Characterization of a highly repetitive family of DNA sequences in the mouse.

A large proportion (0.5-1%) of total mouse DNA is cleaved by Bam HI into fragments whose size is about 500 base pairs. A cloned member of this repetitive family of DNA sequences (BAM5 family) was sequenced by the dideoxy chain termination procedure and shown to contain 507 base pairs. The sequence exhibited no unusual or remarkable features. Repetitive sequences complementary to the cloned BAM5 fragment were found in rat DNA, but not in feline or human DNA. Restriction mapping suggested that many BAM5 sequences were components of much larger repetitive DNAs which were scattered throughout the mouse genome. The BAM5 sequences within the larger repetitive DNAs did not appear to be arranged tandemly or as members of scrambled tandem repeats. RNA homologous to the cloned BAM5 sequence was detected in cultured mouse cells, but not in cultured rat cells.     

Association of two different repetitive DNA elements near immunoglobulin light chain genes.

Sequence studies of repetitive DNA elements approximately 6 kb 3' of the mouse immunoglobulin CK region gene show that the R element located there (Gebhard et al. (1982) J. Mol. Biol. 157, 453-471) is adjacent to a 500 base pair long element which shows 80% homology to the BAM5 element sequenced by Fanning (Nuc. Acids Res. (1982), 10, 5003-5013). Neither the BAM5 element nor the R element itself is surrounded by a direct repeat, but the composite element (BAM5 + R) is surrounded by a 15 base pair direct repeat (with one mismatch). Direct repeats, consisting of target site sequences that surround a repetitive DNA element, are thought to arise during the insertion of the element at that site. It therefore appears that the BAM5 and R elements interacted and inserted as a linked entity. The existence of other BAM5/R composites throughout the mouse lambda chain locus indicates that BAM5-R cooperation is not a rare event.     

Insertion of a long KpnI family member within a mitochondrial-DNA-like sequence present in the human nuclear genome

Structural analysis of a phage lambda Charon 4A clone carrying one of the human nuclear mitochondrial(mut)-DNA-like sequences revealed that a KpnI-family member (KpnI 5.5-kb DNA) is inserted within this sequence. The inserted KpnI 5.5-kb DNA contains several possible polyadenylation signal sequences followed by an A-rich sequence at its 3' end and is flanked by perfect 13-bp direct repeats of the duplicated mtDNA-like sequences. These structures strongly suggest that the KpnI 5.5-kb DNA is a mobile element. Comparison of the 5' terminal sequences of the KpnI 5.5-kb DNA and four other long KpnI-family DNAs so far examined, using the predicted general promoter sequence for eukaryotic tRNAs, indicates that they contain the consensus sequences for the split internal RNA polymerase III control region.     

Structural analysis of a 1.7-kilobase mouse mammary tumor virus-specific RNA

We have detected a mouse mammary tumor virus (MMTV)-specific 1.7-kilobase (kb) polyadenylated RNA in mammary glands of several mouse strains. In BALB/c mice, it is the only MMTV-specific RNA species present. C3H and GR mammary glands and tumors contain, in addition, 3.8- and 7.8-kb MMTV RNAs. Nuclease S1 analysis was performed to map 1.7-kb polyadenylated RNA. It contains predominantly long terminal repeat (LTR) sequences. The 5' end maps approximately 134 nucleotides upstream from the 3' end of the LTR. Colinearity with complete proviral DNA continues to a site about 153 nucleotides downstream from the left (5') LTR. No sequences from the middle part of proviral DNA were found. Colinearity with proviral DNA is resumed 72 nucleotides upstream from the right (3') LTR. The nucleotide sequence in this area is TTCCAGT, which is a splice acceptor consensus sequence. The anatomy of 1.7-kb RNA indicates that it may serve as a messenger for the 36,700-dalton protein encoded by the LTRs of MMTV.     

Analysis of FBJ-MuSV provirus and c-fos (mouse) gene reveals that viral and cellular fos gene products have different carboxy termini

The complete nucleotide sequence of the FBJ-MuSV proviral DNA and the cellular homolog (c-fos) of its oncogene (v-fos) have been determined. The 4026 nucleotide long FBJ-MuSV proviral DNA contains two long terminal repeats, a substitution of 1639 nucleotides of mouse cellular DNA (v-fos) and the 3′ end of the env gene derived from FBJ-MuLV. The sequences of the parental FBJ-MuLV and the cellular c-fos (mouse) gene share five of five nucleotides at the 5′ end and ten of 11 nucleotides at the 3′ end of the v-fos substitution. When compared with the v-fos sequences, the c-fos gene contains four discontinuous regions, three of which are flanked by sequences characteristic of introns. Direct sequence analysis of c-fos (mouse) RNA by primer extension demonstrates that the fourth discontinuity is due to a 104 bp deletion in the v-fos gene. As a consequence of the deletion, the predicted v-fos and c-fos gene products differ at their C termini.     

Isolation and nucleotide sequence of a mouse histidine tRNA gene.

We have sequenced a 1307 base pair mouse genomic DNA fragment which contains a histidine tRNA gene. The sequence of the putative mouse histidine tRNA differs from the published sequence of sheep liver histidine tRNA by a single base change in the D-loop. It does not contain an unpaired 5' terminal G residue, as reported for Drosophila and sheep histidine tRNAs. The gene does not contain introns. The 3' flanking region contains a typical RNA polymerase III termination site of 6 consecutive T residues. 523 residues after the 3' end of the his tRNA coding region, the mouse DNA contains a sequence 72% homologous to part of the consensus sequence of the B1 (alu) family.     

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.     

Linkage map of two HLA-SB beta and two HLA-SB alpha-related genes: an intron in one of the SB beta genes contains a processed pseudogene

Three overlapping cosmid clones contain coding sequences for four HLA Class II genes, provisionally identified as two HLA-SB alpha and two HLA-SB beta genes. The genes are in the order beta, alpha, beta, alpha, inverted with respect to each other. One of the SB beta genes contains a 513 bp sequence that appears to be a processed pseudogene, flanked by direct 17 bp repeat sequences, in the intron upstream of the beta 1 exon. The pseudogene is homologous to a family of sequences of approximately 25-40 members, most of which are not on chromosome 6. A cDNA clone, highly homologous to the pseudogene, except for its 5' end, contains a normal poly(A) addition site and a poly(A) tail. The cDNA clone is homologous to a single-copy gene in both man and mouse, encoded on human chromosome 15. A search of published DNA sequences identified a mouse sequence, with about 77% similarity to the pseudogene sequence, in the negative strand of an intron in a mouse dihydrofolate reductase gene. The second SB beta gene does not contain the pseudogene sequence.     

Poly-dA:dT Tracts Form an In Vivo Nucleosomal Turnstile

Nucleosomes regulate many DNA-dependent processes by controlling the accessibility of DNA, and DNA sequences such as the poly-dA:dT element are known to affect nucleosome binding. We demonstrate that poly-dA:dT tracts form an asymmetric barrier to nucleosome movement in vivo, mediated by ATP-dependent chromatin remodelers. We theorize that nucleosome transit over poly-A elements is more energetically favourable in one direction, leading to an asymmetric arrangement of nucleosomes around these sequences. We demonstrate that different arrangements of poly-A and poly-T tracts result in very different outcomes for nucleosome occupancy in yeast, mouse, and human, and show that yeast takes advantage of this phenomenon in its promoter architecture.     

The structure of HSAG-1, a middle repetitive genetic element which elicits a leukemia-related cellular surface antigen.

HSAG-1 is a cloned member of a heterogeneous middle repetitive family of genetic elements which is capable of eliciting a leukemia-related surface antigen detected with a monoclonal antibody after DNA transformation of mouse cells. HSAG-1 was originally isolated from a Chinese hamster-human leukemia hybrid cell gene library both by sib-selection for antigen producing activity and by hybridization with labelled human genomic human DNA. We show here that the human labelled site is at the right hand end of the insert, while the antigen-eliciting portion is included in a 1450 bp fragment at the left hand end of the insert. We also present the complete nucleotide sequence of the 3369 bp insert. The sequence contains 12 elements which bear a significant resemblance to accepted consensus sequences for Alu repetitive elements. The right hand end contains adjacent elements with close sequence similarity to portions of the human and hamster type I and type II Alu consensus sequences. All of the other Alu-related elements have diverged relative to the Alu consensus sequences by additions, long deletions and substitutions. The left hand portion of the insert which has the antigen-producing activity contains four of these diverged elements representing a relatively high proportion (26%) of the nucleotide sequence. The sequence is thus consistent with our previous observations of a repetitive family with biological function.     

Sequence arrangement of the 16S and 26S rRNA genes in the pathogenic haemoflagellate Leishmania donovani.

Kinetic and chemical analysis show that the haploid genome of Leishmania donovani has between 4.6 and 6.5 X 10(7) Kb pairs of DNA. Cot analysis shows that the genome contains 12% rapidly reassociating DNA, U3% middle repetitive DNA with an average reiteration frequency of 77 and 62% single copy DNA. Saturation hybridization experiments show that 0.82% of the nuclear DNA is occupied by rRNA coding sequences. The average repetition frequency of these sequences is determined to be 166. Sedimentation velocity studies indicate the two major rRNA species have sedimentation values of 26S and 16S, respectively. The arrangement of the rRNA genes and their spacer sequences on long strands of purified rDNA has been determined by the examination of the structure of rRNA:DNA hybrids prepared for electron microscopy by the gene 32-ethidium bromide technique. Long DNA strands are observed to contain several gene sets (16S + 26S). One repeat unit contains the following sequences in the order given: (a) A 16S gene of length 2.12 Kb, (b) An internal transcribed spacer (Spl) of length 1.23 Kb, which contains a short sequence that may code for a 5.8S rRNA, (C) 26S gene with a length of 4.31 Kb which contains an internal gap region of length 0.581 Ib, (d) An external spacer of average length 5.85 Kb.     

Structure of DNA near long tandem arrays of alpha satellite DNA at the centromere of human chromosome 7.

The centromeric regions of human chromosomes contain long tracts of tandemly repeated DNA, of which the most extensively characterized is alpha satellite. In a screen for additional centromeric DNA sequences, four phage clones were obtained which contain alpha satellite as well as other sequences not usually found associated with tandemly repeated alpha satellite DNA, including L1 repetitive elements, an Alu element, and a novel AT-rich repeated sequence. The alpha satellite DNA contained within these clones does not demonstrate the higher-order repeat structure typical of tandemly repeated alpha satellite. Two of the clones contain inversions; instead of the usual head-to-tail arrangement of alpha satellite monomers, the direction of the monomers changes partway through each clone. The presence of both inversions was confirmed in human genomic DNA by polymerase chain reaction amplification of the inverted regions. One phage clone contains a junction between alpha satellite DNA and a novel low-copy repeated sequence. The junction between the two types of DNA is abrupt and the junction sequence is characterized by the presence of runs of A's and T's, yielding an overall base composition of 65% AT with local areas > 80% AT. The AT-rich sequence is found in multiple copies on chromosome 7 and homologous sequences are found in (peri)centromeric locations on other human chromosomes, including chromosomes 1, 2, and 16. As such, the AT-rich sequence adjacent to alpha satellite DNA provides a tool for the further study of the DNA from this region of the chromosome. The phage clones examined are located within the same 3.3-Mb SstII restriction fragment on chromosome 7 as the two previously described alpha satellite arrays, D7Z1 and D7Z2. These new clones demonstrate that centromeric repetitive DNA, at least on chromosome 7, may be more heterogeneous in composition and organization than had previously been thought.     

Structural analysis of type II variants within the mouse intracisternal A-particle sequence family.

Intracisternal A-particle (IAP) elements are present in multiple copies in the mouse and other rodent genomes. The bulk of this sequence family in Mus musculus consists of 7 Kb long elements, but the majority of IAP sequences involved in known transpositions have been deleted forms. The present study describes a subset of deleted IAP sequences (type II IAP) characterized by insertion of a particular short sequence element (AIIins). AIIins are interspersed and the majority occur as part of the type II IAP elements in the mouse genome. AIIins sequences are absent or in low copy number outside Mus musculus. We have isolated clones containing AIIins from a mouse genomic DNA library and have sequenced three isolates of AIIins and their surrounding IAP sequences to define the detailed structure of type II elements. AIIins are 272, 268 and 264 bp long and 90% homologous in sequence. They are bracketed by 9 bp duplications, suggesting they may be inserted elements. A 75 bp region containing a core enhancer sequence is repeated at the 5' end in type II IAP elements. Insertion into the IAP genome, with potential to encode an integrase function, may have played a role in the amplification of AIIins.     

Secondary structure and mutational analysis of the Mason-Pfizer monkey virus RNA constitutive transport element.

The Mason-Pfizer monkey virus (MPMV) genome contains a cis-acting element that serves to facilitate nucleocytoplasmic export of intron-containing RNA. This element, known as the constitutive transport element (CTE), has been mapped to a 154-nt region close to the 3' end of the MPMV genome. The CTE contains a degenerate direct repeat of approximately 70 nt. We have probed the secondary structure of the CTE using double-strand- and single-strand-specific ribonucleases and chemical modification agents. A mutational analysis was also performed to confirm critical features of the CTE structure, as well as to identify regions that contain sequence-specific information required for function. Our results indicate that the CTE forms a long stem structure that contains a 9-nt terminal hairpin loop as well as two identical 16-nt inner loops. The inner loop sequences are rotated 180 degrees relative to each other within the structure. The mutational analysis shows that primary sequences in the loop regions are important for function, suggesting that they may contain binding sites for cellular proteins involved in RNA export. Interestingly, sequences with significant homology to the inner loop regions are found in the genomes of spumaviruses and mouse intracisternal A particles.     

Structure of a processed gene of mouse cytoplasmic gamma-actin transposed into a BAM5 sequence: insertion has created 13 base-pair direct repeats.

We have cloned a processed gene related to cytoplasmic gamma-actin from a phage library of mouse brain DNA. The gene lacks introns, carries a poly(dA) tract of 19-base pairs at the 3' end and is flanked by 13-base-pair direct repeats. Southern blotting experiments indicated that this segment is embedded in highly repetitive and strongly methylated sequences. Direct comparison of the flanking sequences with those of mouse repetitive DNAs revealed that this processed gene is integrated into a BAM5 family, which is one of the repetitive sequences of the mouse genome. In addition, it was shown that the direct repeats were created by a duplication of the corresponding 13-bp stretch of the BAM5 DNA as a consequence of the integration of this segment. These results strongly suggest that the processed gamma-actin gene has been conveyed to this locus through a poly(A)+ RNA intermediate by a transposon-like mechanism.