Intracisternal A-particle Genes Research Articles

Methylation and rearrangement of mouse intracisternal a particle genes in development, aging, and myeloma.

Sequences of DNA that hybridize on Southern blots with cloned intracisternal A-particle (IAP) sequences have been examined in genomic DNAs of neonatal mice, livers of adult mice (3, 6, 12, 18, 24, and 26 months old), and the solid myeloma tumor MOPC-315. The isoschizomers HpaII (CCGG or mCCGG) and MspI (CCGG or CmCGG) were used to assess methylation. All the DNAs produced a major 0.5-kilobase MspI fragment that hybridizes with IAP probe. Only the myeloma DNA, and to a much lesser degree DNA from senescent mouse liver, produced this fragment in HpaII digest; the other DNAs all had IAP sequences resistant to HpaII digestion. These sequences thus become fully methylated to CmCGG early and remain so in adult life, except in the myeloma cells that are expressing the IAP genes. An increase in MspI-sensitive sites in IAP gene-containing DNA was observed in aging mice. The probe used to assess methylation, a 0.8-kilobase fragment produced by BamHI-HindIII double digestion, is common to several cloned IAP genes and is part of a region of DNA which is conserved in genomes of all mouse tissues. The probe hybridized to 1.5- and 1.4-kilobase doublet bands produced by BamHI, HindIII, and EcoRI triple digestions of neonatal DNA. These two bands were found in neonatal livers of Swiss Webster, BALB/c, and C57BL/6J mouse strains, showed less in adult liver, and were barely detectable in senescent livers from C57BL/6J mice.

Methylation and rearrangement of mouse intracisternal a particle genes in development, aging, and myeloma.

Sequences of DNA that hybridize on Southern blots with cloned intracisternal A-particle (IAP) sequences have been examined in genomic DNAs of neonatal mice, livers of adult mice (3, 6, 12, 18, 24, and 26 months old), and the solid myeloma tumor MOPC-315. The isoschizomers HpaII (CCGG or mCCGG) and MspI (CCGG or CmCGG) were used to assess methylation. All the DNAs produced a major 0.5-kilobase MspI fragment that hybridizes with IAP probe. Only the myeloma DNA, and to a much lesser degree DNA from senescent mouse liver, produced this fragment in HpaII digest; the other DNAs all had IAP sequences resistant to HpaII digestion. These sequences thus become fully methylated to CmCGG early and remain so in adult life, except in the myeloma cells that are expressing the IAP genes. An increase in MspI-sensitive sites in IAP gene-containing DNA was observed in aging mice. The probe used to assess methylation, a 0.8-kilobase fragment produced by BamHI-HindIII double digestion, is common to several cloned IAP genes and is part of a region of DNA which is conserved in genomes of all mouse tissues. The probe hybridized to 1.5- and 1.4-kilobase doublet bands produced by BamHI, HindIII, and EcoRI triple digestions of neonatal DNA. These two bands were found in neonatal livers of Swiss Webster, BALB/c, and C57BL/6J mouse strains, showed less in adult liver, and were barely detectable in senescent livers from C57BL/6J mice.

A mouse dispersed repeat sequence showing remarkable similarities to the long terminal repeats of retroviruses

A mouse 1.3-kb EcoRI dispersed repeat sequence that shows homology by hybridisation to the long terminal repeat of intracisternal A-particle genes has been partially sequenced. One of the established sequence regions shows three major features reminiscent of the long terminal repeats of retroviruses. Firstly, a TATA box precedes a putative capping site (G) by 23 bp. Secondly, the TATA box is itself preceded by the sequence CCTAT. Thirdly, following the capping site, the signal sequence AATAA precedes by 20 bp a possible polyadenylation site (CA). Such a retroviral-like sequence may have been responsible for the initial amplification and dispersion of the mouse repeat family.

Intracisternal A-particle genes as movable elements in the mouse genome.

We analyzed two functionally defective mouse kappa light chain gene variants previously shown to contain novel insertions of repetitive DNA in their intervening sequences [Hawley, R. G., Shulman, M. J., Murialdo, H., Gibson, D. M. & Hozumi, N. (1982) Proc. Natl. Acad. Sci. USA 79, 7425-7429]. Heteroduplex analysis of the cloned genes shows that the insertions consist of intracisternal A-particle (IAP) genetic elements. Each insertion includes an IAP 5' long terminal repeat (LTR) sequence and extends to a characteristic IAP internal BamHI site where the IAP sequence is interrupted because the mutant genes were cloned from complete BamHI digests of the cellular DNAs. Restriction enzyme mapping indicates that the 5' LTR boundaries of the inserted IAP elements correspond closely to the previously determined rearrangement sites in the mutant genes. The IAP insertions in the two mutants can be distinguished by restriction-site differences and by the fact that one of them contains a deletion that is absent in the other. Nucleotide sequence data are presented for the LTRs of one full-length IAP gene copy randomly selected from a mouse genomic DNA library. These LTRs show many features typical of known integrated retroviral terminal repeat units, and the entire gene is bracketed by short direct repeats within the adjacent cellular DNA. Thus, the findings show that IAP genetic elements can appear in new locations in mouse cellular DNA and suggest that this may occur through a process of proviral insertion.

Differing populations of intracisternal A-particle genes in myeloma tumors and mouse subspecies.

Intracisternal A-particle genes form a family of endogenous retrovirus-like genetic elements that are transcribed in mouse plasmacytomas (myeloma tumors). Two types of A-particle genes that can be differentiated by a sequence of 0.5 kilobase found in one type but not the other have been identified. Quantitative Southern blot analysis was used to measure the populations of different A-particle genes in DNAs from BALB/c mice, the Japanese subspecies Mus musculus subsp. molossinus, and myeloma tumors. The majority of the genes (715 copies per haploid genome or 76%) were found to be nearly identical except for small changes in conserved restriction enzyme sites. The second type of A-particle gene was much less abundant with 90 copies representing approximately 10%. The A-particle RNA in MOPC104E and MOPC315 was found to be colinear with a small portion of this latter type, comprising only 2% of the endogenous intracisternal A-particle sequences. Myeloma tumor DNA was found to have a two- to fourfold increase in the number of these genes, suggesting that the intracellular viruses have been activated to produce a double-stranded complementary DNA which subsequently integrated into the tumor genome. Analysis of M. musculus subsp. molossinus DNA revealed similar but shifted populations of A-particle genes, when compared with BALB/c DNA, except for the absence of a prominent EcoRI-HindIII band at 3.9 kilobases. This latter band, representing approximately 15% of the A-particle genes in BALB/c DNA, was shown to be a deletion variant of the most abundant gene family.

Intracisternal A-particle genes: structure of adjacent genes and mapping of the boundaries of the transcriptional unit.

Adjacent intracisternal A-particle (IAP) genes were identified in two different recombinant DNA clones, gamma 81 and gamma 19. In clone gamma 81, the most common form of IAP gene was separated by 5.3 kilobases from another IAP gene that had two apparent internal deletions. The two genes were in a head-to-tail configuration. In clone gamma 19, two different types of IAP genes were separated by less than 0.5 kilobase. Blot hybridization analysis of mouse DNA demonstrated that the DNA sequence found in clone gamma 81 is identical to the in vivo configuration. Using isolated DNA fragments from clone gamma 19, we mapped the boundaries of the IAP RNA by S1 digestion of RNA-DNA hybrids and by cDNA extension. With these techniques, both the 5' end and the 3' end of the IAP RNA in two different plasmacytomas (MOPC 315 and TEPC 15) were shown to fall within the long terminal direct repeat of the IAP gene. The fragment sizes generated by S1 digestion of IAP RNAs isolated from the two tumor lines were found to differ, indicating that different IAP genes may be transcribed in these two plasmacytomas.

Terminally redundant sequences in cellular intracisternal A-particle genes.

The sequences coding for intracisternal A-particle RNA form a family of related but not identical genetic elements which are present in 650 to 1,000 copies within the mouse genome. We showed that different intracisternal A-particle genes had a terminally redundant sequence of about 400 base pairs, one-half of which arose from the 3' end of the intracisternal A-particle RNA. A second portion of the redundant region did not contain 3-related sequences and was probably derived from the 5' end of intracisternal A-particle RNA. Thus, there were endogenous intracisternal A-particle genes in the cellular DNA-3'-5'--3'-5'-cellular DNA configuration identified for type B and C retroviruses. This indicated that the initial integration of intracisternal A-particle genes into the Mus musculus genome occurred by the same mechanism as the integration of other retroviruses. Two types of heterogeneity were identified among the 5' sequences of the two genes.

Intracisternal A-particle genes in Mus musculus: a conserved family of retrovirus-like elements.

The structural organization of intracisternal A-particle genes has been studied, using isolates from a mouse gene library in lambda phage Charon 4A. The predominant gene form among the isolates was 7.3 kilobases (kb) in length. R-loops between the 7-kb (35S) A-particle genomic ribonucleic acid and several of these genes were colinear, with no visible evidence of intervening deoxyribonucleic acid sequences. One recombinant was found with an A-particle gene that contained a 1.7-kb deletion. Using the deletion as a reference, the deoxyribonucleic acid and ribonucleic acid homology regions were localized with respect to one another and to the restriction map: the 5' terminus of the ribonucleic acid was several hundred base pairs within the 5' end of the deoxyribonucleic acid homology region. Restriction endonuclease fragments encompassing the 5' and 3' regions of one 7.3-kb gene were separately subcloned into pBR322. Heteroduplexes between the two subclones revealed an approximately 300-base pair segment of terminally redundant sequences. The cloned 3' fragment hybridized with restriction fragments from the 5' end of several other A-particle genes, demonstrating the presence of common (though not necessarily identical) terminally repeated sequences. A-particle genes varied in the occurrence of specific restriction sites at characteristic internal loci. However, heteroduplexes between several variant 7.3-kb genes showed continuous homology regions even when spread under stringent hybridization conditions. The relative abundance of restriction site variants was highly conserved in 12 laboratory strains of Mus musculus, in embryonic and adult tissues of a single inbred strain, and in the SC-1 cell line of feral mouse origin, but appeared to differ in a feral Japanese substrain, Mus musculus molossinus. Some evidence suggests that subsets of A-particle genes may have similar flanking sequences. The results are discussed in terms of the evolution of this multigene family.

Intracisternal A-particle genes in Mus musculus: a conserved family of retrovirus-like elements.

The structural organization of intracisternal A-particle genes has been studied, using isolates from a mouse gene library in lambda phage Charon 4A. The predominant gene form among the isolates was 7.3 kilobases (kb) in length. R-loops between the 7-kb (35S) A-particle genomic ribonucleic acid and several of these genes were colinear, with no visible evidence of intervening deoxyribonucleic acid sequences. One recombinant was found with an A-particle gene that contained a 1.7-kb deletion. Using the deletion as a reference, the deoxyribonucleic acid and ribonucleic acid homology regions were localized with respect to one another and to the restriction map: the 5' terminus of the ribonucleic acid was several hundred base pairs within the 5' end of the deoxyribonucleic acid homology region. Restriction endonuclease fragments encompassing the 5' and 3' regions of one 7.3-kb gene were separately subcloned into pBR322. Heteroduplexes between the two subclones revealed an approximately 300-base pair segment of terminally redundant sequences. The cloned 3' fragment hybridized with restriction fragments from the 5' end of several other A-particle genes, demonstrating the presence of common (though not necessarily identical) terminally repeated sequences. A-particle genes varied in the occurrence of specific restriction sites at characteristic internal loci. However, heteroduplexes between several variant 7.3-kb genes showed continuous homology regions even when spread under stringent hybridization conditions. The relative abundance of restriction site variants was highly conserved in 12 laboratory strains of Mus musculus, in embryonic and adult tissues of a single inbred strain, and in the SC-1 cell line of feral mouse origin, but appeared to differ in a feral Japanese substrain, Mus musculus molossinus. Some evidence suggests that subsets of A-particle genes may have similar flanking sequences. The results are discussed in terms of the evolution of this multigene family.