Differential expression of intracisternal A-particle transcripts in immunogenic versus tumorigenic S49 murine lymphoma cells.

Tumorigenic S49 mouse lymphoma cells (T-25) were compared to their nontumorigenic (immunogenic) substrate-adherent descendants (T-25-Adh), using the differential display technique. A 784-bp fragment with 92% sequence homology to the intracisternal A-particle (IAP) element family was isolated from the latter cells. IAP sequences are endogenous, noninfectious retroviral elements that can undergo transpositions and act as mutagens. Expression of IAP transcripts (as detected by the isolated fragment) was 5- to 10-fold higher in T-25-Adh cells than in T-25 cells. IAP RT-PCR cDNA clones derived from the immunogenic T-25-Adh cells, but not from T-25 cells, contain two distinctive motifs: (i) a motif characteristic of IAP elements expressed in lymphoid cells (lymphocyte specific, LS); (ii) a nonapeptide sequence known to stimulate cytotoxic T lymphocytes in a leukemia cell line expressing IAP sequences. In addition, expression of transcripts containing these motifs is enhanced in the immunogenic cells as opposed to the tumorigenic cells. Furthermore, one of the IAP elements (belonging to the LS1 subfamily) is specifically hypomethylated in the DNA of the immunogenic cells. The above-mentioned relationship was strengthened when tumorigenic revertants derived from T-25-Adh cells, as well as independently selected tumorigenic and immunogenic S49 sublines, were studied. In all cases, enhanced immunogenicity was linked to the up-regulation of specific IAP elements. No transpositions of LS1 elements were observed among the different sublines studied. These findings suggest that, in the S49 lymphoma, selectively expressed IAP retroviral elements may function in a tumor suppressive capacity by affecting the immunogenic potential of these cells.

Genomic organization and mapping of the human and mouse neuronal beta2-nicotinic acetylcholine receptor genes.

As a first step in determining whether there are polymorphisms in the nicotinic acetylcholine receptor (nAChR) genes that are associated with nicotine addiction, we isolated genomic clones of the beta2-nAChR genes from human and mouse BAC libraries. Although cDNA sequences were available for the human gene, only the promoter sequence had been reported for the mouse gene. We determined the genomic structures by sequencing 12 kb of the human gene and over 7 kb of the mouse gene. While the sizes of exons in the mouse and human genes are the same, the introns differ in size. Both promoters have a high GC content (60-80%) proximal to the AUG and share a neural-restrictive silencer element (NRSE), but overall sequence identity is only 72%. Using a 6-Mb YAC contig of Chr 1, we mapped the human beta2-nAChR gene, CHRNB2, to 1q21.3 with the order of markers cen, FLG, IVL, LOR, CHRNB2, tel. The mouse gene, Acrb2, had previously been mapped to Chr 3 in a region orthologous to human Chr 1. We refined mapping of the mouse gene and other markers on a radiation hybrid panel of Chr 3 and found the order cen, Acrb2, Lor, Iv1, Flg, tel. Our results indicate that this cluster of markers on human Chr 1 is inverted with respect to its orientation on the chromosome compared with markers in the orthologous region of mouse Chr 3.

An edited linkage map for the AXB and BXA recombinant inbred mouse strains.

We have updated the history of the AXB and BXA recombinant inbred (RI) strains, typed additional loci, and edited the AXB, BXA RI database. Thirteen of the original 51 AXB and BXA RI strains are either extinct or genetically contaminated, leaving 33 living strains available from The Jackson Laboratory. However, we found a high degree of similarity among three sets of strains, indicating that these strains are not independent, which leaves 27 independent RI strains in the set. Accordingly, we modified the database by combining the AXB and BXA RI sets and eliminating strains that were genetically contaminated or extinct with no available DNA. We added 92 newly typed loci, retyped some questionable genotypings, and removed loci with excessive double crossovers or an insufficient number of typed strains. The edited strain distribution pattern (SDP) is available on the World Wide Web (WWW) (http://www. informatics.jax.org/riset.html) and now includes over 700 loci. Each locus is linked to adjacent loci with a LOD score of at least 3.0 with a few described exceptions. We also carried out a second editing designed for the analysis of quantitative trait loci by deleting extinct strains and loci with identical SDPs; this edited database is also available on the WWW.

Multilocus genomic mapping with intracisternal A-particle proviral oligonucleotide probes hybridized to mouse DNA in dried agarose gels.

Recently, oligonucleotide probes that detect intracisternal A-particle (IAP) gene subfamilies with a limited number of proviral copies have been shown to be useful multilocus markers. A procedure for hybridization of these probes has been developed and is described. In summary, the main features of the method are the following: (i) A pulse controller is used during agarose gel electrophoresis to improve resolution of restriction fragments in genomic DNA. (ii) Hybridization is performed in a dried gel. (iii) The hybridized gel is washed in tetramethylammonium chloride to eliminate differences in oligonucleotide composition on hybrid stability. Use of the procedure is demonstrated by genomic mapping of IAP loci in the AXB BXA recombinant inbred mouse strains, identification of hypomethylated loci in tumor cells, and detection of a transposed IAP provirus previously identified as the basis for a mutation at the agouti locus.

Interacisternal A-particle (IAP) genes show similar patterns of hypomethylation in established and primary mouse plasmacytomas.

Alterations in cell programming associated with neoplastic transformation may involve widespread changes in patterns of DNA methylation. Increased expression of IAP elements in plasmacytomas compared with LPS-stimulated normal B-cells is accompanied by extensive hypomethylation of IAP sequences (Mietz and Kuff 1990), subsets of which are revealed with the LS2, LS3 and T1 probes. Multiple common LS- and PC-specific IAP loci are hypomethylated in established plasmacytomas, showing that hypomethylation does not occur entirely randomly. Many of the same IAP loci are hypomethylated in primary plasmacytomas induced by two different methods, as soon as recognizable tumor tissue can be isolated. In primary tumors hypomethylation frequently appears to occur in DNA flanking the IAP elements. In the established tumors the hypomethylated sites occur primarily in the IAP LTR, suggesting that for these loci hypomethylation begins in the flanking DNA and is extended into the IAP LTRs during progression of the tumors. The newly hypomethylated IAP LTRs in primary plasmacytomas (as compared to normal B cells) may provide a set of reporter genes for chromosomal regions that are characteristically hypomethylated in these transformed cells and that may contain cellular genes whose activation is related to the transformation process.

Mapping of mouse intracisternal A-particle proviral markers in an interspecific backcross.

We present a linkage map of intracisternal A-particle (IAP) proviral loci. The IAP family consists of 2000 endogenous proviral elements that are widely dispersed in the mouse genome. The map was constructed by using an interspecific backcross and markers defined by oligonucleotide probes specific for subclasses of expressed IAP elements. In genomic DNA from C57BL/6J mouse, these probes each detected from 12 to 44 HindIII restriction fragments that represent junctions between proviral and 5'-flanking DNA. The fragments have characteristic strain distribution patterns (SDPs) that are particularly polymorphic in the DNAs of C57BL/6J and Mus spretus mice used for the backcross. IAP loci were placed on the map by comparison of their distribution patterns with those of known genetic markers in the backcross. The map includes 51 IAP loci that have not been previously mapped and 23 IAP proviruses that had been previously mapped in recombinant inbred (RI) strains. Comparable map positions were obtained with the IAP markers in the interspecific backcross and the RI strains. The mapped IAP loci were widely dispersed on the X Chromosome (Chr) and all of the autosomes except Chrs 9 and 19, providing useful genetic markers for linkage studies.

Selective expression of intracisternal A-particle genes in established mouse plasmacytomas.

Mouse plasmacytomas generally express higher levels of RNA transcripts from endogenous intracisternal A-particle (IAP) proviral elements than do lipopolysaccharide-stimulated normal lymphocytes. Lymphocytes express a limited and highly characteristic set of IAP elements (lymphocyte-specific [LS] elements). In this study, we examined whether LS elements are expressed at higher levels after transformation of the cells and/or whether new IAP elements are activated. The IAP elements expressed in plasmacytoma MPC11 were characterized by sequence analysis of 22 cDNA clones. The long terminal repeats (LTRs) of the tumor cDNAs proved to be highly related in sequence. None of the clones was of the LS cDNA type. The MPC11 LTRs were five- to sixfold more active than an LS cDNA LTR when tested for promoter activity by transfection into plasmacytoma cells. The LTRs of the tumor-derived cDNAs contained a canonical ATF core sequence (ATF-PC), while the LS cDNAs contained an altered sequence (ATF-LS). An ATF-PC oligonucleotide probe detected multiple IAP transcripts on Northern (RNA) blots of RNA from several plasmacytoma but gave no reaction with RNA from stimulated B lymphocytes. In contrast, an ATF-LS probe detected higher levels of RNA in lymphocyte than in tumor RNAs. Thus, expression of IAP elements in transformed B cells is selective for a different set of regulatory sequence variants than those expressed in normal B cells. Other oligonucleotide probes representing LS- and PC-specific sequence variants detected multiple common hypomethylated IAP proviral loci in three independently derived plasmacytomas. Overall, the results show that established plasmacytomas exhibit a characteristic pattern of IAP proviral hypomethylation and regulatory sequence selection.

Genomic mapping of intracisternal A-particle proviral elements.

Intracisternal A-particle (IAP) proviral elements are moderately reiterated and widely dispersed in the mouse genome. Oligonucleotide probes have been derived from three distinctive IAP element subfamilies (LS elements) that are transcriptionally active in normal mouse B- and T-cells. In HindIII digests, LS element-specific oligonucleotides each react with a limited number of restriction fragments that represent junctions between proviral and flanking DNA. These fragments have characteristic strain distribution patterns (SDPs) which are polymorphic in the DNAs of different mouse strains. We have established chromosomal assignments for 44 LS proviral loci by comparing their SDPs with those of known genetic markers in the BXD set of RI mouse strains. Some of the loci have also been scored in the CXB RI set. The IAP LS loci can provide a significant number of markers with a recognized genetic organization to the mouse genome map.

Characterization of amplified intracisternal A-particle elements encoding integrase.

Type IIB intracisternal A-particle (IAP) elements have undergone marked amplification and transposition in the genomic DNA of some mouse myelomas. We have made a cDNA library from one such myeloma, MOPC 315, to determine whether some property of the elements themselves has a role in this process. Sequencing of several type IIB cDNAs and one genomic type IIB IAP element has shown that they are nearly identical (greater than 99%) and contain 2 open reading frames (ORFs). ORF2 is capable of encoding the IAP integrase, an enzyme which catalyzes integration of proviral DNA into the genome. An antiserum to a synthetic peptide based on the IAP integrase gene sequence reacted with ORF2 product expressed in bacteria as a fusion protein, and detected a 47 kDa protein, predicted from the size of ORF2, in myeloma cell fractions by Western blotting.

Nucleotide sequence of a complete mouse intracisternal A-particle genome: relationship to known aspects of particle assembly and function.

The 7,095-nucleotide sequence of a mouse genomic intracisternal A-particle (IAP) element, MIA14, is reported. MIA14 is known to be colinear with IAP 35S RNA and to contain functional long terminal repeats. Its internal genetic organization was determined by comparisons with a homologous Syrian hamster element and the related retroviruses simian retrovirus 1 (simian type D) and Rous sarcoma virus (avian type C). MIA14 contains a gag-protease open reading frame of 827 codons and a pol region of 867 codons entered by a frame shift of -1. The env region of 1,100 base pairs has multiple stop codons in all reading frames, consistent with the failure thus far to detect IAP-related glycosylated envelope components. RNA transcribed in vitro from a cDNA clone containing a closely homologous gag-protease open reading frame was translated in a cell-free system. The main product was a 73-kilodalton polypeptide immunoprecipitable with antiserum against the authentic IAP gag-related structural protein p73. Rather than ending at the gag-protease boundary, p73 appears to contain 7 to 8 kilodaltons of peptide encoded by the protease domain, a peculiarity possibly related to the observed impairment of normal protein processing in IAPs. The N-terminal 217 codons of gag are unique to murine IAPs and may have been contributed by recombination with a cellular gene. The mouse-specific region of gag encodes a hydrophobic signal peptide with an atypical cleavage site. Delayed cleavage of this peptide could result in anchoring of newly synthesized p73 to the endoplasmic reticulum membrane and restriction of particle assembly to this site.

Specific association between type-II intracisternal A-particle elements and other repetitive sequences in the mouse genome.

The majority of type-II intracisternal A-particle (IAP) element clones isolated from a mouse genomic library also contained highly repetitive DNA sequences in addition to the moderately repetitive IAP elements. Further analysis revealed that eleven of the twelve clones contained sequences of the mouse L1 family. One clone contained four copies of a limited region of the 3' end of the L1 element in a 12-kb stretch of sequence. This clone also contained a newly identified repetitive sequence which is found associated with type-II IAP elements. Type-II IAP elements were completely methylated in mouse embryo DNA; in myeloma cells, partial demethylation of the sequences correlated with known transcriptional activity of the IAP subclasses. Analysis of genomic DNA showed that association with other repetitive sequences appears to be a general property of many type-II IAP elements and may reflect their location in a particular chromosomal environment.

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.

Chromosome distribution of intracisternal A-particle sequences in the Syrian hamster and mouse.

Metaphase chromosomes of Syrian hamster and BALB/c mice were hybridized in situ with radiolabeled probes derived from cloned intracisternal A-particle (IAP) genes of the corresponding species. The DNAs of these species are known to contain about 900 and 1,000 copies, respectively, of the retrovirus-like IAP sequence elements per haploid genome. Multiple IAP sequences were found on all chromosomes of both hamster and mouse. In the hamster, more than half of the IAP sequences were located in regions of non-centromeric constitutive heterochromatin, at an average concentration per unit chromosome length 5 times greater than in the euchromatic regions. The other dispersed sequences showed marked local variations in concentration along the chromosome lengths; both discrete foci and large grain clusters were observed as well as regions apparently lacking IAP sequences. Within the resolution of the techniques, IAP sequences appeared to be more evenly distributed over the mouse chromosomes; however, some prominent variations in concentration were seen. The number of potentially active IAP genes in the Syrian hamster, and by extension in the mouse, may be restricted by the preferential location of IAP sequences in genetically inert regions of the genome.