Functional domains of wild-type and mutant p53 proteins involved in transcriptional regulation, transdominant inhibition, and transformation suppression.

The wild-type (wt) p53 protein has transcriptional activation functions which may be linked to its tumor suppressor activity. Many mutant p53 proteins expressed in cancers have lost the ability to function as transcriptional activators and furthermore may inhibit wt p53 function. To study the mechanisms by which mutant forms of p53 have lost their transactivation function and can act in a dominant negative manner, a structure-function analysis of both mutant and engineered truncated forms of p53 was carried out. We show that different mutant p53 proteins found in cancers vary in the ability to inhibit the transcriptional transactivation and specific DNA binding activities of wt human p53. This transdominant effect was mediated through the carboxy-terminal oligomerization region. The role of the transactivation activity in transformation suppression by wt p53 was also examined by constructing an N-terminal deletion mutant lacking the transactivation domain. This mutant was unable to transactivate but could bind specifically to DNA. Although it was impaired in its ability to suppress transformation of primary rat embryo fibroblasts by adenovirus E1A plus activated ras, the N-terminal deletion mutant still had some suppression activity, suggesting that additional functions of p53 may contribute to transformation suppression.

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.

The transcriptional transactivation function of wild-type p53 is inhibited by SV40 large T-antigen and by HPV-16 E6 oncoprotein.

The observed interaction between p53 and the oncoproteins encoded by several DNA tumor viruses suggests that these viruses mediate their transforming activities at least in part by altering the normal growth regulatory function of p53. In this study we examined the effect of viral oncoprotein expression on the transcriptional transactivation function of wild-type p53 in human cells. Plasmids expressing human p53 were cotransfected with either SV40 large T-antigen or human papillomavirus (HPV) type 16 E6 expression plasmids and assayed for transactivation function using a reporter gene driven by a p53-responsive promoter containing multiple copies of the consensus p53 DNA binding motif, TGCCT. Both large T-antigen and E6 were able to inhibit transactivation by wild-type p53. Furthermore, SV40 T-antigen mutants that are defective for p53 binding were not able to inhibit transactivation and HPV E6 proteins that were either mutant or derived from non-oncogenic HPV types and unable to bind p53, had no effect on p53 transactivation. These results demonstrate the physiological relevance of the interaction of SV40 T-antigen and HPV E6 oncoproteins with p53 in vivo and suggest that the transforming functions of these viral oncoproteins may be linked to their ability to inhibit p53-mediated transcriptional activation.

Selective activation of a discrete family of endogenous proviral elements in normal BALB/c lymphocytes.

Intracisternal A-particle (IAP) proviral elements are abundant and widely dispersed in the mouse genome. IAP-related transcripts have been detected in normal mouse tissues where expression is under genetic control. In this study, we sought to determine whether IAP expression in BALB/c thymus and lipopolysaccharide-stimulated B cells was due to selective or indiscriminate activation of IAP elements. cDNA libraries were prepared from each source. A total of 86 IAP cDNA clones were isolated from both libraries, and 37 of these were sequenced over a common 0.7- to 1.0-kb region of the IAP genome that included the 3' long terminal repeat (LTR). Three highly related families of elements were found to be expressed in the two cell types examined. All of the related elements had a distinctive U3 regulatory region. Thirteen individual IAP proviral elements were distinguished on the basis of sequence differences within the R region of the LTR. Hybridization of genomic DNA with element-specific oligonucleotide probes confirmed the presence of a restricted number of proviral copies in the lymphocyte-specific family of elements. Most of these copies were found to be methylated in the lymphocyte DNA, but at least seven were hypomethylated in their 5' LTRs. This study shows that activation of IAP elements in normal normal mouse lymphocytes is highly selective. Activation is probably a function of both sequence specificity and methylation status of the proviral LTR.

Intracisternal A-particle-specific oligonucleotides provide multilocus probes for genetic linkage studies in the mouse.

Oligonucleotide probes representing distinct intracisternal A-particle (IAP) subfamilies were derived from the long terminal repeats (LTRs) of transcriptionally active IAP genes in normal mouse cells. These probes were used to examine the distribution of IAP proviral elements in the genomic DNA of several inbred mouse strains. Each oligonucleotide probe identified multiple polymorphisms between the different strains. The distribution of polymorphic restriction fragments among the CXB set of recombinant inbred (RI) strains demonstrates the feasibility of using these probes for chromosome mapping. These and other subset-specific IAP probes can provide a useful series of multilocus markers for genomic mapping and genetic analysis in the mouse.

Tissue and strain-specific patterns of endogenous proviral hypomethylation analyzed by two-dimensional gel electrophoresis.

Proviral sequences related to the intracisternal A particle (IAP) are amplified and dispersed in the mouse genome. Their expression is associated with hypomethylation at CpG sites in the 5' long terminal repeat. We have used two-dimensional agarose gel electrophoresis to examine patterns of IAP hypomethylation in mouse DNA. The method is sensitive to both the methylation status of a conserved Hae II site in the 5' long terminal repeat and the location of the closest BamHI site in the flanking DNA upstream of each hypomethylated long terminal repeat. The method also defects restriction fragments derived from IAP elements that are themselves methylated but have an unmethylated Hae II site in their 5' adjacent DNA. DNAs from each of four inbred mouse strains (BALB/c, C3H/He, C57BL/6, and DBA/2) gave distinctive two-dimensional patterns of BamHI/Hae II restriction fragments detected by hybridization with an IAP probe. This constitutive pattern was largely conserved among several tissues of each strain, but some tissue-specific variations were observed. The site-specific hypomethylations reflected in the two-dimensional patterns were heritable properties, since DNA from progeny of an interstrain cross contained both parental sets of fragments. IAP elements may be useful endogenous reporters of genomic methylation patterns.

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.

Nearly identical members of the heterogeneous IAP gene family are expressed in thymus of different mouse strains.

Poly(A)RNAs prepared from the thymuses of C57BL/6J and DBA/2J mice were used to construct cDNA libraries in the bacterial expression vector lambda gt11. The libraries were scanned first for protein production with polyvalent antiserum prepared against the 73kDa gag protein of mouse intracisternal A-particles (IAP). Reactive plaques were crossed-screened by hybridization with an IAP-specific DNA probe. Two IAP-specific protein-producing plaques were obtained from the C57BL/6 library and 4 from the DBA/2 library. One C57BL/6 cDNA clone (B12) and two DBA cDNA clones (D8 and D20) were sequenced in their entirety. Clones B12 and D8 were remarkably similar, particularly when compared to the 6 other IAP elements that have been sequenced thus far. We discuss the evidence which leads us to suggest that these clones may be derived from allelic IAP elements expressed in mouse thymus.

cDNA clones encoding murine IgE-binding factors represent multiple structural variants of intracisternal A-particle genes.

Previously [Moore, K. W., Jardieu, P., Mietz, J. A., Trounstine, M. L., Kuff, E. L., Ishizaka, K. & Martens, C. L. (1986) J. Immunol. 136, 4283-4290], we examined a T-hybridoma-derived cDNA clone, 8.3, that encodes a biologically active murine IgE-binding factor (IgE-BF), and we showed that it was a variant member of the endogenous retroviral gene family related to mouse intracisternal A particles (IAPs). We have now characterized four more IgE-BF cDNA clones by heteroduplex and restriction enzyme analysis and found that they all represent different structural variants of the full-size IAP genomic element. In clones 8.3 and 10.2, which have been fully sequenced, the open reading frames span deletions 3.4 and 1.9 kilobases (kb) long, respectively, and specify different gag-pol fusion polypeptides. Clone 9.5 contains a 2.1-kb deletion entirely within the pol region. Two other clones (4.2 and 11.7) contain no internal deletion and may represent truncated cDNA copies of full-size (7.2 kb) IAP gene transcripts. Structural variants very similar to clone 10.2 are common in the mouse genome, and clone 9.5 is also probably not a unique gene form. The sequences of clones 8.3 and 10.2 are different in detail, but each is closely homologous to a randomly cloned mouse genomic IAP element throughout the gag-related portions of their open reading frames. Antibodies against two oligopeptides specified by the sequence of clone 8.3 immunoprecipitated IAP-related proteins from mouse neuroblastoma and myeloma cells, confirming that the IgE-BF produced by this clone shares sequence with expressed IAP elements in different cell types. Thus, information related to the IgE-BF is an integral part of the murine IAP retrotransposon gag gene.

Rodent IgE-binding factor genes are members of an endogenous, retrovirus-like gene family.

Synthesis of IgE by B lymphocytes can be regulated by soluble lymphocyte factors which have affinity for the Fc region of IgE (IgE-binding factors). In previous studies, we identified cDNA clones encoding rodent IgE-binding factors by direct expression in transfected mammalian cells. Here we show that IgE-binding factor cDNA clone 8.3 is a member of the endogenous, retrovirus-like intracisternal A-particle gene family of the mouse. This conclusion is supported by blot hybridization, DNA sequence comparisons, heteroduplex analysis, and immunochemical cross-reactivity of the encoded proteins. The results identify a member of this highly reiterated gene family with a role in regulation of the allergic immune response.

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.