Single copy sequences in galago DNA resemble a repetitive human retrotransposon-like family

Summary Galago DNA contains a few single copy sequences that are homologous to the human THE 1 family of repeats. Two of these galago loci have been isolated as genomic clones and their structures are compared to the THE 1 consensus sequence. Whereas the human sequence resembles a proretroviral transposon, the galago sequences provide no evidence for a proretroviral sequence organization. The two galago clones share a common repeat sequence, which is homologous to the U5 region of the THE 1 long terminal repeat. Immediately 3 to this repeat, each galago clone contains sequences that are homologous to mutually exclusive regions of the internal THE 1 sequence. Thus, the human THE 1 sequence can be represented as a mosaic of the two ancestrally related galago loci. The galago loci are transcribed in vivo, so that their conservation in the primate genome could be selected. Human THE 1 repeats apparently resulted by recruiting preexisting cellular sequences via a retrovirally mediated process.     

Nucleotide sequences of mouse genomic loci including a gene or pseudogene for U6 (4.8S) nuclear RNA.

We have isolated four clones which hybridize with U6 (4.8S) nuclear RNA, a mammalian small nuclear RNA(nRNA), from DNA of BALB/C mouse liver. Their restriction maps are totally different from each other, indicating that they derived from different loci in the mouse genome. The nucleotide sequences around the hybridizing region in the three clones have been determined. One clone gives a gene that is co-linear with the U6 RNA. There is a sequence TATAAAT beginning 31 nucleotides upstream of the gene, which may suggest that the U6 RNA is transcribed by RNA polymerase II. The other two clones contain a pseudogene for the U6 RNA which has 7 or 9 nucleotide changes from the RNA. The pseudogenes are surrounded by radically different sequences from those surrounding the gene, and they are closely linked to a pseudogene for another snRNA, 4.5S-I RNA, or a part of highly repetitive an interspersed sequence B1.     

Molecular cloning and characterization of murine leukemia virus-related DNA sequences from C3H/HeN mouse DNA.

Ten murine leukemia virus (MuLV)-related DNA sequences were isolated from C3H/HeN mouse genomic DNA by cloning of EcoRI fragments in a Charon 4A vector. Detailed restriction endonuclease maps of four of the clones were developed by using AKR MuLV [32P]cDNA as a probe. C3H clone 14-9 contains approximately 7 kilobase pairs of MuLV-related DNA, one copy of an MuLV long terminal repeat-like sequence, and a region of flanking mouse DNA. C3H clones 34.2 and 36.1 contain approximately 2 kilobase pairs of MuLV-related DNA, one copy of a MuLV LTR-like sequence, and differing lengths of flanking mouse DNA sequences. C3H clone 8.13 was found to contain an insert of 5.7 kilobase pairs of MuLV-related DNA with two long terminal repeat-like regions and sequences which are partially homologous to AKv-1. Comparison fo the restriction endonuclease cleavage maps of these C3H clones with maps recently developed for ecotropic and xenotropic MuLV DNAs indicates that C3H clone 14-9 corresponds to the 5'-terminal portion of a genomic DNA sequence related to xenotropic MuLVs, whereas C3H clones 34.2 and 36.1 correspond to the 3' terminal portions of genomic DNA sequences related to xenotropic MuLVs. Clone 8.13 represents a deleted, xenotropic MuLV-related provirus. C3H clones 14-9, 34.2, 36.1, and 8.13 provide defined DNA sequence probes with which to characterize the organization and expression of endogenous MuLV-related DNA sequences in the mouse genome.     

Loci for human U1 RNA: structural and evolutionary implications

Three clones U1-1, U1-6, and U1-8 containing sequences related to human U1 RNA have been studied by sequence analysis. The results show that each of the three clones represents a distinct locus. The U1-6 locus is closely related to the HU1-1 locus, which is believed to represent a functional U1 gene. The U1-1 and U1-8 loci are pseudogenes by definition, since they contain sequences that are closely related to but not identical with the human U1 RNA sequence. The U1-6 locus contains the sequence T-A-T-A-T close to the 5'-end of the U1 sequence but it is unclear if this represents the promoter. When the U1-8 locus was compared to the U1-6 locus, it was observed that the 5'-flanking sequences, except in the immediate vicinity of the pseudogene, are as well-conserved as the U1-related sequence itself, at least up to position -220. The high degree of homology in the 5'-flanking region suggests that U1 genes have a much more strict sequence requirement with regard to 5'-flanking sequences than most other eukaryotic genes. The U1-6 and U1-8 loci contain the sequence T-A-T-G-T-A-G-A-T-G-A between positions -211 and -221. An identical sequence is present in the equivalent position in the HU1-1 locus, and may represent the promoter. The high degree of conservation in the postulated promoter region indicates that pseudogenes like U1-8 possibly could be expressed. A truncated U1-related sequence is present between 106 to 150 nucleotides upstream from the U1 gene/pseudogene in the U1-6, the U1-8 and the HU1-1 loci, suggesting that the U1 genes may have been clustered early in evolution. The U1-1 locus has a strikingly different structure from the U1-8 locus; the pseudogene itself is as closely related to the U1 RNA sequence as is the U1-8 pseudogene but the flanking sequences, both on the 5' and the 3' side, share no detectable homology with the corresponding regions in the U1-6 or U1-8 loci. It may therefore be postulated that small nuclear RNA pseudogenes are created by several different mechanisms.     

Characterization of rat ribosomal DNA. The highly repetitive sequences that flank the ribosomal RNA transcription unit are homologous and contain RNA polymerase III transcription initiation sites

The non-transcribed spacers (NTS) of the ribosomal genes of a number of organisms have been studied and were found to contain repetitive sequences. In these studies with plasmid subclones of NTS, designated p3.4, p2.6 and p1.7, which come from both 5' and 3' flanking regions of the rat ribosomal genes, respectively, it has been determined that these sequences are found elsewhere within the genome. Southern hybridization analysis has demonstrated that the 5' and 3' NTS subclones cross-hybridize, and that the cross-hybridizing regions are synonymous with the highly repetitive regions. Sequences homologous to the rat NTS were specifically localized to both 5' and 3' flanking regions as well as to a number of the introns of cloned genes including rat serum albumin, rat alpha-fetoprotein, rat casein and human serum albumin. No hybridization was detected of the 5' NTS subclone to the human Alu sequence clone, Blur 8, or to the rodent equivalent, a clone containing Chinese hamster ovary type I and II Alu sequences. However, as reported for type II Alu sequences, the subcloned rat NTS sequences contain RNA polymerase III initiation sites and also hybridize to a number of small RNAs, but not 4.5 S or 7 S RNA. Sequence analysis of two distinct repetitive regions in p1.7 has revealed a region of alternating purine-pyrimidine nucleotides, potentially of Z DNA, and stretches of repetitive sequences. The possible roles for these repetitive sequences in recombination and in maintaining a hierarchical structure for the ribosomal genes are discussed.     

Human U1 RNA pseudogenes may be generated by both DNA- and RNA-mediated mechanisms.

Analysis of cloned human genomic loci homologous to the small nuclear RNA U1 established that such sequences are abundant and dispersed in the human genome and that only a fraction represent bona fide genes. The majority of genomic loci bear defective gene copies, or pseudogenes, which contain scattered base mismatches and in some cases lack the sequence corresponding to the 3' end of U1 RNA. Although all of the U1 genes examined to date are flanked by essentially identical sequences and therefore appear to comprise a single multigene family, we present evidence for the existence of at least three structurally distinct classes of U1 pseudogenes. Class I pseudogenes had considerable flanking sequence homology with the U1 gene family and were probably derived from it by a DNA-mediated event such as gene duplication. In contrast, the U1 sequence in class II and III U1 pseudogenes was flanked by single-copy genomic sequences completely unrelated to those flanking the U1 gene family; in addition, short direct repeats flanked the class III but not the class II pseudogenes. We therefore propose that both class II and III U1 pseudogenes were generated by an RNA-mediated mechanism involving the insertion of U1 sequence information into a new chromosomal locus. We also noted that two other types of repetitive DNA sequences in eucaryotes, the Alu family in vertebrates and the ribosomal DNA insertions in Drosophila, bore a striking structural resemblance to the classes of U1 pseudogenes described here and may have been created by an RNA-mediated insertion event.     

Nucleotide sequence of the brome mosaic virus genome and its implications for viral replication

The nucleotide sequences of brome mosaic virus (BMV) RNAs 1 (3234 bases) and 2 (2865 bases) have been determined, completing the primary structure of the 8200 base tripartite BMV genome. cDNA clones covering 99% of BMV RNA1 and a full-length cDNA clone of BMV RNA2 were isolated in the course of this work. Extensive sequence homology and known interaction with several proteins suggest that the 3' ends of the BMV RNAs are the major regulatory regions of the genome. Smaller regions at the 5' ends of RNAs 1 and 2 show strong homology to each other and lesser homology to RNA3. These and other features of the sequences are discussed in relation to replication, regulation and evolution of the BMV genome.     

Human DNA sequences homologous to a protein coding region conserved between homeotic genes of Drosophila

Several human DNA sequences were isolated by virtue of homology to a highly conserved region that has been identified in a number of homeotic genes in Drosophila. Structural analysis of the human DNAs indicate that two separate and distinct regions sharing a high degree of homology with the homeo box sequences of Drosophila are separated by only 5 kb in the human genome. Sequence determination of these regions reveals that both human DNA sequences contain a region capable of coding 61 amino acids, which shares greater than 90% homology with the peptide sequences specified by the homeo box domain of Drosophila homeotic genes, Antennapedia, fushi tarazu, and Ultrabithorax. By contrast, the human DNA sequences lying outside of the 190 nucleotide homeo box region share virtually no sequence homology, either with the flanking sequences of the other human clones or with flanking regions of the known Drosophila homeotic genes.     

Analysis of integrated human papillomavirus type 16 DNA in cervical cancers: amplification of viral sequences together with cellular flanking sequences.

We have isolated four clones of integrated human papillomavirus type 16 (HPV-16) DNA from four different primary cervical cancer specimens. All clones were found to be monomeric or dimeric forms of HPV-16 DNA with cellular flanking sequences at both ends. Analysis of the viral sequences in these clones showed that E6/E7 open reading frames and the long control region were conserved and that no region specific for the integration was detected. Analysis of the cellular flanking sequences revealed no significant homology with any known human DNA sequences, except Alu sequences, and no homology among the clones, indicating no cellular sequence specific for the integration. By probing with single-copy cellular flanking sequences from the clones, it was demonstrated that the integrated HPV-16 DNAs, with different sizes in the same specimens, shared the same cellular flanking sequences at the ends. Furthermore, it was shown that the viral sequences together with cellular flanking sequences were amplified. The possible process of viral integration into cell chromosomes in cervical cancer is discussed.     

DNA sequences complementary to human 7 SK RNA show structural similarities to the short mobile elements of the mammalian genome

A complementary DNA clone of 7 SK RNA from HeLa cells was used to study the genomic organization of 7 SK sequences in the human genome. Genomic hybridizations and genomic clones show that 7 SK is homologous to a family of disperse repeated sequences most of which lack the 3' end of the 7 SK RNA sequence. Only few of the genomic K sequences are homologous to both 3' and 5' 7 SK probes and presumably include the gene(s) for 7 SK RNA. The sequence of four genomic 7 SK clones confirms that they are in most cases pseudogenes. Although Alu sequences are frequently found near the 3' and 5' end of K DNA, the sequences immediately flanking the pseudogenes are different in all clones studied. However, direct repeats were found flanking directly the K DNA or the K-Alu unit, suggesting that the K sequences alone or in conjunction with Alu DNA might constitute a mobile element.     

Identification of putative homology between horse microsatellite flanking sequences and cross-species ESTs, mRNAs and genomic sequences

In this study the flanking sequences of 1534 horse microsatellites were used in a BLAST search to identify putative human-horse homologies. BLAST searches revealed 129 flanking sequences with significant blastn matches [alignment scores (S) > or = 60 and sum probability values (E) < or = 3.0E-6], also, 25 of these produced significant blastx matches. To provide a reference point in the human genome the flanking sequences with matches were subjected to a BLAT search of the University of California Santa Cruz (UCSC) human genome assembly (July 2003 freeze). Eighty-three of the flanking sequences showed high similarity to sequence of known or putative human genes and the remaining 46 demonstrated high similarity to human intragenic regions. Interestingly, 87 of the microsatellites showed conservation of the tandem repeat in addition to flanking regions. Overall, 41 of the microsatellites had been mapped in the horse and of these 37 localized to the expected syntenic location. The other four did not and represent new putative regions of human-horse synteny. The results of this study contribute 79 new putative human-horse homologies, increasing the density of markers on the human-horse comparative map.     

Integrated structures of duck hepatitis B virus DNA in hepatocellular carcinoma.

The structure of integrated viral DNA in a hepatocellular carcinoma of a duck from Chi-tung county in China was analyzed. Three different clones of integrated viral DNA, lambda DHS 6-1, lambda DHS 6-2, and lambda DHE 6-2, were obtained from the neoplastic portion of the liver by molecular cloning. One of the three clones, lambda DHS 6-1, showed inverted repetition of integrated viral DNA with chromosomal flanking sequences. Another clone, lambda DHS 6-2, showed a head-to-head configuration of the core and surface gene regions of duck hepatitis B virus (DHBV) DNA. The virus-chromosome junctions were often located near direct repeat 1 or 2 of DHBV DNA in three independent clones. Nucleotide sequences at the virus-virus junctions in two clones, lambda DHS 6-1 and 6-2, indicated the possible rearrangement of chromosomal DNA and recombination of viral DNA. DHBV DNA appears to be integrated into the genome of hepatocytes in a manner similar to that of human and woodchuck hepatitis viruses. Thus, the duck system may serve as a useful animal model for the study of human hepatocarcinogenesis.     

Cloned embryonic DNA sequences flanking the mouse immunoglobulin C gamma 3 and C gamma 1 genes

To investigate the DNA surrounding genes for immunoglobulin heavy chain constant (CH) regions, we have isolated two clones bearing a C gamma 3 gene and two bearing a C gamma 1 gene from a library of mouse embryo DNA fragments. The C gamma 3 clones span 8.6 kilobase pairs (kb) on the 5' side of the gene and 6.7 kb on its 3' side, while the C gamma 1 clones together span 13 kb of 5' flanking sequence and 2.5 kb of 3' flanking sequence. Restriction mapping of the C gamma 3 gene indicates that intervening sequences divide the gene into segments of domain size, as in other CH genes. Hybridization of clone fragments to restriction digests of mouse DNA indicates that both the C gamma 1 and C gamma 3 genes probably occur as single copies in the genome. Moreover, the entire cloned sequences on the 5' side of both genes appear to be unique in the genome, indicating that no large common sequences flank CH genes. Restriction data suggest that the C gamma 3 gene is 37-40 kb 5' to the C gamma 1 gene.     

Multiple,dispersed human U6 small nuclear RNA genes with varied transcriptional efficiencies

Vertebrate U6 small nuclear RNA (snRNA) gene promoters are among the founding members of those recognized by RNA polymerase III in which all control elements for initiation are located in the 5'-flanking region. Previously, one human U6 gene (U6-1) has been studied extensively. We have identified a total of nine full-length U6 loci in the human genome. Unlike human U1 and U2 snRNA genes, most of the full-length U6 loci are dispersed throughout the genome. Of the nine full-length U6 loci, five are potentially active genes (U6-1, U6-2, U6-7, U6-8 and U6-9) since they are bound by TATA-binding protein and enriched in acetylated histone H4 in cultured human 293 cells. These five all contain OCT, SPH, PSE and TATA elements, although the sequences of these elements are variable. Furthermore, these five genes are transcribed to different extents in vitro or after transient transfection of human 293 cells. Of the nine full-length U6 loci, only U6-7 and U6-8 are closely linked and contain highly conserved 5'-flanking regions. However, due to a modest sequence difference in the proximal sequence elements for U6-7 and U6-8, these genes are transcribed at very different levels in transfected cells.