Kira K. Lueders

The intracisternal A-particle gene family: structure and functional aspects

Publisher Summary This chapter reviews the various aspects of research on intracisternal type A particles (IAPs)—expression in early development, genomic sequences as chromosomal genes and transposable elements, general molecular biology, and proteins as neoantigen in autoimmune diabetes. IAPs are defective retrovirions encoded by the members of a large family of endogenous proviral elements. The particles assemble on the membranes of the endoplasmic reticulum and bud into the cisternae. IAP genetic elements transposes in the genome of mouse tumor cells and also in the germ line of several mouse strains. Most of the tranpositions are found because they affect the function of genes at the target sites. IAP transpositions are a source of genetic variability and as such may contribute to the process of neoplastic transformation. The chapter is useful to investigators who encounter IAP-related proteins and are concerned directly with the role of endogenous transposable elements in normal development or neoplasia. Although IAP expression is a common accompaniment of transformation in mouse cells, there is no evidence for a direct causal relationship between the two phenomena of transformation or tumor progression—that is, IAP proviral elements are not known to have acquired cellular oncogenes or to integrate regularly at preferred sites near cellular transforming elements.

Spontaneous and detergent activation of a glucuronyltransferase in vitro

Abstract The microsomal glucuronyltransferase catalyzing formation of p-nitrophenyl glucuronide in rat, guinea pig, and mouse livers, and transplantable rat hepatomas, was activated spontaneously during storage at 0 ° and by low concentrations (less than 0.05%) of deoxycholate and Triton X-100 added in vitro. Activity in liver microsomes was increased 5- to 10-fold, in hepatoma microsomes 20-fold, and in homogenates 2-to 5-fold. Higher concentrations of detergent were inhibitory to activity in fresh preparations, and preparations which had been fully activated spontaneously were inhibited by all concentrations of detergents. The activation was irreversible and not accompanied by solubilization of the enzyme. The Michaelis constants (for uridine diphosphate glucuronic acid) for detergent- and spontaneously activated liver and hepatoma microsomes appeared to be the same. Attempts to determine the mechanism of activation by kinetic analysis were unsuccessful, but the data were compatible with an increase in the relative site number per milligram microsomal protein during activation.

Sequences associated with intracisternal a particles are reiterated in the mouse genome

Using a 3H-cDNA for RNA sequences specifically associated with murine intracisternal type A particles, we have found multiple copies of this information in high molecular weight nuclear DNA from tissues of both Mus muscules (BALB/c, NIH Swiss, A/Jax and feral) and Mus cervicolor. Reiteration frequencies varied from 1050-1800 per haploid genome, except that fewer copies (450) were found in BALB/3T3 cells. In the series studied, the reiteration frequencies in the DNA of A particle-rich tumor cells (myeloma and neuroblastoma) were not higher than those in normal tissues (liver and sperm). Multiple copies were retained when cellular DNAs were sedimented through alkaline sucrose gradients, indicating that the sequences are integrated in the mouse genome. In situ hybridization with cDNA showed that the sequences were associated with many chromosomes and were concentrated over certain regions of some chromosomes. Only low levels of homologous sequences were detected in rat, hamster and guinea pig DNA under stringent conditions of hybridization. The presence of reiterated sequence transcripts in poly(A) RNA from a neuroblastoma A particle fraction was confirmed by direct hybridization of the RNA with cellular DNA.

Homology between an endogenous viral LTR and sequences inserted in an activated cellular oncogene

Recently, some of us reported1 the detection and molecular cloning of a rearranged cellular oncogene, designated rc-mos, from a non-virally-induced mouse myeloma, XRPC24. Recombinant λ phage DNA containing the rc-mos gene was active in transforming NIH 3T3 cells in a transfection assay, whereas recombinant DNA containing the unrearranged c-mos gene was not. In rc-mos, coding sequences from the 5′ end of c-mos were found to have been displaced by a novel cellular element whose nucleotide sequence was reported. We now document the fact that a 349-base pair (bp) segment of the novel DNA immediately adjacent to the retained c-mos sequences in rc-mos has close homology with the long terminal repeat (LTR) of a known intracisternal A-particle gene. This homology was mentioned in Nature recently2 after it had been brought to the attention of the editors (N. Hozumi and R. Hawley, personal communication).

Comparison of the sequence organization of related retrovirus-like multigene families in three evolutionary distant rodent genomes

Sequences related to mouse intracisternal A-particle (IAP) genes have been isolated from rat and Syrian hamster gene libraries as recombinants in lambda phage. The sequences are moderately reiterated in both these genomes but their sequence organization in the hamster genome is different from that in the rat genome. Restriction analysis and electron microscopy indicate that the Syrian hamster IAP sequences represent a family of relatively homogeneous well-conserved units; in this, they resemble the mouse IAP genes. The rat sequences, in contrast, are heterogeneous. Both the hamster and rat IAP sequences contain regions homologous to mouse IAP genes interspersed with regions of apparent non-homology. The interspersed regions range in size from 0.5-1.0 kilobases (Kb). The regions of homology among the mouse, rat and Syrian hamster IAP sequences have been mapped to a 5-6 Kb internal region on the mouse IAP genes. Mouse IAP long terminal repeat (LTR) sequences were not detected in the rat and Syrian hamster genomes. We used the thermal stability of hybrids between cloned and genomic IAP sequences to measure family homogeneity. Mouse and Syrian hamster IAP sequences are homogeneous by this criterion, but the rat IAP sequences are heterogeneous with a Tm 6 degrees C below the self-hybrid. The contrasting organization of IAP-related elements in the genomes of these rodents indicates that amplification or homogenization of this sequence family has occurred independently and at different periods of time during their evolution.

AN EDITED LINKAGE MAP FOR THE AXB AND BXA RECOMBINANT INBRED MOUSE STRAINS

Abstract. 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.

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.

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.

RNA sequences specifically associated with mouse intracisternal A particles

Abstract Intracisternal type A particles were purified from mouse myeloma line MOPC-104E, and the particle-associated polyadenylated RNA was transcribed with AMV DNA-polymerase. The kinetics of hybridization of the 3 H-cDNA with its template RNA revealed an abundant class of sequences comprising about 55% of the total A particle cDNA. The fraction of 3 H-cDNA representing this class of sequences was isolated by selective low Crt hybridization and used to quantify the sequences in other RNA preparations. The entire class of sequences, having a genetic complexity of 2-2.5 × 10 6 daltons, was many-fold more concentrated in A particle-rich tumors of three different cell types (myeloma, rhabdomyosarcoma and neuroblastoma) than in several cell lines that are apparently devoid of particles. In MOPC-104E, the A particle-specific sequences comprised nearly 8% of the total cytoplasmic poly(A) RNA. These sequences were further concentrated in A particle fractions prepared from each of the various tumors. In A particle-containing fractions prepared rapidly from exponentially growing neuroblastoma cells, the specific sequences were associated principally with RNA molecules sedimenting in the range of 26S–32S. There was no detectable homology between the class of specific A particle sequences defined here and a set of sequences common to several murine type C viruses.

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.