Epigenetic inhibition of lysyl oxidase transcription after transformation by ras oncogene.

Lysyl oxidase is an extracellular enzyme involved in connective tissue maturation that also acts as a phenotypic suppressor of the ras oncogene. To understand how this suppressor is controlled, gene transcription was studied and the promoter was characterized. Nuclear runoff transcription assays indicated that the markedly reduced amounts of lysyl oxidase message detected after ras transformation resulted from inhibition of lysyl oxidase transcription. Interferon-mediated phenotypic reversion of ras transformed cells, in which the ras oncogene continued to be expressed, was accompanied by the restoration of lysyl oxidase transcription. Reporter gene assay of a transfected mouse lysyl oxidase promoter indicated that it was active in the transformed background, despite the silencing of the endogenous lysyl oxidase promoter. The detection of comparable amounts of mRNA for transcription factors IRF-1 and IRF-2 in normal and ras-transformed cell lines suggests that the differential transcription of lysyl oxidase was not due to regulation of IRFs. Lysyl oxidase promoter activity was localized to a 126 bp region that includes two consensus TATA boxes with associated confirmed cap signals. Analysis of a human lysyl oxidase promoter sequence indicated similar promoter elements and extensive sequence identity with the mouse promoter. The binding of transcription factor AP2 to sites predicted in the control region was confirmed by DNase footprinting. Lysyl oxidase transcription was stimulated by dexamethasone treatment of cells, but this effect could not be assigned within the approximately 3 kb region tested in reporter gene constructs. The promoter activity of the lysyl oxidase reporter gene construct was completely abolished by in vitro DNA methylation, suggesting that the transcriptional suppression after transformation by the ras oncogene may involve DNA methylation.

Reversion by deletion of transforming oncogene following interferon-beta and retinoic acid treatment.

We have previously shown that prolonged interferon-beta (IFN-beta) treatment of RS485 cells (NIH3T3 cells transformed with multiple copies of an LTR-cHa-ras oncogene) resulted in the phenotypic reversion of 1%-5% of the culture, depending on the conditions used. This reversion persisted after IFN-beta was discontinued, although the revertants retained the LTR-cHa-ras and continued to express ras mRNA and p21. Clones were prepared of such persistent revertant cell lines (PRs). Expression of lysyl oxidase (LOX), which appears to act as a suppressor of ras transformation, was downregulated in RS485 and upregulated in the PRs. When retinoic acid (RA) was combined with IFN-beta treatment of the RS485 cultures, a different mechanism of reversion predominated. Following 60 days of treatment with 20 IU/ml of IFN-beta and 10 microM RA, all of the multiple (3-5) copies of the transforming LTR-c-Ha-ras originally present in RS485 cells were deleted from the genome in 72% of 54 revertant cell lines isolated. As in the case of revertants observed after treatment with IFN-beta alone, LOX mRNA expression was upregulated in all of the revertants that resulted from the treatment with IFN plus RA. The level of LOX mRNA expression acts, therefore, as an indicator of transformation in this system.

Modulation of ras transformation affecting chromatin supraorganization as assessed by image analysis.

Changes in chromatin supraorganization defined in terms of patterns of chromatin texture were studied by video image analysis in Feulgen-stained revertants of LTR-ras-transformed NIH 3T3 cells and in cell lines obtained by transfection of these revertants with sense and antisense constructs of the lysyl oxidase gene (also named Lox or "ras recision gene"). The objective was to determine whether changes in expression of the Lox gene, which have been assumed to modulate cell transformation by ras, could also affect the chromatin supraorganization changes known to be elicited in NIH 3T3 cells by ras transformation. The image analysis results revealed that, although a nuclear phenotype visually similar to the most frequent one (III) in ras-transformed NIH 3T3 cells also appeared in the revertant, it contained a remarkably less tight chromatin packing state. This situation was also found in the revertant transfected with the sense construct of the Lox gene, but in the revertant transfected with the Lox antisense constructs the chromatin texture of the III phenotype was equal to or close to that of the ras-transformed cells. With regard to the nuclear phenotype characterized by abundant loosely packed chromatin and less represented in the transformed cell lines (I'), changes in the various cell lines, although detectable, were not as drastic as those reported for the III phenotype. The enhancement in chromatin condensation of the type III nuclei, which affects euchromatin, is probably associated with a limited transcription of the genome. Although the image analysis results are mostly in agreement with previously published data on the molecular biology and tumorigenicity of the same cell lines, it appears that the phenomenon of chromatin condensation once established in NIH 3T3 cells by LTR-ras transformation could not be totally reverted by simply affecting Lox expression.

A novel human cDNA with a predicted protein similar to lysyl oxidase maps to chromosome 15q24-q25.

A novel human cDNA with a predicted protein homologous to the carboxyl end of lysyl oxidase, an extracellular enzyme involved in the maturation of collagen and elastin, has been isolated. The homology to lysyl oxidase begins exactly at the position of the exon 1/exon 2 boundary in the mouse gene (Contente, S., Csiszar, K., Kenyon, K., and Friedman, R. M. (1993) Genomics 16, 395-400). This lysyl oxidase-like gene, which appears to be no larger than 22.1 kilobases, codes for a single polyadenylated RNA species of 2.48 kilobases and has been mapped to chromosome 15q24-q25.

Structure of the mouse lysyl oxidase gene.

Lysyl oxidase, an extracellular enzyme involved in the maturation of collagen and elastin, also appears to function as a phenotypic suppressor of transformation by the ras gene product, p21. Genomic clones of the mouse lysyl oxidase gene have been isolated, analyzed, and sequenced. Lysyl oxidase appears to be a single-copy gene, organized into seven exons and six introns, and spans approximately 14 kb of the mouse genome. The gene encodes two messages, sized at about 4.8 and 3.8 kb, that differ in the length of the untranslated sequence at the 3' end of the gene. All of the 3' untranslated sequence and the polyadenylation signals are contained in exon VII; there is no evidence of alternate splicing. Primer extension and ribonuclease protection experiments revealed two sites of transcription initiation in a region with sequence motifs characteristic of a promoter, which was upstream and adjacent to the 5' untranslated sequence found in the cDNA.

Expression of gene rrg is associated with reversion of NIH 3T3 transformed by LTR-c-H-ras.

A partial complementary DNA was isolated for a gene (rrg) that is normally expressed in mouse NIH 3T3 cells, but is down-regulated after cellular transformation by long terminal repeat (LTR)-activated c-H-ras (LTR-c-H-ras). This gene was reexpressed in a nontumorigenic persistent revertant cell line created by prolonged treatment of the transformed cells with mouse interferon alpha/beta. Persistent revertants stably transfected with rrg complementary DNA antisense expression vectors appeared transformed, had decreased amounts of rrg messenger RNA, and were tumorigenic in nude mice. Stable transfection with sense constructs did not alter the normal morphology, message level, or nontumorigenicity of the persistent revertant cell line.

Differential stringent control of the tandem E. coli ribosomal RNA promoters from the rrnA operon expressed in vivo in multicopy plasmids.

The tandem P1, P2 promoter region of the rrnA ribosomal operon has been fused to the t1, t2 terminator region of the rrnB operon in pBR322 plasmid derivatives. This deletes most internal RNA structural elements ordinarily processed out of ribosomal operon transcripts. In vivo as well as in vitro transcripts arising from both promoters terminate predominantly in the t1 terminator region about 40 base pairs beyond the mature rrnB 5S RNA gene. Stringent control of the P1 and P2 promoted transcripts has been assessed in vivo. In these plasmid fusions, the upstream (P1) promoter activity was subject to stringent control, while the downstream (P2) promoter activity was inhibited by amino acid starvation in both stringent and relaxed hosts. A plasmid with an additional deletion of the P2 region also showed stringent regulation of the P1 promoter.

Replication and incompatibility properties of plasmid pE194 in Bacillus subtilis.

pE194, a 3.5-kilobase multicopy plasmid, confers resistance to the macrolide-lincosamide-streptogramin B antibiotics in Bacillus subtilis. By molecular cloning and deletion analysis we have identified a replication segment on the physical map of this plasmid, which consists of about 900 to 1,000 base pairs. This segment contains the replication origin. It also specifies a trans-acting function (rep) required for the stable replication of pE194 and a negatively acting copy control function which is the product of the cop gene. The target sites for the rep and cop gene products are also within this region. Two incompatibility determinants have been mapped on the pE194 genome and their properties are described. One (incA) resides within the replication region and may be identical to cop. incB, not located in the replication region, expresses incompatibility toward a copy control mutant (cop-6) but not toward the wild-type replicon.

Molecular cloning of heterologous chromosomal DNA by recombination between a plasmid vector and a homologous resident plasmid in Bacillus subtilis.

The difficulty experienced in the shotgun cloning of chromosomal DNA on plasmid vectors in Bacillus subtilis is analyzed and an explanation for this difficulty is offered based on an inherent property of competent cells which imposes a requirement of plasmid multimers in transformation of plasmid-free recipients (Canosi et al., 1978). A stratagem which uses cloning by recombination between the vector and a resident homologous plasmid is tested and shown to be successful. Several recombinant plasmids are obtained containing Bacillus licheniformis DNA fragments which complement aromatic amino acid mutants of Bacillus subtilis. The yield of recombinant clones ranges from 6.7 to 210 per microgram of chromosomal DNA, depending on the selection and the restriction endonuclease. The various trp clones obtained after cutting chromosomal DNA with BglII and BclI do not complement trpE and exhibit both orientations with respect to the vector. The location of several restriction endonuclease cleavage sites in the cloned trp fragments is presented, and their relationship to the genetic map of Bacillus licheniformis is described.

Characterization of plasmid transformation in Bacillus subtilis: kinetic properties and the effect of DNA conformation.

Transformation of competent cells of Bacillus subtilis with antibiotic resistance plasmid DNA has shown that (a) competence for plasmid and chromosomal DNA develops with similar kinetics; (b) DNA linearized with a variety of restriction endonucleases does not transform; (c) CCC plasmid DNA is inactivated for transformation by a single nick; (d) T4 ligase restores transforming activity to both nicked and linearized DNA; (E) CCC relaxed DNA is fully active in transformation; (f) the DNA concentration-dependence of plasmid transformation is first order; and (g) plasmid transformation proceeds with a low efficiency, requiring the uptake of 10(3) to 10(4) DNA molecules per transformant. Based on this information, a model for the processing of chromosomal, plasmid and transfecting DNA is proposed.

Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis.

Covalently closed circular DNA from five Staphylococcus aureus plasmids has been introduced into Bacillus subtilis. Four of these plasmids (pUB110, pCM194, pSA2100, and pSA0501) have been selected for further study. These plasmids replicate as multicopy autonomous replicons in both Rec+ and Rec- B. subtilis strains. They may be transduced between B. subtilis strains or transformed at a frequency of 10(4) to 10(5) transformants per microgram of DNA. The molecular weights of these plasmids were estimated, and restriction endonuclease cleavage site maps are presented. Evidence is given that pSA2100, an in vivo recombinant of pSA0501 and pCM194 (S. Iordanescu, J. Bacteriol. 124:597-601, 1975), arose by a fusion of the latter plasmids, possibly by insertion of one element into another as a translocatable element. Genetic information from three other S. aureus plasmids (pK545, pSH2, and pUB101) has also been introduced into B. subtilis, although no covalently closed circular plasmid DNA was recovered.