Sense, antisense, and common sense.
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DNA↗
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Cooperative and non-cooperative DNA binding modes of catabolite control protein CcpA from Bacillus megaterium result from sensing two different signals.
Carbon catabolite repression (CCR) of several operons in Bacillus subtilis and Bacillus megaterium is mediated by the cis-acting cre sequence and trans-acting catabolite control protein (CcpA). We describe purification of CcpA from B. megaterium and its interaction with regulatory sequences from the xyl operon. Specific interaction of CcpA with cre as scored by DNase I footprints at concentrations similar to the in vivo situation requires the presence of effectors. We have found two molecular effectors for CcpA activity, which lead to different recognition modes of DNA. The heat-stable phosphotransfer protein HPr from the PTS sugar uptake system triggers non-cooperative binding of CcpA to cre when phosphorylated at Ser46 (HPr-Ser46-P). Glucose 6-phosphate (Glc-6-P) triggers cooperative binding of CcpA to cre and two auxiliary cre* sites, one of which overlaps the -35 box of the xyl promoter. Binding to cre* depends on the presence of the functional cre sequence. A mutation in cre abolishes carbon catabolite repression in vivo and binding of CcpA to cre and cre* in vitro, indicating looping of the intervening DNA. The two triggers are not simultaneously active. The acidity of the buffer determines which of them activates CcpA when both are present in vitro. Glc-6-P is preferred at pH values below 5.4, and HPr-Ser46-P is preferred at neutral pH. The Ccpa dimers present at neutral pH form tetramers and higher oligomers at pH 4.6, explaining cooperativity of binding to DNA. CcpA is the first member of the LacI/GalR family of regulators, for which oligomerization without the leucine zipper at the C terminus is demonstrated.
Bacillus megaterium↗
Banana bunchy top virus DNA-2 to 6 are monocistronic.
Banana bunchy top virus (BBTV) DNA-3 to 6 have each previously been shown to contain one large open reading frame in the virion sense, whereas no large ORF had been identified in BBTV DNA-2. RNAs transcribed from the BBTV genome were mapped using northern hybridisation and 3' RACE. One mRNA was transcribed from each of BBTV DNA-2 to 6 and four of these mRNAs mapped to the ORFs previously identified in BBTV DNA-3 to 6. The mRNA of BBTV DNA-2 was transcribed from a virion sense ORF probably using a TATA box sequence different to that in BBTV DNA-1, and DNA-3 to 6. This ORF encoded a 10 kDa protein of unknown function. The 3' untranslated region of the five mRNAs varied from 25 nucleotides (BBTV DNA-6) to 167 nucleotides (BBTV DNA-4) and each contained putative polyadenylation signals with associated GT rich sequence together with a possible termination signal (C/T/A)TGTAA conserved in all five mRNAs.
3' Untranslated Regions↗
Simultaneous mutagenesis of multiple sites: application of the ligase chain reaction using PCR products instead of oligonucleotides.
A method is described for preparing mutants with multiple, site-directed mutations by ordered coupling of PCR-generated fragments catalyzed by a thermostable DNA ligase. Annealing of the sense strands of the fragments to a single-stranded (antisense) template created a full-length sense strand leaving only nicks that were closed by ligation. Mutations were introduced in the PCR primers. Following 40 cycles of denaturation and annealing, tags on the flanking primers of the ligase chain reaction product were used specifically to amplify the mutated product with specific primers that could not amplify the original template. The amplified ligation product was cloned and was found to contain the desired restriction sites introduced by way of the mutagenic primers.
Bacillus↗
Order and disorder in biophysical systems: a study of the correlation between structure and function of DNA.
The concept of order and disorder as complementary in the quantum-mechanical sense is presented. Negative entropy is introduced as a measure of the order of the system under study--namely, the DNA chain--and temperature as a measure of its disorder. A Heisenberg-like uncertainty relationship for order and disorder phenomena is derived. The concept of quasi-equilibrium is introduced because living matter is peculiar in that under physiological conditions there is no equilibrium in the strict sense. A new approach for DNA activity is suggested in terms of local or general disorder of the chain.
Biophysical Phenomena↗
Increased growth of NIH/3T3 cells by transfection with human p120 complementary DNA and inhibition by a p120 antisense construct.
The human nucleolar antigen p120 was detected with an anti-p120 monoclonal antibody in most human malignant tumors but not in most resting human tissues (J. W. Freeman et al., Cancer Res., 48: 1244-1251, 1988) and has been used as a prognostic tumor marker in breast cancer patients (J. W. Freeman et al., Cancer Res., 51: 1973-1978, 1991). After the complementary DNA and gene for the human p120 protein were isolated and sequenced (review: H. Busch, Cancer Res., 50: 4830-4838, 1990), constructs were prepared to study the expression of the sense p120 and its antisense, p021 message. NIH/3T3 cells were transfected by electroporation with pSVX plasmids containing either the p120 complementary DNA (pSVX120) or the antisense, p021 DNA (pSVX021), and clones containing these constructs were selected. The expression of p120 or p021 in these constructs was regulated by Moloney murine leukemia virus long terminal repeats. In pSVX120-transfected NIH/3T3 cells, the expressed human p120 protein was localized to the nucleoli as shown by anti-p120 monoclonal antibody immunofluorescence. Expression of the p120 message and protein was confirmed by Northern (mRNA) and Western (protein) blots. Transfection of the p120 complementary DNA in sense orientation caused malignant transformation of NIH/3T3 cells in vitro and produced rapidly growing tumors in nude mice. Transfection of the antisense p120 constructs markedly delayed the growth of these tumors in vitro and in vivo (L. Perlaky et al., Proc. Am. Assoc. Cancer Res., 32: 1682, 1991). When transformed 3T3/pSVX120 cells were transfected with an inducible antisense p120 construct (pMSG021), dexamethasone induction decreased the growth rate by 62%, and the cell line returned to its normal phenotype. Northern blot analysis showed a decreased level of p120 mRNA, and the immunofluorescence was also markedly reduced.
3T3 Cells↗
The primary structure of chicken B-creatine kinase and evidence for heterogeneity of its mRNA.
cDNA clones for chicken B-CK were isolated by immunoscreening from a gizzard cDNA library constructed in the expression vector lambda gtll. The entire coding portion in addition to the complete 3' untranslated region and 42 bp of the 5' noncoding part are represented in the clone H4. On RNA blots H4 insert DNA hybridized to a 1600 bp poly(A)+ RNA from gizzard, brain and heart but not to breast or skeletal muscle RNA. In vitro generated sense strand transcripts of H4 insert DNA were translated in vitro into a protein indistinguishable from isolated, authentic B-CK. The distinct nucleotide sequences of H4 insert DNA and M-CK cDNA were translated into 82% homologous amino acid sequences. Sequence heterogeneity among the B-CK cDNA clones within both the 3' noncoding and even in the coding region indicates the existence of multiple B-CK mRNA species.
Amino Acid Sequence↗
Design of asymmetric DNAzymes for dynamic control of nanoparticle aggregation states in response to chemical stimuli.
Dynamic control of nanomaterial assembly states in response to chemical stimuli is critical in making multi-component materials with interesting properties. Previous work has shown that a Pb2+-specific DNAzyme allowed dynamic control of gold nanoparticle aggregation states in response to Pb2+, and the resulting color change from blue aggregates to red dispersed particles can be used as a convenient way of sensing Pb2+. However, a small piece of DNA (called invasive DNA) and low ionic strength (approximately 30 mM) were required for the process, limiting the scope of application in assembly and sensing. To overcome this limitation, a series of asymmetric DNAzymes, in which one of the two substrate binding regions is longer than the other, has been developed. With such a system, we demonstrated Pb2+-induced disassembly of gold nanoparticle aggregates and corresponding color change at room temperature without the need for invasive DNA, while also making the system more tolerant to ionic strength (33-100 mM). The optimal lengths of the long and short arms were determined to be 14 and 5 base pairs, respectively. In nanoparticle aggregates, the activity of the DNAzyme increased with decreasing ionic strength of the reaction buffer. This simpler and more versatile system allows even better dynamic control of nanoparticle aggregation states in response to chemical stimuli such as Pb2+, and can be used in a wider range of applications for colorimetric sensing of metal ions.
Base Sequence↗
Transcriptional analysis and genome expression of chicken anaemia virus.
Strand-specific riboprobes representative of either strand of the chicken anaemia virus (CAV) replicative form (RF) DNA indicated that only one strand of the RF was transcribed to produce a major 2.0 kb transcript and that the encapsidated DNA strand was of negative sense. Primer extension analysis located a single transcriptional start site at nucleotide position 360 of the CAV sequence. Amplification, cloning and sequencing of the 3' end of the major transcript revealed the polyadenylation site at nucleotide position 21. Northern blot analysis using a series of genomic probes indicated that the 2.0 kb transcript was devoid of splicing and identified a non-transcribed region of the genome. This non-transcribed region was shown to possess promoter activity, enhancing the expression of the human growth hormone reporter gene in a transient gene expression assay. These observations suggest a simple strategy of genome expression involving a functional polycistronic message.
Animals↗
The antiactivator TraM interferes with the autoinducer-dependent binding of TraR to DNA by interacting with the C-terminal region of the quorum-sensing activator.
Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by quorum sensing via the transcriptional activator TraR and the acyl-homoserine lactone Agrobacterium autoinducer (AAI). Unique to this system, the activity of TraR is negatively modulated by an antiactivator called TraM. Analyses from yeast two-hybrid studies suggest that TraM directly interacts with the activator, but the conditions under which these components interact and the region of TraR responsible for this interaction are not known. Induction of traM in a strain in which TraR was activating transcription of a reporter system led to rapid cessation of gene expression. As assessed by a genetic assay that measures AAI-dependent DNA binding, TraM inhibited TraR function before and after the transcription factor had bound to its DNA recognition site. Consistent with this observation, in gel retardation assays, purified TraM abolished the DNA binding activity of TraR in a concentration-dependent manner. Such inhibition occurred independent of the order of addition of the reactants. As assessed by far Western analyses TraM interacts with TraR by directly binding the activator. TraM in its native form interacted with native TraR and also with heat-treated TraR but only when SDS was included with the denatured protein. TraM interacted with TraR on blots prepared with total lysates of cells grown in the presence and absence of AAI. Far Western analysis of N- and C-terminal deletion mutants localized a domain of TraR contributing to TraM binding to the C-terminal portion of the activator protein. Random mutagenesis by hydroxylamine treatment and error-prone polymerase chain reaction identified several residues in this region of TraR important for interacting with TraM as well as for transcriptional activation or/and DNA binding. We conclude that TraM inhibits TraR by binding to the activator at a domain within or close to the helix-turn-helix motif located at the C terminus of the protein.
Agrobacterium tumefaciens↗
ATM signaling and 53BP1.
The ATM (mutated in Ataxia-Telangiectasia) protein kinase is an important player in signaling the presence of DNA double strand breaks (DSBs) in higher eukaryotes. Recent studies suggest that ATM monitors the presence of DNA DSBs indirectly, through DNA DSB-induced changes in chromatin structure. One of the proteins that sense these chromatin structure changes is 53BP1, a DNA damage checkpoint protein conserved in all eukaryotes and the putative ortholog of the S. cerevisiae RAD9 protein. We review here the mechanisms by which ATM is activated in response to DNA DSBs, as well as key ATM substrates that control cell cycle progression, apoptosis and DNA repair.
Ataxia Telangiectasia Mutated Proteins↗
Characterisation of independent DNA and multiple Zn-binding domains at the N terminus of human DNA-(cytosine-5) methyltransferase: modulating the property of a DNA-binding domain by contiguous Zn-binding motifs.
We report here a detailed mapping and characterisation of a DNA-binding domain at the N terminus of human DNA-(cytosine-5) methyltransferase. A small region, B1 (codon 202 to 369), was first identified by its Zn- and gross DNA-binding properties. Further fine-mapping using deletion and point mutation analysis shows that the DNA- and Zn-binding domains involve separate peptide motifs, KRRKTTPKEPTEKK (codons 202 to 215) for a bipartite DNA-binding oligopeptide (DB1) and CX2CX13HX2D(X)23EX2EX13CX3H (codons 232 to 297) for possibly two contiguous Zn-binding domains (AZn), which can function independently. However, B1 (containing DB1 and AZn) differs from DB1 because it does not bind to a 30 base-pair duplex. Interestingly, H3 codons 202 to 974, which encloses B1 and B2 (containing the Zn-binding CX2CX2CX4CX2CX2C motif from codon 533 to 550) binds preferentially to 0.8 kb duplexes, as compared with 0.4 to 0.6 kb duplexes. As the homologous murine B1, which targets the murine methylase to replication foci, also binds to DNA and Zn, it is possible that the N terminus of mammalian methylase may be involved in sensing the appropriate length of newly synthesized DNA before methylation by its C terminus. This may enable a time delay for the transient existence of hemi-methylation sites for their unknown biological functions in mammals.
Amino Acid Sequence↗
A PCR-derived, non-isotopic labeled prolactin cRNA probe suitable for in situ hybridization.
We have developed a novel vector-free method for the synthesis of nonisotopic (digoxigenin) labeled prolactin (PRL)-gene specific cRNA probe based on the direct in vitro transcription of DNA template amplified by polymerase chain reaction (PCR). The T7 and T3 RNA polymerase promoters were incorporated into the amplified DNA by including the promoter sequences in the '5 end of the oligonucleotides used to prime the PCR. These promoters allowed the subsequent transcription of digoxigenin labeled antisense and sense cRNA probes from the amplified DNA. We successfully utilized these probes to detect specific PRL mRNA in human pituitary and decidua tissues by in situ hybridization (ISH) which can provide identification and localization of PRL-gene expression at a single cell level. This approach avoided time consuming steps which required subcloning of target DNA into the vectors that contains bacteriophage RNA polymerase promoter as well as the need for radioactive materials. This non-isotopic ISH procedure takes less than 72 h from specimen preparation to microscopic analysis and should prove to be useful for molecular biological studies of hormones and clinical diagnosis.
Base Sequence↗
Loss of function and p53 protein stabilization.
Wild-type (wt) p53 protein is rapidly degraded, has a short half-life and low intracellular levels. Stabilization of wt p53 protein following an appropriate stimulus (for example DNA damage) is a physiological regulation to increase function. In contrast, stabilization of p53 protein in the absence of a stimulus is always a hallmark of loss of function secondary to a mutation, or interaction with viral or cellular oncoproteins. It is generally accepted that stability of p53 protein depends on its intrinsic biochemical properties such as conformation or protein/protein interactions. However, I will discuss evidence that the stability of p53 is not a consequence of its intrinsic properties, but instead is determined by feedback control of its function. In the absence of an appropriate stimulus, a cell needs to keep p53 levels low, since increased levels can lead to apoptosis. To precisely regulate p53 levels, a cell must sense its level; and sensing its transactivating function, is the simplest way to sense p53. Following an appropriate stimulus (for example, DNA damage), the cell senses a state of 'relative' p53 deficiency and adapts by reducing p53 degradation. When the state of p53 deficiency is a consequence of a mutation or interaction with viral oncoproteins, the cell does not sense p53, and again attempts to adapt by reducing p53 degradation. However, in the latter case, the increase in levels does not restore function, and the adaptation continues until degradation of p53 protein is maximally inhibited. In this case, no further inhibition of degradation is possible after DNA-damage or pharmacological inhibition of proteasomes. Thus lack of wt p53 function always results in increased p53 levels and nonregulation.
Hydrolysis↗
A neo-Darwinian algorithm: asymmetrical mutations due to semiconservative DNA-type replication promote evolution.
Evolution is, in a sense, to resolve optimization problems. Our neo-Darwinian algorithm based on the mechanics of inheritance and natural selection uses double-stranded DNA-type genetic information to resolve the "knap-sack problem." The algorithm with asymmetrical mutations due to semiconservative DNA-type replication most effectively resolved the problem. Our results strongly suggest that disparity in mutations caused by the asymmetric machinery of DNA replication promotes evolution, in particular of diploid organisms with a high mutation rate, in a small population, and under strong selection pressure.
Algorithms↗
Forkhead/winged-helix transcription factor Whn regulates hair keratin gene expression: molecular analysis of the nude skin phenotype.
The molecular characteristics of the nude phenotype (alopecia and thymic aplasia) in humans and rodents are unknown. The nude locus encodes Whn, a transcription factor of the forkhead/winged-helix class. Expression of Whn in HeLa cells induced expression of human hair keratin genes Ha3-II and Hb5. Correspondingly, in nude mice, which are homozygous for a loss-of-function mutation of Whn, expression of mouse mHa3 and mHb5 hair keratin genes is severely reduced. Characterization of a previously identified nude allele, nu(Y), revealed a mis-sense mutation (R320C) in the DNA binding domain of Whn. This mutant protein is unable to activate hair keratin gene expression in HeLa cells. When the Whn transcription factor was expressed in two parts, one containing the N-terminal DNA binding domain and the other the C-terminal activation domain, no activation of hair keratin genes in HeLa cells was observed. However, when these two proteins were noncovalently linked by means of synthetic dimerizers, hair keratin gene expression was induced. This finding suggests that target gene activation by Whn depends on the structural integrity and physical proximity of DNA binding and activation domains, providing a molecular framework to explain the loss-of-function phenotypes of all previously characterized nude mutations. Our results implicate Whn as a transcriptional regulator of hair keratin genes and reveal the nude phenotype as the first example of an inherited skin disorder that is caused by loss of expression rather than mutation of keratin genes.
Animals↗
Viral infection. I. Regulation of protein synthesis during vaccinia viral infection of animal cells.
Regulation of vaccinia viral infection was studied using three animal cell lines: KRC-7 (rat hepatoma), L929 (mouse fibroblast), and CV-1 (African green monkey kidney). KRC-7 is highly enriched in p67, a glycoprotein which protects eIF-2 alpha-subunit from phosphorylation by eIF-2 kinases. We report: (i) At 5 pfu per cell of the virus, KRC-7 is resistant to the virus. Other cells are sensitive. At 25 pfu per cell of the virus, KRC-7 is also sensitive to the virus. After productive viral infection, the cell extracts showed strong p67-DG activity and actively deglycosylated exogenous p67. After p67-deglycosylation, the cell extracts also phosphorylated eIF-2. (ii) The rate of synthesis of a major host protein (approximately 45 kDa) in infected L929 cells measured after 2 h of viral infection declined more than 50%. The rate declined thereafter. The rate of synthesis of host proteins in viral-resistant KRC-7 cells (infected with 5 pfu per cell of the virus) remained unchanged. The mechanism of resistance of KRC7 cells to vacinia virus at 5 pfu per cell of the virus was investigated. The p67 level in these cells was varied by growing the cells under different physiological conditions such as serum starvation and expression of p67-sense and p67-antisense DNA. At low p67 level in the cells, p67-DG is activated. This deglycosylates p67 and inactivates p67. This accompanies eIF-2 phosphorylation and shutoff of host protein synthesis. At high p67 level in the cells, activation of p67-DG is prevented. This prevents shut-off of host protein synthesis and viral growth.
Acetylglucosaminidase↗
Expression of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) in inflammation-associated corneal neovascularization.
Matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) are all implicated in the development of neovascularization. To investigate the possible role of these factors in corneal neovascularization we have analysed the expression of MMP-2, MMP-9 and VEGF in a rat model of inflammation-associated corneal neovascularization. In this model, corneal neovascularization was induced in Long-Evans rats by krypton laser photocoagulation whereafter eyes were enucleated at 1, 4, 7, 10 and 20 days. Slit-lamp biomicroscopy and histologic analysis revealed a gradual development of corneal neovascularization that peaked 7-10 days after treatment when newly formed vessels could be seen throughout the corneal surface reaching deep into the stroma. Antisense and sense riboprobes were generated using DNA complementary to MMP-2, MMP-9 and VEGF, and mRNA expression was analysed using in situ hybridization. The expression of MMP-2 and MMP-9 in untreated corneas was low or absent whereas VEGF was weakly expressed in the corneal epithelium. MMP-2 expression was increased during corneal neovascularization and was mainly localized to the cells infiltrating areas of new vessel formation. Many of these cells appeared to be inflammatory cells. VEGF expression had a similar overall distribution to MMP-2 during neovascularization with the exception that its expression in the corneal epithelium remained and even increased slightly. MMP-9 was prominently expressed at the border of regenerating corneal epithelium in areas with epithelial wounding but was not detected in the vascularized stroma. Together, the results of the present study support a role for MMP-2 and VEGF in inflammation-associated corneal neovascularization whereas MMP-9 instead appears to be involved in corneal epithelial wound-healing.
Animals↗