New EMBO members' review: the double life of HMGB1 chromatin protein: architectural factor and extracellular signal.
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Animals↗
Biomedical subjects
M Beltrame
Publications and source records attributed to M Beltrame.
Hyperpolarization-activated cyclic nucleotide-gated channel 1 is a molecular determinant of the cardiac pacemaker current I(f).
The pacemaker current I(f) of the sinoatrial node (SAN) is a major determinant of cardiac diastolic depolarization and plays a key role in controlling heart rate and its modulation by neurotransmitters. Substantial expression of two different mRNAs (HCN4, HCN1) of the family of pacemaker channels (HCN) is found in rabbit SAN, suggesting that the native channels may be formed by different isoforms. Here we report the cloning and heterologous expression of HCN1 from rabbit SAN and its specific localization in pacemaker myocytes. rbHCN1 is an 822-amino acid protein that, in human embryonic kidney 293 cells, displayed electrophysiological properties similar to those of I(f), suggesting that HCN1 can form a pacemaker channel. The presence of HCN1 in the SAN myocytes but not in nearby heart regions, and the electrophysiological properties of the channels formed by it, suggest that HCN1 plays a central and specific role in the formation of SAN pacemaker currents.
Amino Acid Sequence↗
Cloning and expression pattern of a zebrafish homolog of forkhead activin signal transducer (FAST), a transcription factor mediating Nodal-related signals.
Forkhead activin signal transducer (FAST) is a member of the winged-helix family of DNA-binding proteins that has been implicated in mesoderm induction and left-right axis specification during embryonic development in Xenopus and mouse. We have cloned and characterized a zebrafish FAST homolog. Zebrafish fast is expressed maternally and zygotically. Transcripts start regionalizing and decline in level during gastrulation. During somitogenesis, fast is expressed bilaterally in the lateral plate mesoderm, like its mouse homolog. In addition, zebrafish fast is also expressed bilaterally in the dorsal diencephalon, where the nodal-related cyclops gene is only expressed on the left side. It remains to be demonstrated whether FAST expression in the brain can mediate Nodal-induced asymmetric development.
Amino Acid Sequence↗
Upwardly mobile proteins. Workshop: the role of HMG proteins in chromatin structure, gene expression and neoplasia.
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Amino Acid Motifs↗
The human gene coding for HCN2, a pacemaker channel of the heart.
Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, underlying 'pacemaker' currents (I(f)/Ih), are involved in pacemaker activity of cardiac sinoatrial node myocytes and central neurons. Several cDNAs deriving from four different genes were recently identified which code for channels characterized by six transmembrane domains and a cyclic nucleotide binding domain. We report here the identification of the human HCN2 gene and show that its functional expression in a human kidney cell line generates a current with properties similar to the native pacemaker f-channel of the heart. The hHCN2 gene maps to the telomeric region of chromosome 19, band p13.3. This is the first identification of a genetic locus coding for an HCN channel.
Amino Acid Sequence↗
Expression patterns of zebrafish sox11A, sox11B and sox21.
We have cloned three sox genes in zebrafish (Danio rerio), one related to human and chicken SOX21, and two related to mammalian and chicken Sox-11. Zebrafish sox21, sox11A and sox11B transcripts are accumulated in the egg, are present in all cells until gastrulation and become restricted later to the developing central nervous system (CNS); expression in adults is undetectable. sox21 is expressed in the forebrain, midbrain and hindbrain, but maximally at the midbrain-hindbrain junction; sox11A,B have a widespread and dynamic expression in the CNS, but in contrast to sox21 are absent at the midbrain-hindbrain boundary.
Amino Acid Sequence↗
The RAG1 homeodomain recruits HMG1 and HMG2 to facilitate recombination signal sequence binding and to enhance the intrinsic DNA-bending activity of RAG1-RAG2.
V(D)J recombination is initiated by the specific binding of the RAG1-RAG2 (RAG1/2) complex to the heptamer-nonamer recombination signal sequences (RSS). Several steps of the V(D)J recombination reaction can be reconstituted in vitro with only RAG1/2 plus the high-mobility-group protein HMG1 or HMG2. Here we show that the RAG1 homeodomain directly interacts with both HMG boxes of HMG1 and HMG2 (HMG1,2). This interaction facilitates the binding of RAG1/2 to the RSS, mainly by promoting high-affinity binding to the nonamer motif. Using circular-permutation assays, we found that the RAG1/2 complex bends the RSS DNA between the heptamer and nonamer motifs. HMG1,2 significantly enhance the binding and bending of the 23RSS but are not essential for the formation of a bent DNA intermediate on the 12RSS. A transient increase of HMG1,2 concentration in transfected cells increases the production of the final V(D)J recombinants in vivo.
Binding Sites↗
Hmg4, a new member of the Hmg1/2 gene family.
High mobility group (HMG) proteins are abundant components of mammalian nuclei and fall into three families. The members of one such family, HMG1 and HMG2, are ubiquitously expressed and facilitate the formation of nucleoprotein complexes where the DNA is sharply bent. We have identified a mouse cDNA that codes for a novel 200-amino-acid protein of the HMG1/2 family, which we called HMG4. The mouse Hmg4 gene is highly expressed in the embryo; Hmg4 transcripts are barely detectable in adult tissues. The human HMG4 gene, which is extremely similar to its mouse homolog, has been sequenced as part of chromosome X, band q28. HMG4, HMG1, and HMG2 proteins have been highly conserved during vertebrate evolution, suggesting that each has at least some unique property. It is possible that HMG4 is required during development.
Amino Acid Sequence↗
Flexing DNA: HMG-box proteins and their partners.
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Animals↗
Base pairing between U3 and the pre-ribosomal RNA is required for 18S rRNA synthesis.
The nucleolus, the site of pre-ribosomal RNA (pre-rRNA) synthesis and processing in eukaryotic cells, contains a number of small nucleolar RNAs (snoRNAs). Yeast U3 snoRNA is required for the processing of 18S rRNA from larger precursors and contains a region complementary to the pre-rRNA. Substitution mutations in the pre-rRNA which disrupt this base pairing potential are lethal and prevent synthesis of 18S rRNA. These mutant pre-rRNAs show defects in processing which closely resemble the effects of genetic depletion of components of the U3 snoRNP. Co-expression of U3 snoRNAs which carry compensatory mutations allows the mutant pre-rRNAs to support viability and synthesize 18S rRNA at high levels. Pre-rRNA processing steps which are blocked by the external transcribed spacer region mutations are largely restored by expression of the compensatory U3 mutants. Pre-rRNA processing therefore requires direct base pairing between snoRNA and the substrate. Base pairing with the substrate is thus a common feature of small RNAs involved in mRNA and rRNA maturation.
Base Composition↗
Analysis of muscle cell culture medium by size-exclusion chromatography.
The application of both low-pressure (preparative) and high-performance (analytical) size-exclusion chromatography to the fingerprinting of muscle cell culture supernatant is reported. The chromatograms showed significant differences between fresh media and muscle cell culture media. In addition, only one fraction derived from muscle culture medium contained factor(s) of proteic nature able to interfere with the cell cycle, of a continuous proliferating cell line.
Animals↗
Mutational analysis of an essential binding site for the U3 snoRNA in the 5' external transcribed spacer of yeast pre-rRNA.
The small nucleolar RNA U3 is essential for viability in yeast. We have previously shown that U3 can be cross-linked in vivo to the pre-rRNA in the 5' external transcribed spacer (ETS), at +470. This ETS region contains 10 nucleotides of perfect complementarity to U3. In a genetic background where the mutated rDNA is the only transcribed rDNA repeat, the deletion of the 10 nt complementary to U3 is lethal. Cells lacking the U3 complementary sequence in pre-rRNA fail to accumulate 18S rRNA: pre-rRNA processing is inhibited at sites A0 in the 5' ETS, A1 at the 5' end of 18S rRNA and A2 in ITS1. We show here that effects on processing at site A0 are specific for U3 and its associated proteins and are not seen on depletion of other snoRNP components. The deletion of the sequence complementary to U3 in the ETS therefore mimics all the known effects of the depletion of U3 in trans. This indicates that we have identified an essential U3 binding site on pre-rRNA, required in cis for the maturation of 18S rRNA.
Base Sequence↗
Mutational analysis of an essential binding site for the U3 snoRNA in the 5' external transcribed spacer of yeast pre-rRNA.
The small nucleolar RNA U3 is essential for viability in yeast. We have previously shown that U3 can be cross-linked in vivo to the pre-rRNA in the 5' external transcribed spacer (ETS), at +470. This ETS region contains 10 nucleotides of perfect complementarity to U3. In a genetic background where the mutated rDNA is the only transcribed rDNA repeat, the deletion of the 10 nt complementary to U3 is lethal. Cells lacking the U3 complementary sequence in pre-rRNA fail to accumulate 18S rRNA: pre-rRNA processing is inhibited at sites A0 in the 5' ETS, A1 at the 5' end of 18S rRNA and A2 in ITS1. We show here that effects on processing at site A0 are specific for U3 and its associated proteins and are not seen on depletion of other snoRNP components. The deletion of the sequence complementary to U3 in the ETS therefore mimics all the known effects of the depletion of U3 in trans. This indicates that we have identified an essential U3 binding site on pre-rRNA, required in cis for the maturation of 18S rRNA.
Base Sequence↗
Protein HU binds specifically to kinked DNA.
We have purified the main four-way junction DNA-binding protein of Escherichia coli, and have found it to be the well-known HU protein. HU protein recognizes with high-affinity one of the angles present in the junction, a molecule with the shape of an X. Other DNA structures characterized by sharp bends or kinks, like bulged duplex DNAs containing unpaired bases, are also bound. HU protein appears to inhibit cruciform extrusion from supercoiled inverted repeat (palindromic) DNA, either by constraining supercoiling or by trapping a metastable interconversion intermediate. All these properties are analogous to the properties of the mammalian chromatin protein HMG1. We suggest that HU is a prokaryotic HMG1-like protein rather than a histone-like protein.
Amino Acid Sequence↗
Identification and functional analysis of two U3 binding sites on yeast pre-ribosomal RNA.
It has long been known that U3 can be isolated hydrogen bonded to pre-ribosomal RNAs, but the sites of interaction are poorly characterized. Here we show that yeast U3 can be cross-linked to 35S pre-rRNA both in deproteinized extracts and in living cells. The sites of cross-linking were localized to the 5' external transcribed spacer (ETS) and then identified at the nucleotide level. Two regions of U3 near the 5' end are cross-linked to pre-rRNA in vivo and in vitro; the evolutionarily conserved box A region and a 10 nucleotide (nt) sequence with perfect complementarity to an ETS sequence. Two in vivo cross-links are detected in the ETS, at +470, within the region complementary to U3, and at +655, close to the cleavage site at the 5' end of 18S rRNA. A tagged rDNA construct was used to follow the effects of mutations in the ETS in vivo. A small deletion around the +470 cross-linking site in the ETS prevents the synthesis of 18S rRNA. This region is homologous to the site of vertebrate ETS cleavage. We propose that this site may be evolutionarily conserved to direct the assembly of a pre-rRNA processing complex required for the cleavages that generate 18S rRNA.
Animals↗
Pharmacokinetics and tumor concentration of intraarterial and intravenous cisplatin in patients with head and neck squamous cancer.
Tumor-tissue platinum levels and major pharmacokinetic parameters were determined in 11 patients with head and neck squamous cancer (HNSC) who were given cisplatin (50 mg/m2 daily x 2 days) and 5-fluorouracil (5-FU; 1000 mg/m2, continuous infusion x 5 days) either i.a. or i.v. The plasma peak platinum concentrations (cmax) and the areas under the curve for total platinum concentration versus time (AUC) during i.a. infusions were lower than the i.v. cmax (mean, 1.92 +/- 0.28 and 4.08 +/- 2.80 mg/l, for i.a. and i.v. infusions, respectively) and AUC values (mean, 22.55 +/- 4.96 and 40.66 +/- 10.71 mg h-1 l-1 for i.a. and i.v. treatment, respectively), suggesting a first-passage extraction of the drug by the tumor mass during i.a. infusion. However, no statistically significant difference was found in platinum tumor concentrations after i.a. administration versus i.v. infusion. The lack of a difference in tumor platinum concentrations between the i.a. and the i.v. administration routes might be explained either by a relatively high blood supply to the tumor area, enabling efflux of the surplus free platinum from the tissue, or by the delay between drug infusion and biopsy. After three cycles of i.a. treatment good tumor remission was obtained with minimal local toxicity. Larger clinical studies testing the advantages of the i.a. administration route over i.v. infusion appear to be necessary.
Adult↗
A gene family for acidic ribosomal proteins in Schizosaccharomyces pombe: two essential and two nonessential genes.
We have cloned the genes for small acidic ribosomal proteins (A-proteins) of the fission yeast Schizosaccharomyces pombe. S. pombe contains four transcribed genes for small A-proteins per haploid genome, as is the case for Saccharomyces cerevisiae. In contrast, multicellular eucaryotes contain two transcribed genes per haploid genome. The four proteins of S. pombe, besides sharing a high overall similarity, form two couples of nearly identical sequences. Their corresponding genes have a very conserved structure and are transcribed to a similar level. Surprisingly, of each couple of genes coding for nearly identical proteins, one is essential for cell growth, whereas the other is not. We suggest that the unequal importance of the four small A-proteins for cell survival is related to their physical organization in 60S ribosomal subunits.
Amino Acid Sequence↗
The RPC31 gene of Saccharomyces cerevisiae encodes a subunit of RNA polymerase C (III) with an acidic tail.
The RPC31 gene encoding the C31 subunit of Saccharomyces cerevisiae RNA polymerase C (III) has been isolated, starting from a C-terminal fragment cloned on a lambda gt11 library. It is unique on the yeast genome and lies on the left arm of chromosome XIV, very close to a NotI site. Its coding sequence perfectly matches the amino acid sequence of two oligopeptides prepared from purified C31. It is also identical to the ACP2 gene previously described as encoding an HMG1-like protein (W. Haggren and D. Kolodrubetz, Mol. Cell. Biol. 8:1282-1289, 1988). Thus, ACP2 and RPC31 are allelic and encode a subunit of RNA polymerase C. The c31 protein has a highly acidic C-terminal tail also found in several other chromatin-interacting proteins, including animal HMG1. Outside this domain, however, there is no appreciable homology to any known protein. The growth phenotypes of a gene deletion, of insertions, and of nonsense mutations indicate that the C31 protein is strictly required for cell growth and that most of the acidic domain is essential for its function. Random mutagenesis failed to yield temperature-sensitive mutants, but a slowly growing mutant was constructed by partial suppression of a UAA nonsense allele of RPC31. Its reduced rate of tRNA synthesis in vivo relative to 5.8S rRNA supports the hypothesis that the C31 protein is a functional subunit of RNA polymerase C.
Amino Acid Sequence↗