Gene regulation by BCL3 in a cervical cancer cell line.

BCL3 is a putative proto-oncogene deregulated in haematopoieitic and solid tumours. It has been suggested that its oncogenic effects could be mediated, at least in part, by inducing proliferation and inhibiting cell death. To provide more insight into the mediators of these effects, we used an unbiased approach to analyse the mRNA expression changes after knocking-down BCL3 using specific shRNAs. One hundred eighty genes were up-regulated and sixtynine genes were down-regulated after knocking down BCL3. Function analyses showed enrichment in genes associated with cellular growth and proliferation, cell death and gene expression. We found that STAT3, an important oncogene in human cancer, was the central node of one of the most significant networks. We validated STAT3 as a bona fide target of BCL3 by additional interference RNA and in silico analyses of previously reported lymphoma patients.

ERK 1,2 and p38 pathways are involved in the proliferative stimuli mediated by urokinase in osteoblastic SaOS-2 cell line.

Bone metastases from prostate origin generate an osteoblastic reaction that is expressed in vitro by increased osteoblast proliferation. The urokinase-like plasminogen activator (u-PA) present in the media conditioned by tumoral prostatic cells acting as a ligand of the cellular membrane receptor (u-PAR), has been identified as the specific factor that modulates this proliferative reaction. The present study represents an effort to unravel the intracellular pathway by which u-PA activates osteoblastic proliferation and to evaluate the role of cellular receptor u-PAR in this proliferative phenomenon. Our results show that in vitro u-PA stimulates proliferation of SaOS-2 osteoblastic cells by activating the MAP kinase route of ERK 1 and 2 and the p38 pathway. These results are in accordance with the inhibition of intermediate activation and cell proliferation by PD 098059 and SB 203580, specific inhibitors of MEK and p38, respectively. We also show that SaOS-2 cells increase their proliferative response when cells are plated onto vitronectin, the second natural ligand of u-PAR, and that culturing SaOS-2 cells in the presence of u-PA represents a stimuli for u-PAR expression. On the basis of these results we propose that osteoblastic cells respond to the prostate-derived u-PA stimuli in a very efficient manner that includes the utilization of two different signaling routes and the stimulation of the expression of the u-PA receptor.

Mitochondrial DNA rearrangements: intracellular information system.

The extent of mtDNA rearrangements has been analyzed in nDNA preparations of rat and human with a statistically representative group of oligonucleotides directed to two regions of mtDNA: genes for cytochrome oxidase subunits I and III. Human PCR preparations generated with oligonucleotides directed 'normally' showed the expected fragment for mtDNA and the presence of a plethora of fragments with rearrangements (deletions and insertions), in contrast to rat PCR preparations under the same reaction conditions in which these kinds of rearranged fragments were rarely observed. Both human and rat PCR preparations generated with oligonucleotides directed 'inversely' showed numerous fragments, some of which showed differences in copy number correlating with distinct phases during development/aging. Sequence analysis of some normal and rearranged fragments demonstrated in all cases DNA sequences 99% homologous with other mtDNA sequences at rearranged fragments. No evidence of nuclear DNA sequences was found. The following scheme is proposed for mtDNA rearrangements during the lifetime of an organism: variation in copy number of some fragments with inversions of mtDNA depends on the specific developmental/aging period; in old cells there is an increase in higher molecular weight mtDNA deletions. These findings strongly suggest that the mtDNA rearrangements play a role as an intracellular 'information system'.

Analysis of the polyadenylation consensus sequence context in the genes of nuclear encoded mitochondrial proteins.

A compilation of the pre-mRNA ends of the genes of nuclear encoded mitochondrial proteins resulted in a consensus sequence of the type (T/A)NTTNNNNNTTTNAATAAA. Nucleotide positions +8, +13, +14, +16 and +17 downstream of the AATAAA sequence show also a predominance of nucleotide T. This consensus sequence suggests the importance of the immediate surroundings of the cannonical polyadenylation signal sequence AATAAA on the efficiency of the cleavage and polyadenylation of this specific group of pre-mRNAs.

Structure of the genes of two homologous intracellularly heterotopic isoenzymes. Cytosolic and mitochondrial aspartate aminotransferase of chicken.

The genes of mitochondrial and cytosolic aspartate aminotransferase of chicken were cloned and sequenced. In both genes nine exons encode the mature enzyme. The additional exon for the N-terminal presequence that directs mitochondrial aspartate aminotransferase into the mitochondria is separated by the largest intron from the rest of the gene. A comparison of the two genes of chicken with the aspartate aminotransferase genes of mouse [Tsuzuki, T., Obaru, K., Setoyama, C. & Shimada, K. (1987) J. Mol. Biol. 198, 21-31; Obaru, K., Tsuzuki, T., Setoyama, C. & Shimada, K. (1988) J. Mol. Biol. 200, 13-22] reveals closely similar structures: in the gene of both the mitochondrial and the cytosolic isoenzyme all but one intron positions are conserved in the two species and five introns out of nine are placed at the same positions in all four genes indicating that the introns were in place before the genes of the two isoenzymes diverged. The variant consensus sequence (T/C)11 T(C/T)AG at the 3' splice site of the introns of the genes for nuclear-encoded mitochondrial proteins, which had been deduced from a total of 34 introns [Juretic, N., Jaussi, R., Mattes, U. & Christen, P. (1987) Nucleic Acids Res. 15, 10,083-10,086], was confirmed by including an additional 22 introns into the comparison. The position -4 at the 3' splice site is occupied by base T in 43% of the total 56 introns and appears to be subject to a special evolutionary constraint in this particular group of genes. The following course of evolution of the aspartate aminotransferase genes is proposed. Originating from a common ancestor, the genes of the two isoenzymes intermediarily evolved in separate lineages, i.e. the ancestor eukaryotic and ancestor endosymbiontic cells. When endosymbiosis was established, part of the endosymbiontic genome, including the aspartate aminotransferase gene, was transferred to the nucleus. This process probably led to the conservation of certain splicing factors specific for nuclear-encoded mitochondrial proteins. The presequence for the mitochondrial isoenzyme was acquired by DNA rearrangement. In the eukaryotic lineage, the mitochondrial isoenzyme evolved more slowly than its cytosolic counterpart.

Evolutionary and biosynthetic aspects of aspartate aminotransferase isoenzymes and other aminotransferases.

The mitochondrial and cytosolic isoenzymes of aspartate aminotransferase are homologous proteins. Both are encoded by nuclear DNA and synthesized on free polysomes. The organization of their genes is very similar, five out of a total of eight introns are located at the same nucleotide position. A variant consensus sequence was observed at the 3' splice site of introns of genes of imported mitochondrial proteins which may reflect the existence of splicing factors specific for the genes of this particular group of nuclear-encoded proteins. To date the amino acid sequences of 22 aminotransferases are known. A rigorous analysis yielded clear evidence that aspartate, tyrosine, and histidinol-phosphate aminotransferases are homologous proteins despite their low degree of sequence identity. The evolutionary relationship among the vitamin B6-dependent enzymes in general appears less clear. Conceivably, their common structural and mechanistic features are dictated by the chemical properties of pyridoxal 5'-phosphate rather than being due to a common ancestor of their protein moieties. In agreement with this notion, the ubiquitous active-site lysine residue that forms a Schiff base with the coenzyme can be replaced in the case of aspartate aminotransferase by a histidine residue without complete loss of catalytic competence.

Structure of cDNA of cytosolic aspartate aminotransferase of chicken and its expression in E. coli.

The structure of the mRNA of chicken cytosolic aspartate aminotransferase has been determined by analysis of cDNA and genomic clones. Two transcripts of different length were found that appear to arise from the alternate use of 2 polyadenylation signals in the 3' untranslated region. The expression product of the full-length construct in E. coli proved to be catalytically active and possessed the expected molecular weight.

Genes of nuclear encoded mitochondrial proteins: evidence for a variant of the 3' splice-site consensus sequence.

The introns of animal nuclear genes and of viral genes encoding protein possess at their 3' splice-site the consensus sequence (CT)11NTCAG (Mount, S.M. (1982) Nucl. Acids Res. 10, 459-472; Green, M.R. (1986) Ann. Rev. Genet. 20, 671-708). However, the total 39 introns of the 5 imported mitochondrial proteins of higher eucaryotes whose gene structure has been determined to date show a predominance of 44% for base T at position -4. Apparently, a variant consensus sequence, i.e. (CT)11TTCAG, characterizes the genes of nuclear encoded mitochondrial proteins.

The primary structure of the precursor of chicken mitochondrial aspartate aminotransferase. Cloning and sequence analysis of cDNA.

The mitochondrial isoenzyme of aspartate amino-transferase (mAspAT; subunit Mr 45,000) is synthesized on free polysomes in the cytosol as a precursor of higher Mr (pre-mAspAT; Sonderegger, P., Jaussi, R., Christen, P., and Gehring, H. (1982) J. Biol. Chem. 257, 3339-3345). We have isolated three overlapping cDNA clones that correspond almost to the full length of pre-mAspAT mRNA with 100 nucleotides at the 5' end missing. The mRNA is 2.1 kilobase pairs long and has a 3' noncoding region of 0.7 kilobase pairs. The cDNAs code for the 401 amino acid residues of mAspAT plus an NH2-terminal pre-piece. Deviations from the reported amino acid sequence were found at positions 154 and 202 where the cDNA specifies Gln instead of Glu. The pre-piece consists of 22 amino acid residues, among them 4 arginine and no acidic residues.

Application of single radial immunodiffusion for qualitative comparison of plant virus particles.

The three fundamental types of precipitin patterns obtained with plant viruses by double radial immunodiffusion (DRID) tests can also be obtained by single radial immunodiffusion (SRID) tests. Using virus antigens that were serologically identical or unrelated, the reaction patterns were readily recognized. However, the precipitin reaction corresponding to the spur in DRID tests using virus antigens that were partially identical serologically was difficult to distinguish. It seems, therefore, that when virus isolates are serologically closely related, DRID should be preferred to SRID. Different viruses in a mixed infection can be successfully distinguished by SRID and also by 'rocket' immunoelectrophoresis.