Nucleotide sequence of the intracisternal A-particle genome inserted 5'' to the interleukin-3 gene of the leukemia cell line WEHI-3B.

The nucleotide sequence of the intracisternal A-particle genome IAP-IL3 is presented. This IAP element was found to have inserted upstream of the promoter of the interleukin-3 gene of the leukemia cell line WEHI-3B. IAP-IL3 is 5095 bp in length, with identical long terminal repeats (LTRs) of 337 bp. The LTRs show many of the conserved sequence elements identified in other retroviruses. Comparison with other available sequences of IAP genomes indicates that IAP-IL3 is a deleted type I element. It carries a deletion covering the 3' end of the putative IAP gag gene and extending into the 5' end of the putative IAP pol gene. IAP-IL3 has extensive sequence homology with an IgE-binding factor cDNA and evidence is presented indicating that it was derived from a member of the mouse IAP sequence family. Comparison between the pol region of IAP-IL3 and other retroviruses suggests that IAP-IL3 is most closely related to type B and type D retroviruses.     

Isolation and analysis of a rat genomic clone containing a long terminal repeat with high similarity to the oncomodulin mRNA leader sequence

The structure of a novel long terminal repeat (LTR) from an intracisternal A particle (IAP) DNA element in the rat (Sprague-Dawley) genome was determined. This LTR has a total length of 313 base pairs (bp). Several structural features typical for retroviral LTR promoters were identified, including a "CCAAT" box, a "TATA" box, a polyadenylation signal, and a polyadenylation site. The LTR is flanked by 3-bp inverted repeats, and it consists of the three typical LTR regions, U3, R, and U5. U3 contains 213 bp, R 46 bp, and U5 54 bp, which is within the usual size range of IAP LTRs. A sequence of 60 bp in the U3 region reveals considerable similarity to a murine IAP LTR U3 element, which is known to interact with nuclear proteins. A sequence of 69 bp in the U5 and R regions has 83 and 93% similarities to an endogenous retroviral LTR from Syrian hamster and to the cDNA leader sequence of (Buffalo) rat oncomodulin, respectively. Oncomodulin is an "EF-hand" Ca2+-binding protein and appears in many human and rodent tumors and in cells with tumor-like properties but not in normal tissues. We postulate that in the rat the tumor-specific expression of oncomodulin is controlled by a retroviral LTR promoter.     

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.     

5' LTR complete nucleotide sequence of Chinese hamster intracisternal A-particle.

Retrovirus like sequences homologous to mouse IAP are present in Chinese hamster genome (Lueders K.K. and Kuff, E.L., 1981, 1983, Servenay et al., 1990). Murine IAP long terminal repeats (LTRs) can function as effective promoters in different cell types (Horowitz M. et al., 1984, Howe, C.C. et al., 1986). Thus CHO IAP sequences could act as retrotransposons in the cellular genome, and in this way affect the expression of other genes at the target sites. We had sequenced previously a Chinese hamster IAP genomic region corresponding mainly to the gag gene and including 57 nucleotides of U5 5' LTR (Servenay et al., 1988). In this paper, we present the 5' LTR complete nucleotide sequence of the Chinese hamster IAP element and its comparison with those of mouse and Syrian hamster.     

In vitro methylation inhibits the promotor activity of a cloned intracisternal A-particle LTR.

We studied the relation between LTR methylation and expression of the family of endogenous retrovirus-like elements related to mouse intracisternal A-particles (IAP). Comparative HpaII/MspI and HhaI restriction analysis of genomic DNA's showed that in cells and tissues with a low level of IAP gene expression, HpaII and HhaI sites within the 5' LTR were heavily methylated, while in cells abundantly expressing IAP's 20 to 30% of the 5' LTRs were demethylated at these sites. The effects of methylation on the promoter activity of a cloned IAP 5' LTR was studied directly, using the plasmid pMIA5' L-cat in which this LTR was linked to the chloramphenicol acetyl transferase (CAT) gene. In vitro methylation of three HhaI sites located between -137 and -205 bp from the RNA start site of this LTR completely inactivated the promoter activity of pMIA5' L-cat transfected into COS7 cells. Methylation of a HpaII site located 94 bp downstream from the RNA start site reduced the promoter activity by 75%. The results show that methylation at sites both upstream and downstream from the RNA start site profoundly effects the promoter activity of this LTR and suggest that methylation within the 5' LTR can serve to regulate IAP gene expression in vivo.     

ART-CH, a new chicken retroviruslike element.

A 3' region of a previously unknown retroviruslike element named ART-CH (avian retrotransposon from chicken genome) was obtained in the course of polymerase chain reaction-mediated cloning of avian leukosis virus long terminal repeats (LTRs) from DNAs of infected chicken cells. About 50 copies of ART-CH are present in the genome of chickens of different breeds. ART-CH is not found in DNA of quails, ducks, turkeys, or several other birds tested. The ART-CH element is about 3 kb in size, including 388 bp LTRs. The major class of ART-CH-specific RNA, also 3 kb in size, is detected in various organs of chickens. An ART-CH polypurine tract, a tRNA(Trp)-binding site, regions around the TATA box and polyadenylation signal, and the beginning of the putative gag gene strongly resemble the corresponding regions of avian leukosis viruses and EAV, the two described classes of chicken retroviruses. An open reading frame capable of encoding a polypeptide with a putative transmembrane domain is located upstream of the right ART-CH LTR. This sequence, as well as the U3 and U5 regions of the ART-CH LTR, has no obvious similarities with the corresponding parts of other known vertebrate retroviruses and retrotransposons. A short sequence upstream of the right LTR of ART-CH is very similar to sequences which flank the 3' ends of the oncogenes v-src, v-myc, v-fps, and v-crk in four different recombinant avian retroviruses and which are absent from the genomes of other studied avian retroviruses. Thus, ART-CH is a new endogenous chicken provirus that may participate in the formation of recombinant oncogenic retroviruses.     

The mitochondrial genome of the brachiopod Laqueus rubellus

The complete nucleotide sequence of the 14,017-bp mitochondrial (mt) genome of the articulate brachiopod Laqueus rubellus is presented. Being one of the smallest of known mt genomes, it has an extremely compact gene organization. While the same 13 polypeptides, two rRNAs, and 22 tRNAs are encoded as in most other animal mtDNAs, lengthy noncoding regions are absent, with the longest apparent intergenic sequence being 54 bp in length. Gene-end sequence overlaps are prevalent, and several stop codons are abbreviated. The genes are generally shorter, and three of the protein-coding genes are the shortest among known homologues. All of the tRNA genes indicate size reduction in either or both of the putative TPsiC and DHU arms compared with standard tRNAs. Possession of a TV (TPsiC arm-variable loop) replacement loop is inferred for tRNA(R) and tRNA(L-tag). The DHU arm appears to be unpaired not only in tRNA(S-tct) and tRNA(S-tga), but also in tRNA(C), tRNA(I), and tRNA(T), a novel condition. All the genes are encoded in the same DNA strand, which has a base composition rich in thymine and guanine. The genome has an overall gene arrangement drastically different from that of any other organisms so far reported, but contains several short segments, composed of 2-3 genes, which are found in other mt genomes. Combined cooccurrence of such gene assortments indicates that the Laqueus mt genome is similar to the annelid Lumbricus, the mollusc Katharina, and the octocoral Sarcophyton mt genomes, each with statistical significance. Widely accepted schemes of metazoan phylogeny suggest that the similarity with the octocoral could have arisen through a process of convergent evolution, while it appears likely that the similarities with the annelid and the mollusc reflect phylogenetic relationships.     

Three divergent mitochondrial genomes from California populations of the copepod Tigriopus californicus

Previous work on the harpacticoid copepod Tigriopus californicus has focused on the extensive population differentiation in three mtDNA protein coding genes (COXI, COXII, Cytb). In order to get a more complete understanding of mtDNA evolution in this species, we sequenced three complete mitochondrial genomes (one from each of three California populations) and compared them to two published mtDNA genomes from an Asian congener, Tigriopus japonicus. Several features of the mtDNA genome appear to be conserved within the genus: 1) the unique order of the protein coding genes, rRNA genes and most of the tRNA genes, 2) the genome is compact, varying between 14.3 and 14.6 kb, and 3) all genes are encoded on the same strand of the mtDNA. Within T. californicus, extremely high levels of nucleotide divergence (>20%) are observed across much of the mitochondrial genome. Inferred amino acid sequences of the proteins encoded in the mtDNAs also show high levels of divergence; at the extreme, the three ND3 variants in T. californicus showed >25% amino acid substitutions, compared with <3% amino acid divergence at the previously studied COXI locus. Unusual secondary structures make functional assignments of some tRNAs difficult. The only apparent tRNA(trp) in these genomes completely overlaps the 5' end of the 16S rRNA in all three T. californicus mtDNAs. Although not previously noted, this feature is also conserved in T. japonicus mtDNAs; whether this sequence is processed into a functional tRNA has not been determined. The putative control region contains a duplicated segment of different length (from 88 to 155 bp) in each of the T. californicus sequences. In each case, the duplicated segments are not tandem repeats; despite their different lengths, the distance between the start of the first and the start of the second repeat is conserved (520 bp). The functional significance, if any, of this repeat structure remains unknown.     

Mechanism of activation of the mouse c-mos oncogene by the LTR of an intracisternal A-particle gene.

In the mouse myeloma XRPC-24 the DNA of an intracisternal A-particle (IAP) is inserted within the coding region of c-mos. This insertion splits the c-mos into a 3' rc-mos and a 5' rc-mos separated by approximately 4.7 kb of IAP DNA. The insertion is in a head-to-head orientation and brings the 5' LTR of the IAP in juxtaposition to the 3' rc-mos such that the IAP and the 3' rc-mos are transcribed in opposite directions. The intact c-mos gene is usually dormant, whereas the 3' rc-mos is actively transcribed and is capable of transforming NIH3T3 cells. In an effort to understand the nature of this activation we mapped the 5' ends of the 3' rc-mos mRNA present in XPRC-24. We found two main mRNA start sites, one mapping to the junction of the 3' rc-mos and the 5' LTR, and the other located 10 nucleotides upstream to this junction, within the 5' LTR. This result indicates that the 3' rc-mos in XRPC-24 was activated by insertion of a promoter provided by the LTR of an IAP genome. Furthermore, the 5' LTR appears to possess promoter activities in two directions. This conclusion was confirmed by the fact that this 5' LTR, in both orientations, was able to activate the bacterial gene coding for chloramphenicol acetyltransferase (CAT) in the modular vector pSVOCAT.     

A novel selection system for recombinational and mutational events within an intron of a eucaryotic gene.

In order to identify a poison sequence that might be useful in studying illegitimate recombination of mammalian cell chromosomes, several DNA segments were tested for their ability to interfere with gene expression when placed in an intron. A tRNA gene and its flanking sequences (267 bp) were shown to inhibit SV40 plaque formation 100-fold, when inserted into the intron in the T-antigen gene. Similarly, when the same DNA segment was placed in the second intron of the adenosine phosphoribosyl transferase (APRT) gene from CHO cells, it inhibited transformation of APRT-CHO cells 500-fold. These two tests indicated that the 267-bp DNA segment contained a poison sequence. The poison sequence did not affect replication since the replication of poisoned SV40 genomes was complemented by viable SV40 genomes and poisoned APRT genes were stably integrated into cell chromosomes. Cleavage of the poison sequence in the SV40 T-antigen intron by restriction enzymes indicated that the tRNA structural sequences and the 5' flanking sequences were not required for inhibition of SV40 plaque formation. Sequence analysis of viable mutant SV40, which arose after transfection of poisoned genomes, localized the poison sequence to a 35 bp segment immediately 3' of the tRNA structural sequences.     

Genetic relationship between the Mus cervicolor M432 retrovirus and the Mus Musculus intracisternal type A particle

The genetic relationship between the retrovirus-like intracisternal type A particle (IAP) from Mus musculus and the novel retrovirus (M432) from M. cervicolor has been determined by heteroduplex and restriction endonuclease analyses of molecular clones of the respective genomes. We have found a major homology region (3.7 kilobase pairs) which probably begins near the 3' end of the M432 gag gene, spans the pol gene, and ends in the env gene. A second region (0.6 kilobase pairs) of weak homology was also observed adjacent to the 3' long terminal repeats of the respective genomes. The IAP genome is well conserved in the cellular DNA of all species of the genus Mus. In contrast, cellular DNA sequences related to the 5' end of the M432 genome, which shares no homology with the IAP genome, are found only in M. cervicolor and the closely related species M. cookii. These results suggest that the infectious M432 retroviral genome arose as a result of a recombinational event(s) between the IAP genome and another, as yet unidentified, class of retrovirus-related sequences or other cellular sequences.