Proteins tightly bound to DNA: new data and old problems.

Proteins tightly bound to DNA (TBP) comprise a group of proteins that remain bound to DNA after usual deproteinization procedures such as salting out and treatment with phenol or chloroform. TBP bind to DNA by covalent phosphotriester and noncovalent ionic and hydrogen bonds. Some TBP are conservative, and they are usually covalently bound to DNA. However, the TBP composition is very diverse and significantly different in different tissues and in different organisms. TBP include transcription factors, enzymes of the ubiquitin-proteasome system, phosphatases, protein kinases, serpins, and proteins of retrotransposons. Their distribution within the genome is nonrandom. However, the DNA primary structure or DNA curvatures do not define the affinity of TBP to DNA. But there are repetitive DNA sequences with which TBP interact more often. The TBP distribution within genes and chromosomes depends on a cell's physiological state, differentiation type, and stage of organism development. TBP do not interact with DNA in the sites of its association with nuclear matrix and most likely they are not components of the latter.

Familial form of hirschsprung disease: nucleotide sequence studies reveal point mutations in the RET proto-oncogene in two of six families but not in other candidate genes.

Hirschsprung disease (HSCR; McKusick 142623) or aganglionic megacolon is a frequent (1 in 5,000 live births) heritable disorder of the enteric nervous system. By haplotyping with a variety of microsatellite markers, by amplifying all 20 exons of the RET proto-oncogene and by applying a direct DNA sequencing protocol, we have analyzed the DNA from HSCR patients in 6 different families. In one family with a joint occurrence of HSCR and FMTC (follicular medullary thyroid carcinoma), we have identified a mutation in codon 609 in one out of 6 cysteine residues encoded in exon 10 of the RET gene. This C609R point mutation has not previously been reported to cause HSCR. In 2 of the HSCR patients described here from different families, we have found a mutation in exon 2 (R77C) and a silent mutation in exon 3 (Y204Y), respectively, in the extracellular part of the RET proto-oncogene. In introns 2 and 17 of the RET proto-oncogene in 2 families, we have detected single nucleotide exchanges that are probably polymorphisms with unknown, if any, relations to HSCR. The DNA sequences of 5 further genes (GDNF, GDNFRalpha, EDN3, EDNRB, and NTN), that may contribute to the development of HSCR, have not shown mutations in the patients analyzed so far. In 2 of the reported families with several affected children and one grandchild, sequence analyses revealed no mutations in the coding regions of any of the candidate genes analyzed.

Treatment of experimental autoimmune encephalomyelitis with adenylate deaminase from Penicillium lanoso-viride.

The effect of intramuscularly administered immunomodulator, adenylate deaminase (E.C. 3.5.4.6), from Penicillium lanoso-viride on the clinical score of acute experimental autoimmune encephalomyelitis (EAE), a T cell-mediated autoimmune disease, was examined by inoculation of guinea pigs with rabbit brain and spinal cord homogenate (encephalitogen) and complete Freund's adjuvant. Adenylate deaminase (ADA) was effective in delaying the onset of clinical disease. ADA inhibited the severity of EAE. There was a significant decrease in clinical signs. A decrease in the number of morbid and dead animals was observed. Of ADA treated animals, 50-80% developed no clinical manifestations of EAE. The optimal version of treatment was a single preventive injection of ADA 1 day before the sensitization and then every second day after immunization for 20 days. ADA treatment of immunized animals diminished the activity of 2', 3'-cyclic nucleotide 3'-phosphodiesterase in the cerebrospinal fluid, as well the amount of complement fixing antiencephalitogenic antibodies in the blood serum. The mechanism of ADA cerebroprotective action is discussed. Significant skin-allergic cross-reaction of delayed-type hypersensitivity between ADA and encephalitogen was observed.

Efficient control of raf gene expression by CAP and two Raf repressors that bend DNA in opposite directions.

The plasmid-borne raf operon of Escherichia coli encodes proteins involved in the uptake and utilisation of the trisaccharide raffinose. The operon is subject to dual regulation; to negative control by the binding of RafR repressor to twin operators, O1 and O2, and to positive control by the cAMP-binding protein, CAP. We have identified the CAP binding site (CBS) as a 22 bp palindromic sequence with incomplete dyad symmetry by deletion analysis, DNasel footprinting and electrophoretic mobility shift assays (EMSA) of CAP-DNA complexes. The CBS is centred 60.5 bp upstream of the transcription start point and partially overlaps O1. In vivo, CAP increases rafA (alpha-galactosidase) gene expression up to 50-fold. The 28 bp spacing between the centres of CBS and the - 35 box is essential, since insertions of 4, 8, 12 or 16 bp completely eliminated rafA gene expression. In vitro binding studies revealed that the CBS, O1 and O2 sites, can be simultaneously occupied by their cognate proteins. However, no cooperativity between binding of CAP and RafR was detected. EMSA with circularly permuted DNA fragments demonstrated that CAP and RafR proteins bend raf promoter (rafP) DNA by 75 degrees +/- 5 degrees and 95 degrees +/- 5 degrees, respectively, in opposite directions. Among sugar catabolic operons, the compact arrangement of three protein-binding sites, a CBS and two operators bounding the - 35 promoter box, is unique and provides a sensitive and highly efficient device for transcriptional control.

Phytoremediation of trichloroethylene with hybrid poplars.

Axenic tumor cultures of poplar cells, clone H11-11, were grown in the presence of [14C]-trichloroethylene (TCE) (uniformly labeled). The cells were capable of metabolizing TCE to produce trichloroethanol, di- and trichloroacetic acid. Some of the carbon from TCE was found in insoluble, nonextractable cell residue, and small amounts were mineralized to [14C]CO2. Poplar cuttings grown in soil and exposed to TCE produced the same metabolites. In field trials, trees were planted in soil in test cells and exposed to TCE via underground water injection during the growing season. During the growing season, at least 95% of the TCE was removed from the influent water stream in cells containing trees. Mass balance studies conducted in the laboratory indicated that 70 to 90% of the TCE was transpired; however, greenhouse and field study results showed that less than 5% of the total TCE taken up by the plants is transpired. These results show that significant TCE uptake and degradation occur in poplars. Poplars appear to be useful for in situ remediation of TCE-contaminated sites under proper conditions.

DNA fragments with specific nucleotide sequences in their single-stranded termini exhibit unusual electrophoretic mobilities.

DNA restriction fragments, 120-650 base pairs (bp) in length, with 5'-GCGC-3', 5'-GGCC-3' or 3'-GCGC-5' single-stranded overhanging termini, give rise to diffuse bands of unusual electrophoretic mobility in non-denaturing polyacrylamide gels. This shift in electrophoretic mobility can be observed at 4-12 degreesC, not at higher temperatures, but is stabilized by 5-10 mM Mg2+, even at 37 degreesC. The nucleotide sequence in the abutting double-stranded part of the fragment does not affect this phenomenon, which is not caused by dimerization. The altered mobility may be due to the unusual terminal DNA structure, which is dependent on co-operative interactions among more than two neighboring G and C residues. The structure is stabilized by cytidine methylation. The biological role of such fragment structures in DNA repair and recombination is presently unknown.

Protective effect of adenylate deaminase (from Penicillium lanoso-viride) against acute infections in mice.

We examined the effects of the immunomodulator-adenylate deaminase (E.C. 3.5.4.6) from Penicillium lanoso-viride on experimental mice infections. Prophylactic intraperitoneal administration of adenylate deaminase (ADA) increased survival time and numbers of survivors after infection with Salmonella typhimurium, Pseudomonas aeruginosa and influenza A (H3N2) virus. Protection against influenza virus after intranasal ADA application was also observed. The influence of ADA was time and dose dependent. The most pronounced protection was obtained by administration of 3 U ADA/mice 24 h prior to infection. ADA had no antibiotic effect against these bacterial strains. Protective effects of ADA were studied in immunosuppressed mice under different regimes of treatment including cyclosporin A and trypan blue. The results indicated, that the protective effect of ADA is of a complex nature and probably depends on both T-cell and macrophage components.

The topology of the promoter of RNA polymerase II- and III-transcribed genes is modified by the methylation of 5'-CG-3' dinucleotides.

In eukaryotic cells, RNA polymerase II- and III-transcribed promoters can be inactivated by sequence-specific methylation. For some promoter motifs, the introduction of 5-methyldeoxycytidine (5-mC) residues has been shown to alter specific promoter motif-protein interactions. To what extent does the presence of 5-mC in promoter or regulatory DNA sequences affect the structure of DNA itself. We have investigated changes in DNA bending in three naturally occurring DNA elements, the late E2A promoter of adenovirus type 2 (Ad2) DNA, one of our main model systems, the VAI (virus-associated) RNA gene of Ad2 DNA, and an Alu element associated with the human angiogenin gene. Alterations in electrophoretic mobility of differently permuted promoter segments in non-denaturing polyacrylamide gels have been used as assay system. In the late E2A promoter of Ad2 DNA, a major and possibly some minor DNA bending motifs exist which cause deviations in electrophoretic mobility in comparison to coelectrophoresed marker DNA fragments devoid of DNA bending motifs. DNA elements have been specifically in vitro methylated by the HpaII (5'-CCGG-3'), the FnuDII (5'-CGCG-3'), or the CpG DNA methyltransferase from Spiroplasma species (M-SssI; 5'-CG-3'). Methylation by one of these DNA methyltransferases influences the electrophoretic mobility of the three tested promoter elements very strikingly, though to different extents. It cannot be predicted whether sequence-specific promoter methylation increases or decreases electrophoretic mobility; these changes have to be experimentally determined. Methylation of the E. coli dcm (5'-CCA/TGG-3') sites in some of the DNA constructs does not make a contribution to mobility changes. It is concluded that sequence-specific methylations in promoter or regulatory DNA elements can alter the bending of DNA very markedly. This parameter may contribute significantly to the silencing of promoters, probably via altering spatial relationships among DNA-bound transcription factors.

The impact of 5'-CG-3' methylation on the activity of different eukaryotic promoters: a comparative study.

The inhibiting or inactivating effects of position-specific promoter methylation in different viral or human cellular promoters Ad2 E2AL, SV40, LTR-MMTV, HSV-tk, TNF alpha) have been compared by in vitro 5'-CCGG-3' methylation by M-HpaII or the M-SssI DNA-methyltransferase, respectively. In most promoters, 5'-CG-3' methylation reduces activity to a few percent of that of mock-methylated controls. The number of 5'-CG-3' dinucleotides in a promoter does not strictly correlate with the extent of methylation inhibition. The LTR-MMTV promoter, which lacks 5'-CG-3' dinucleotides, is not affected by methylation. The late E2A promoter of Ad2 DNA cannot be inactivated by 5'-CCGG-3' methylation when the construct carries the strong cytomegalovirus enhancer devoid of this sequence. In contrast, 5'-CG-3' methylation shuts this promoter off even in the presence of this enhancer.

Role of two operators in regulating the plasmid-borne raf operon of Escherichia coli.

The plasmid-borne raf operon encodes functions required for the inducible uptake and utilization of raffinose in Escherichia coli K12. The expression of three structural genes for alpha-galactosidase (rafA), Raf permease (rafB) and sucrose hydrolase (rafD) is negatively controlled by the binding of RafR repressor (rafR) to two operator sites, O1 and O2, that flank the -35 sequence of the raf promoter, PA. In vitro, O1 and O2 are occupied on increasing the concentration of RafR, without detectable preference for one site or the other or any indication of cooperative binding. Nucleotide substitutions at positions 3, 4 or 5 in an operator half-site prevented repressor binding, supporting a model that postulates specific interactions of these base pairs with the recognition helix of RafR. To study the role of each operator site, we have compared by gel shift analysis the binding of purified RafR repressor to DNA fragments containing the original O1O2 configuration or mutant O1 or O2. When either one of the two operators was inactivated by site-directed mutagenesis, both O1 and O2 exhibited the same affinity for repressor and the same sensitivity to arrest of repressor binding by the natural inducer, melibiose. However, in the native O1O2 configuration, simultaneous binding of RafR to both operators was sterically hindered, leading to a 13-fold decrease in the intrinsic affinity of an operator site for repressor, once the other site had been occupied. To assess the role of each operator in vivo, rafA was used as a reporter gene. A 1200-fold repression (100%) was exerted by RafR binding to the native O1O2 configuration, whereas O2 alone exerted 45% and O1 alone 6% repression of rafA transcription. The differential effects of O1 versus O2 on transcription (despite matching affinities of O1 and O2 for repressor) suggest that positioning of the O2-repressor complex between the -35 and -10 signals is crucial for transcription control and that repressor binding to the upstream O1 serves to enhance this effect.

Successive binding of raf repressor to adjacent raf operator sites in vitro.

The raf repressor negatively regulates the transcription of the raf operon which encodes functions required for the uptake and hydrolysis of raffinose in Escherichia coli. Overexpression of the repressor gene under lac promoter control led to the formation of inclusion bodies. These were partially purified by centrifugation, solubilized in 0.1% SDS and reactivated by dilution. DNase I protection and gel retardation experiments demonstrated the specific binding of raf repressor to DNA fragments that contained the previously identified raf operator, an element comprising two 18 bp palindromic nucleotide sequences that flank the -35 raf promoter box. By using DNA fragments with one, two, or four copies of the 18 bp palindrome, these experiments revealed concentration dependent, successive occupation of all available binding sites by raf repressor. Melibiose released the repressor from the operator complexes, whereas raffinose and other alpha-galactosides did not, indicating that melibiose is the actual inducer in vivo. We suggest that successive occupation by repressor of two strategically located operator sites is a specific type of stepwise down-regulation of gene expression in response to repressor concentration.

Ultracentrifugal analysis of the quaternary structure of the raf repressor from Escherichia coli.

The raf repressor from Escherichia coli regulates the expression of the plasmid-borne raf operon by switching between active and inactive conformational states. Ultracentrifugal analysis of the largely purified repressor proves the DNA-free protein to undergo concentration-dependent dissociation-association. High-speed sedimentation equilibria show that the 72 kDa dimer prevails under meniscus depletion conditions. At intracellular concentrations the 144 kDa dimer-of-dimers is the dominating species. It is suggested that the tetrameric structure of the raf repressor is involved in the recognition of the 18-basepair operator DNA.