bp region between nt positions 23 and 47 and a 16-bp region between nt positions 245 and 260. The 16-bp region corresponds to the region of 7SL RNA that is claimed to fold and become paired with the internal promoter sequence. Two A-rich regions, one located at the right end of the first monomer and the other at the right end of the second monomer, are variable. No defined property was found with direct repeats flanking the Alu repeats.     

Human plasminogen activator inhibitor-1 gene. Promoter and structural gene nucleotide sequences

We have determined the nucleotide sequence of the human plasminogen activator inhibitor-1 (PAI-1) gene and significant stretches of DNA which extend into its 5'-and 3'-flanking DNA regions; a total sequence of 15,867 base pairs (bp) is presented. The sequenced 5'-flanking DNA (1,520 bp) contains the essential eukaryotic cis-type proximal regulatory elements CCAAT and TATAA; the more distal 5'-flanking DNA region, as well as some introns, contain sequence elements which share identities with known eukaryotic enhancer elements. A major finding is the identification of a large region of shared nucleotides (comprising of about 520 bp) between the 5'-flanking DNAs of PAI-1 and tissue-type plasminogen activator genes. The length of the PAI-1 5'-untranslated region was found to be 145 bp as determined by nuclease analysis. The remaining PAI-1 structural gene consists of amino acid coding regions (containing a total of 1,206 bp, coding for the 23 amino acids of the signal peptide and 379 amino acids of the mature PAI-1 protein), 8 intron regions (a total of 8,978 bp), and a long 3'-untranslated region of about 1,800 bp which contains several polyadenylation sites. Two types of repetitive DNA elements are located within the PAI-1 structural gene and flanking DNAs: we have found 12 Alu elements and 5 repeats of a long poly (Pur) element. These Alu-Pur elements may represent a subset of the more abundant Alu family of repetitive sequence elements.     

Analysis of TR-DNA/plant junctions in the genome of a Convolvulus arvensis clone transformed by Agrobacterium rhizogenes strain A4

A Charon 4A phage library, containing insert DNA isolated from a morning glory (Convolvulus arvensis) plant genetically transformed by Ri T-DNA from Agrobacterium rhizogenes strain A4, was used to isolate a lambda clone that contains part of the Ri TL-DNA and the complete TR-DNA. The two Ri T-DNAs were recovered adjacent to each other in a tail-to-tail configuration (i.e. with the TR-DNA inverted with respect to the TL-DNA). Comparison of nucleotide sequences from this lambda clone with the corresponding sequences from the Ri plasmid allowed us to determine the location of the T-DNA/plant junction for the right end of the TL-DNA and the left and right ends of the TR-DNA. We located, near each of these borders, a 24 bp sequence that is similar to the 24 bp consensus sequence found near the pTi T-DNA extremities. In addition, sequences similar to the core overdrive sequence from pTi are located near each right border. Hybridization and nucleotide sequence analysis of the DNA adjacent to the TL/TR junction shows that no plant DNA is located between the TL and TR-DNAs and suggests that the plant DNA adjacent to the end of the TR-DNA may have been rearranged during the integration into the plant genome.     

Characterization of the primate-specific repetitive DNA element MER1.

Computer analyses of the 3'-flanking DNA sequence of the human elastase I gene revealed a significant degree of similarity with seven human gene sequences in the GenBank and EMBL databases. Genomic Southern analysis indicates that the shared nucleotide sequences are a primate-specific family of short interspersed elements. These elements are members of MER1 sequences (medium reiteration frequency sequences). The consensus sequence of MER1 repeats spans 543 nucleotides and contains several inverted repeats. Since the copy number of MER1 elements seems to be much smaller than that of Alu and L1 repeats, MER1 elements may provide useful landmarks marks for human genome mapping.     

The human THE-LTR(O) and MstII interspersed repeats are subfamilies of a single widely distributed highly variable repeat family.

Fifteen examples of the transposon-like human element (THE) LTR and thirteen examples of the MstII interspersed repeat are aligned to generate new consensus sequences for these human repetitive elements. The consensus sequences of these elements are very similar, indicating that they compose subfamilies of a single human interspersed repetitive sequence family. Members of this highly polymorphic repeat family have been mapped to at least 11 chromosomes. Seven examples of the THE internal sequence are also aligned to generate a new consensus sequence for this element. Estimates of the abundance of this repetitive sequence family, derived from both hybridization analysis and frequency of occurrence in GenBank, indicate that THE-LTR/MstII sequences are present every 100-3000 kb in human DNA. The widespread occurrence of members of this family makes them useful landmarks, like Alu, L1, and (GT)n repeats, for physical and genetic mapping of human DNA.