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PubMed2001

Agrin differentially regulates the rates of axonal and dendritic elongation in cultured hippocampal neurons.

In the present study, we examined the role of agrin in axonal and dendritic elongation in central neurons. Dissociated hippocampal neurons were grown in the presence of either recombinant agrin or antisense oligonucleotides designed to block agrin expression. Our results indicate that agrin differentially regulates axonal and dendritic growth. Recombinant agrin decreased the rate of elongation of main axons but induced the formation of axonal branches. On the other hand, agrin induced both dendritic elongation and dendritic branching. Conversely, cultured hippocampal neurons depleted of agrin extended longer, nonbranched axons and shorter dendrites when compared with controls. These changes in the rates of neurite elongation and branching were paralleled by changes in the composition of the cytoskeleton. In the presence of agrin, there was an upregulation of the expression of microtubule-associated proteins MAP1B, MAP2, and tau. In contrast, a downregulation of the expression of these MAPs was detected in agrin-depleted cells. Taken collectively, these results suggest an important role for agrin as a trigger of the transcription of neuro-specific genes involved in neurite elongation and branching in central neurons.

Agrin↗
PubMed1977

Poly(A)-binding RNAs from nuclei and polysomes of BHK-21 cells.

The presence in nuclei and cytoplasm of cultured BHK-21/C13 cells (baby hamster fibroblasts) of RNA species with high affinity for poly(A) was detected using either a Millipore-poly(A)-binding assay or columns of poly(A)-Sepharose 4B. The nuclear species which labels rapidly is metabolically unstable and appears to be a specific subclass of heterogenous nuclear RNA. Digestion with T1 RNAase gives rise to a low level (1%) of oligonucleotide fragments of a disperse size range which are relatively rich in uridylate residues. The cytoplasmic species with affinity for poly(A) is similar in size to polyadenylated messenger RNAs and also associates with polysomes. However it appears to be distinct from the polyadenylated messenger RNAs by virtue of an unusual base composition and relative metabolic instability.

Binding Sites↗
PubMed1989

A protein target site in an early replicated human DNA sequence: a highly conserved binding motif.

We have previously reported that a human nuclear factor, probably corresponding to the USF/MLTF protein [1,2], is able to bind specifically to a DNA sequence present in DNA replicated at the onset of S-phase [3]. Here we demonstrate that the same factor binds also to several other similar sequences, present in eukaryotic and viral genomes. Mutations or methylation in a CpG dinucleotide, central in the palindromic binding site, completely abolish binding. Furthermore, we present evidence for the existence of at least two other nuclear proteins in human cells with the same DNA binding specificity. The data presented suggest a strong evolutionary conservation, among distantly related organisms, of the binding motif, which is probably the target of a number of nuclear factors that share the same DNA binding specificity albeit in the context of different functions.

Base Composition↗
PubMed1994

Equilibrium analysis of ethidium binding to DNA containing base mismatches and branches.

In the processes of DNA replication, recombination, and repair, duplex DNA can transiently form branched structures, such as Holliday junctions, as well as base pair mismatches and bulges. These stages have altered ligand and protein binding properties from normal double helical DNA. A variety of ligands have been reported to interact more tightly at branches and bulges than to normal duplex sites. The stoichiometry, structural basis, and thermodynamics of this effect have not been determined. We have investigated the binding of the intercalator, ethidium bromide, to several DNA constructs including base mismatches, bulges, and three- and four-arm branched structures, using chemical footprinting, titration calorimetry, and fluorescence lifetime measurements. Two classes of binding sites are detected in three- and four-arm junctions in our high ionic strength conditions: one class is characterized by a small number of ligands (2-4 per DNA), with high binding affinity (K > 10(5)), and the second by a larger number of sites (10-12 per DNA) with lower affinity (K approximately 10(4)). By use of appropriate control experiments, the former appear to be associated with sites at or near the branch point or mismatch, while the latter are consistent with binding to the normal duplex DNA region(s) of the molecule. Titration calorimetry indicates an enthalpy of -10 to -13 kcal/mol for binding of ethidium to a mismatch or three- and four-arm branch point. The tight binding class is associated with a fluorescence lifetime of 12-16 ns, distinct from that of free ethidium (ca. 2 ns) and the longer lifetime observed for ethidium intercalated in duplex DNA (22-26 ns).

Base Composition↗
PubMed1990

Single-strand DNA triple-helix formation.

Chemical modification studies provide evidence that single-stranded oligodeoxyribonucleotides can form stable intrastrand triple helices. Two oligonucleotides of opposite polarity were synthesized, each composed of a homopurine-homopyrimidine hairpin stem linked to a pyrimidine sequence which is capable of folding back on the hairpin stem and forming specific Hoogsteen hydrogen bonds. Using potassium permanganate as a chemical modification reagent, we have found that two oligodeoxyribonucleotides of sequence composition type 5'-(purine)8(N)4(pyrimidine)8(N)6(pyrimidine)8-3' and 5'-(pyrimidine)8N6(pyrimidine)8N4(purine)8-3' undergo dramatic structural changes consistent with intrastrand DNA triple-helix formation induced by lowering the pH or raising the Mg2+ concentration. The intrastrand DNA triple helix is sensitive to base mismatches.

Base Sequence↗
PubMed1991

Cloning of cDNAs encoding a rabbit renal brush border membrane protein immunologically related to band 3. Sequence similarity with microsomal dipeptidase.

Distinct anion transport processes have been identified in the mammalian renal proximal tubule, but none of the responsible proteins or genes have been isolated. A 43 kDa rabbit microvillus membrane protein that is immunologically related to the erythroid anion exchanger (band 3) was a candidate for a renal anion transporter. To examine the structural relationship with band 3, we cloned cDNAs encoding the 43 kDa protein. The 43 kDa band-3-like protein was purified, and a novel sequence of 24 amino acids was obtained from the N-terminus. Degenerate oligonucleotides were synthesized based on this sequence, and the polymerase chain reaction with single-sided specificity was used to amplify and clone a 1330 bp cDNA from rabbit renal cortex. Additional overlapping 272 bp and 1123 bp cDNAs were obtained by synthesizing and screening a rabbit renal cortical cDNA library. The composite sequence was 1483 bp, terminated with (A)16, and was similar in size to the principal transcript expressed in rabbit renal cortex. The single long open reading frame was predicted to encode a protein composed of 410 amino acids with a molecular mass of 45,193 Da; 15 amino acids predicted to reside at the N-terminus were absent in the mature protein and may constitute a signal peptide. There was only limited sequence similarity with human erythroid band 3. Rather, the sequence was highly similar to microsomal dipeptidase, including the presence of a signal peptide and a consensus sequence for covalent linkage to glycosylphosphatidylinositol. In summary, the 43 kDa protein from rabbit renal cortex that is recognized by a monospecific antibody to erythroid band 3 is most likely a microvillus membrane dipeptidase.

Amino Acid Sequence↗
PubMed1990

Identification and characterization of a regulated promoter element in the epidermal growth factor receptor gene.

We have identified a 36-base-pair proximal element (-112 to -77 relative to the AUG translation initiation codon) in the epidermal growth factor receptor 5' region that functions as a promoter; mediates inductive responses to epidermal growth factor, phorbol 12-myristate 13-acetate, and cyclic AMP; and acts in an orientation-independent manner. This region functions as an enhancer when transferred to a heterologous promoter containing a TATA box. Mutations within the 36-base-pair region alter function as assayed by reporter gene expression in recipient cells. A protein has been identified that demonstrates appropriate binding specificity to mutant DNA sequences that correlates with promoter activity observed in vivo. On the basis of DNA binding characteristics and size, the identified protein appears distinct from several previously identified transcription factors known to bind to G+C-rich regions.

Base Composition↗
PubMed1989

Comparison of RNase A, a chemical cleavage and GC-clamped denaturing gradient gel electrophoresis for the detection of mutations in exon 9 of the human acid beta-glucosidase gene.

Gaucher disease (GD), which results from mutations in the human acid beta-glucosidase (beta-Glc) gene, was used as a model system to compare the utility of three methods capable of detecting single base substitutions. PCR-amplified beta-Glc exon 9 sequences of GD patients were screened for single base mutations by GC-clamped denaturing gradient gel electrophoresis (DGGE) and RNase A cleavage of RNA-DNA heteroduplexes, and by chemical (hydroxylamine/osmium tetroxide) cleavage of dsDNA heteroduplexes. PCR products showing abnormal behaviour were cloned and sequenced. Three new point mutations were detected by this strategy. A G to C (Asp409 to His409) substitution was present in two Type 1 and one Type 3 GD patients; an A to T transversion (Asp409 to Val409) was detected in only a single Type 3 individual, and a G to T mutation (Val394 to Leu394) was present in one Type 1 and one Type 3 patient. GD thus exhibits extensive molecular heterogeneity, with at least five single base mutations in beta-Glc exon 9. In every case verified by ASO hybridization, DGGE had correctly identified the presence of the three new mutations, as well as the two previously described exon 9 mutations. In comparison, although RNase A and the chemical method were both able to detect some of these mutations, neither method reproducibly detected all of them. Additionally, DGGE was the only method that was able to reliably determine whether a given mutation was present homozygously or heterozygously. These results suggest that GC-clamped DGGE may be a more reliable and informative screening method for point mutation detection.

Base Composition↗
PubMed1996

Secondary structure of the r(CUUCGG) tetraloop.

The oligonucleotide r(GGACUUCGGUCC) has been observed to adopt a hairpin conformation by solution NMR and a double helical conformation by X-ray diffraction. In order to understand this apparent conflict, we used time-resolved fluorescence depolarization and 19fluorine NMR to follow the secondary structure of this dodecamer as the solution composition was changed stepwise from the NMR experimental conditions to those used for crystallization. Calculation of the dodecamer concentration in the crystal (180 mM strands) and the cation concentration needed for neutrality (>2 M) prompted investigation of a tethered species, in which two dodecamers are connected by a string of 4 nt, geometrically equivalent to approximately 100 mM strands, in 2.5 M NaCl. The RNA tetraloop and its DNA analog maintain a single-strand hairpin conformation in solution, even under the conditions used to grow the crystal. Under high salt conditions, the tethered RNA and DNA analogs of this sequence yield secondary components which could be the double helical conformation. Crystal contacts in addition to solvent changes and high RNA concentrations are needed to obtain the double helix as the predominant species.

Chemical Phenomena↗
PubMed1991

Critical chemical features in trans-acting-responsive RNA are required for interaction with human immunodeficiency virus type 1 Tat protein.

The human immunodeficiency virus type 1 Tat protein binds to an RNA stem-loop structure called TAR which is present at the 5' end of all human immunodeficiency virus type 1 transcripts. This binding is centered on a bulge within the stem of TAR and is an essential step in the trans-activation process which results in a dramatic increase in viral gene expression. By analysis of a series of TAR derivatives produced by transcription or direct chemical synthesis, we determined the structural and chemical requirements for Tat binding. Tat binds well to structures which have a bulge of two to at least five unpaired bases bounded on both sides by a double-stranded RNA stem. This apparent flexibility in bulge size is in contrast to an absolute requirement for an unpaired uridine (U) in the 5'-most position of the bulge (+23). Substitution of the U with either natural bases or chemical analogs demonstrated that the imido group at the N-3 position and, possibly, the carbonyl group at the C-4 position of U are critical for Tat binding. Cytosine (C), which differs from U at only these positions, is not an acceptable substitute. Furthermore, methylation at N-3 abolishes binding. While methylation of U at the C-5 position has little effect on binding, fluorination reduces it, possibly because of its effects on relative tautomer stability at the N-3 and C-4 positions. Thus, we have identified key moieties in the U residue that are of importance for the binding of Tat to TAR RNA. We hypothesize that the invariant U is involved in hydrogen bond interactions with either another part of TAR or the TAR-binding domain in Tat.

Base Composition↗
PubMed1991

Skeletal actin mRNA increases in the human heart during ontogenic development and is the major isoform of control and failing adult hearts.

Expression of the two sarcomeric actins, alpha-skeletal and alpha-cardiac, is regulated in the rodent heart in response to developmental, hormonal, and hemodynamic stimuli. Little is known in man, except that both isogenes were found to be coexpressed in three adult ventricles. In this report, we investigated the isoactin mRNA composition in ventricles from 21 control patients (4 fetal, 5 juvenile, 12 adult) and from 15 patients undergoing cardiac transplantation (5 idiopathic dilated cardiomyopathies, 5 ischemic myopathies with myocardial infarcts, 5 diverse etiologies) by two different and complementary techniques: RNA dot blot analysis with specific cDNA probes, and primer extensions with an oligonucleotide common to alpha-cardiac and alpha-skeletal actins. In the case of dot blot analysis, quantification of each isoform was performed by using as standards RNA transcripts obtained from cloned human alpha-actin sequences, and the total amount of sarcomeric actin mRNA was evaluated as a function of total poly(A+)RNA. We found that both isogenes are always coexpressed, and that the isoactin pattern changes during development. In utero and in neonatal hearts, alpha-skeletal actin mRNA represents less than or equal to 20% of sarcomeric actins, it increases to 48 +/- 6% during the first decade after birth and becomes the predominant isoform of adult hearts (60.4 +/- 8.5%). The 15 adult failing hearts exhibited the same isoactin pattern as the control ones (62.84 +/- 11.06%), and there was no difference in expression between patients with dilated cardiomyopathy or ischemic heart disease. These observations demonstrate that cardiac development in man, in contrast to rodent heart, is characterized by an up-regulation of the skeletal actin gene, the expression of which does not change in hypertrophied and failing hearts, and suggest that the actin and myosin heavy chain families are independently regulated in human heart.

Actins↗
PubMed1991

Evidence that an adrenal-specific nuclear protein regulates the cAMP responsiveness of the human CYP21B (P450C21) gene.

Based on competition analysis of gel shift assays utilizing nuclear extracts from the mouse adrenal Y1 cell line, the cAMP-responsive element of the human CYP21B gene (-129/-96 base pairs (bp] is found to contain two overlapping nuclear protein binding elements. One, -126/-113 bp, interacts specifically with an adrenal-specific protein factor (ASP) while the other, -119/-110 bp, contains a GC boxlike sequence and binds Sp1. The nucleotide replacements (GG----CC at -113 and -112 bp) introduced within the sequence -129/-96 bp greatly decreased the binding affinities of both Sp1 and ASP to the fragment and resulted in a loss of cAMP-enhanced transcription of a reporter gene in transient transfection experiments. When the 14-bp ASP-binding sequence was inserted in front of the reporter gene, induced transcription by cAMP was observed that could be increased by multimerization of this -126/-113-bp sequence. The -126/-113-bp fragment revealed specific binding only with nuclear extracts from adrenal Y1 cells among various types of cells tested, indicating that the protein factor ASP is specifically expressed in adrenal cells. The nucleotide replacement (G----C at -117 bp) of the -126 -113-bp sequence not only abolishes the binding to ASP but also the cAMP-dependent transcription, strongly supporting the hypothesis that ASP is a tissue-specific transacting protein that directly functions as a transcription factor essential to the cAMP-dependent transcription. Furthermore, the -129/-115-bp sequence of the bovine cytochrome P450C21 gene corresponding to the human -126/-113-bp sequence is demonstrated to be functionally conserved with respect to both cAMP-dependent enhancement of transcription and ASP binding.

Adrenal Glands↗
PubMed1999

Development and validation of an automated, microscopy-based method for enumeration of groups of intestinal bacteria.

An automated microscopy-based method using fluorescently labelled 16S rRNA-targeted oligonucleotide probes directed against the predominant groups of intestinal bacteria was developed and validated. The method makes use of the Leica 600HR image analysis system, a Kodak MegaPlus camera model 1.4 and a servo-controlled Leica DM/RXA ultra-violet microscope. Software for automated image acquisition and analysis was developed and tested. The performance of the method was validated using a set of four fluorescent oligonucleotide probes: a universal probe for the detection of all bacterial species, one probe specific for Bifidobacterium spp., a digenus-probe specific for Bacteroides spp. and Prevotella spp. and a trigenus-probe specific for Ruminococcus spp., Clostridium spp. and Eubacterium spp. A nucleic acid stain, 4',6-diamidino-2-phenylindole (DAPI), was also included in the validation. In order to quantify the assay-error, one faecal sample was measured 20 times using each separate probe. Thereafter faecal samples of 20 different volunteers were measured following the same procedure in order to quantify the error due to individual-related differences in gut flora composition. It was concluded that the combination of automated microscopy and fluorescent whole-cell hybridisation enables distinction in gut flora-composition between volunteers at a significant level. With this method it is possible to process 48 faecal samples overnight, with coefficients of variation ranging from 0.07 to 0.30.

Adult↗
PubMed1993

A simple physical model for the gel electrophoretic manifestations of sequence-dependent DNA superstructures.

A simple physical model of the gel electrophoretic manifestations of sequence-dependent DNA superstructures is illustrated. The model is based on the calculation of the curvature dispersion as evaluated by integrating the orientational parameters of the base pairs along the chain. Such a quantity is proportional to the straightening activation energy of the curved DNA in the direction of the average orientation of the helical axis and was found to be proportional to the logarithm of the gel electrophoretic retardation. The model is capable of consistently explaining the different electrophoretic manifestations of DNA superstructures, such as the retardation of a large ensemble of multimeric oligonucleotides with different sequences, periodicities, and lengths, and the permutation gel assays as well as the mobility changes consequent to extensive point mutations in a DNA tract.

Base Composition↗
PubMed1993

Compositional changes of AP-1 DNA-binding proteins are regulated by light in a mammalian circadian clock.

Recent reports have shown that the nuclear phosphoprotein Fos is induced by light in a mammalian circadian clock, the suprachiasmatic nucleus. To learn how light and circadian phase affect the binding of Fos to DNA, we analyzed the photic and temporal regulation of immunoreactive Jun protein expression and AP-1 DNA-binding activity in the rat suprachiasmatic nucleus. Immunohistochemistry and gel mobility shift assays suggest that AP-1 activity during the night and after a light pulse consists of constant, as well as variable, protein components; JunD could be identified as a constituent of both dark- and light-activated binding complexes, whereas binding by JunB and Fos could be implicated only after photic stimulation. Since JunD or JunB could be colocalized with Fos in individual suprachiasmatic nucleus cell nuclei, light may be acting in at least some suprachiasmatic nucleus cells by altering AP-1 protein composition rather than binding site occupancy.

Animals↗
PubMed1988

Interaction of DNA hairpin loops and a complementary strand by a triplet of base pairs.

Hypothesis of non-enzymatic recognition of primordial tRNA and mRNA precursors is experimentally approached. DNA hairpins containing a different number of deoxyguanosine residues in the loop are analyzed for their binding ability to a chemically fixed single-strand of oligo(dC). In presence of small Mg2+ concentration a hairpin with five dG residues in the loop is adsorbed to affinity matrix. Comparison of elution temperatures of hairpin oligonucleotides with those of single-stranded oligoguanylic acids with length of the loop indicates, that smallest loop able to bind forms a triplet of base pairs.

Adsorption↗
PubMed1981

Abortive initiation and long ribonucleic acid synthesis.

In vitro transcription assays have been used to study the rate of ribonucleic acid (RNA) synthesis from the Escherichia coli lactose promotor mutant lacL8UV5 contained on a 203-bp (base pair) restriction fragment. The half-life of long (63-base) RNA production from heparin-resistant RNA polymerase-promotor complexes was found to be related to the amount of oligonucleotides released during the initiation process (abortive initiation). Studies indicate that once a ternary complex between the promoter, RNA polymerase, and a newly synthesized RNA seven and nine nucleotides long is formed, abortive initiation is reduced and the rate of synthesis of long RNAs is increased. The promoter for the left inverted repeat of the transposable element Tn5 was also examined. It was observed to have a much slower rate of production of long RNAs, and it released oligonucleotides 4 times as often as the lactose promoter. The correlation between the amount of abortive initiation and the half-time of long RNA production is discussed.

Base Composition↗
PubMed2005

Regulation of Mycobacterium tuberculosis cell envelope composition and virulence by intramembrane proteolysis.

Mycobacterium tuberculosis infection is a continuing global health crisis that kills 2 million people each year. Although the structurally diverse lipids of the M. tuberculosis cell envelope each have non-redundant roles in virulence or persistence, the molecular mechanisms regulating cell envelope composition in M. tuberculosis are undefined. In higher eukaryotes, membrane composition is controlled by site two protease (S2P)-mediated cleavage of sterol regulatory element binding proteins, membrane-bound transcription factors that control lipid biosynthesis. S2P is the founding member of a widely distributed family of membrane metalloproteases that cleave substrate proteins within transmembrane segments. Here we show that a previously uncharacterized M. tuberculosis S2P homologue (Rv2869c) regulates M. tuberculosis cell envelope composition, growth in vivo and persistence in vivo. These results establish that regulated intramembrane proteolysis is a conserved mechanism controlling membrane composition in prokaryotes and show that this proteolysis is a proximal regulator of cell envelope virulence determinants in M. tuberculosis.

Animals↗