Differential binding of HMG1, HMG2, and a single HMG box to cisplatin-damaged DNA

The HMG box domain is a DNA binding domain present in the nonhistone chromosomal proteins HMG1 and HMG2 and in other proteins involved in the regulation of gene expression. Previous studies have demonstrated that HMG1 and HMG2 bind with high affinity to DNA modified with the cancer chemotherapeutic drug cisplatin (CDDP). In this report, we compare the binding of full-length HMG1 and HMG2 and the HMG boxes present in these proteins to that of CDDP-DNA. Complexes between HMG1, HMG2, or HMG Box A + B and CDDP-DNA were stable at > or = 500 mM salt, while complexes between a single HMG box and CDDP-DNA exhibited decreased stability. Analysis of a series of HMG1 Box A mutant constructs revealed different affinities for CDDP-DNA. Two constructs containing a Phe to Ala substitution at position 19 and a Tyr to Gly substitution at position 71, are noteworthy; these peptides exhibited reduced affinity for CDDP-DNA. We have generated a structure of HMG1 Box A and used it, along with the results of our binding studies, to model its interaction with CDDP-DNA. HMG1 Box A binds in the minor groove of CDDP-DNA, in agreement with earlier studies. Our model predicts that Tyr71 partially intercalates and forms an H bond with the sugar-phosphate backbone. The model also suggests that Phe 19 does not directly interact with DNA, and hence an Ala substitution at position 19 may alter protein structure. This model should provide a framework for future studies examining HMG Box-DNA interactions.     

Cloning of cDNAs encoding xenopus neuregulin: expression in myotomal muscle during embryo development

The ontogeny of the myotome was investigated using [3H]thymidine or Brdu treatment in conjunction with 1,1', di-octadecyl-3, 3, 3', 3',-tetramethylindo-carbocyanine perchlorate (DiI) labeling and expression of specific markers. We have identified a subset of early post-mitotic cells that is present in the dorsomedial aspect of epithelial somites and is homogeneously distributed along their entire rostrocaudal extent. The post-mitotic quality of this cell subset enabled us to trace their fate in time-course experiments. Following initial somite dissociation, this epithelial post-mitotic layer bends underneath the medial portion of the nascent dermomyotome. Then, these cells progressively lose epithelial arrangement and migrate in a rostral direction where they accumulate temporarily. Subsequently, these early post-mitotic precursors extend processes that reach both rostral and caudal edges of each segment. Medial somite-derived myofibers also fill the entire mediolateral extent of the segment and reach the dorsomedial lip of the dermomyotome, thus forming the primary myotome. During this process, their large nuclei localize to a narrow stripe in the middle of the nascent myotome. Consistent with the proliferation studies, DiI labeling of the medial epithelial somite cells gave rise to a primary myotomal structure, and continuous pulsing of the DiI-injected embryos with radioactive thymidine revealed that these fibers indeed developed from post-mitotic progenitors. As these early post-mitotic cells that arise prior to somite dissociation are the first wave of progenitors that constitutes the myotome, we have termed them avian muscle pioneers. We propose that the primary myotome formed by the muscle pioneers constitutes a longitudinal scaffold that serves as a substrate for the addition of subsequent waves of myotomal cells.     

Differential expression of the TEF family of transcription factors in the murine placenta and during differentiation of primary human trophoblasts in vitro

Konjac (konnyaku) glucomannan was examined for its degradation in human intestines and fermentation products. The konjac glucomannan was degraded almost 100% by soluble enzymes in human feces to give 4-O-beta-D-mannopyranosyl-D-mannopyranose (beta-1,4-D-mannobiose), 4-O-beta-D-glucopyranosyl-D-glucopyranose (cellobiose), 4-O-beta-D-glucopyranosyl-D-mannopyranose, and small amounts of glucose and mannose. These three disaccharides were further degraded by a cell-associated enzyme(s) to glucose or mannose, or to both. Konjac glucomannan underwent fermentation by intestinal anaerobic bacteria and produced formic acid, acetic acid, propionic acid, and 1-butyric acid. These fatty acids were different in their proportions among test subjects, their total amounts ranging from 17.1% to 48.8% of the initial konjac glucomannan.     

Seven zinc-finger transcription factors are expressed sequentially during the development of anthers in petunia

BACKGROUND: Little is known about the value of heart rate variability in patients with symptomatic coronary artery disease with a preserved left ventricular function. We hypothesized that in these patients heart rate variability might be a helpful adjunct to conventional parameters to predict clinical events. METHODS: In a prospective 2-year follow-up study ambulatory electrocardiographic recordings were performed in 263 consecutive male patients (mean age 56+/-8 years) with stable angina pectoris and a mean left ventricular ejection fraction of 71%+/-12%. Clinical events consisted mainly of coronary events such as percutaneous transluminal angioplasty or coronary artery bypass graft operation. RESULTS: Low measures of standard deviation of normal R-R intervals, standard deviation of the mean R-R intervals of 5 minutes, and two spectral components of heart rate variability were found in patients who had had an event compared with patients with no event. Adjusted for severity of angina, the presence of a previous myocardial infarction, and the use of beta-blockers in a logistic regression model this relation remained statistically significant for SDNN. Healthy volunteers appeared to have the highest measures of heart rate variability. CONCLUSION: In patients with ischemic heart disease and normal or near normal ventricular function decreased heart rate variability is associated with adverse clinical events.     

Selection of a cDNA clone for chicken high-mobility-group 1 (HMG1) protein through its unusually conserved 3'-untranslated region, and improved expression of recombinant HMG1 in Escherichia coli

Screening of cDNA libraries for the homologous vertebrate proteins high mobility group (HMG) 1 and 2 using DNA probes based on the coding sequences is likely to result in isolation of both HMG1 and HMG2 clones, as well as pseudogenes, which may be transcribed at low levels. However, the 3'-untranslated regions (UTRs) of HMG1 and 2 are quite distinct, and unusually conserved across species. We have used this property to select the true chicken HMG1 cDNA clone from a chicken lymphocyte cDNA library in lambdagt11, using a probe based on the 3'-UTR of rat HMG1 cDNA. The chicken HMG1 cDNA clone is very similar to all the complete HMG1 cDNA clones isolated so far. We suggest that the sequence designated chicken HMG1 in the GenBank Data Library (Accession number D14314) is, in fact, that of HMG2a [and moreover that the recently reported mouse clone (Accession number AF022465), proposed to encode a new HMG protein, HMG4, is also likely to encode an HMG2a, based on the translated amino-acid sequence and 3'-UTR]. We also report much improved expression of intact recombinant HMG1 in Escherichia coli by the use of chloramphenicol rather than ampicillin selection and conditions that limit cell growth. This should be general for all members of the HMG1 (and 2) family which may be toxic to cells (possibly because of the long acidic tail), and may also prove useful in the production of other such proteins.     

Solution structure of the sequence-specific HMG box of the lymphocyte transcriptional activator Sox-4

Two groups of HMG box proteins are distinguished. Proteins in the first group contain multiple HMG boxes, are non-sequence-specific, and recognize structural features as found in cruciform DNA and cross-over DNA. The abundant chromosomal protein HMG-1 belongs to this subgroup. Proteins in the second group carry a single HMG box with affinity for the minor groove of the heptamer motif AACAAAG or variations thereof. A solution structure for the non-sequence-specific C-terminal HMG box of HMG-1 has recently been proposed. Now, we report the solution structure of the sequence-specific HMG-box of the SRY-related protein Sox-4. NMR analysis demonstrated the presence of three alpha-helices (Val10-Gln22, Glu30-Leu41 and Phe50-Tyr65) connected by loop regions (Ser23-Ala49 and Leu42-Pro49). Helices I and II are positioned in an antiparallel mode and form one arm of the HMG box. Helix III is less rigid, makes an average angle of about 90 degrees with helices I and II, and constitutes the other arm of the molecule. As in HMG1B, the overall structure of the Sox-4 HMG box is L-shaped and is maintained by a cluster of conserved, mainly aromatic residues.     

Regulation of Raf-1-dependent signaling during early Xenopus development

The Raf-1 gene product is activated in response to cellular stimulation by a variety of growth factors and hormones. Raf-1 activity has been implicated in both cellular differentiation and proliferation. We have examined the regulation of the Raf-1/MEK/MAP kinase (MAPK) pathway during embryonic development in the frog Xenopus laevis. We report that Raf-1, MEK, and MAPK activities are turned off following fertilization and remain undetectable up until blastula stages (stage 8), some 4 h later. Tight regulation of the Raf-1/MEK/MAPK pathway following fertilization is crucial for embryonic cell cycle progression. Inappropriate reactivation of MAPK activity by microinjection of oncogenic Raf-1 RNA results in metaphase cell cycle arrest and, consequently, embryonic lethality. Our findings demonstrate an absolute requirement, in vivo, for inactivation of the MAPK signaling pathway to allow normal cell cycle progression during the period of synchronous cell divisions which occur following fertilization. Further, we show that cytostatic factor effects are mediated through MEK and MAPK.     

Interactions between the subfractons of calf thymus H1 and nonhistone chromosomal proteins HMG1 and HMG2

The nonhistone chromosomal proteins, HMG1 and HMG2, interact with the various subfractions of calf thymus H1 with a high degree of specificity. Subfractions 1b and 2 interact very strongly with HMG1 to form heterodimers. In contrast, subfractions 3a and 3b interact much more weakly. HMG2 interacts with 3a and 3b but shows no detectable complexing with 1a and 2.     

Interaction of non-histone chromosomal proteins HMG1 and HMG2 with DNA

Interaction between non-histone protein HMG1 or HMG2 and DNA has been studied by using thermal denaturation and circular dichroism (CD) spectroscopy. We have made the following observations. 1. The binding of each of these two proteins to DNA stabilizes the latter, as shown by an increase in melting temperature of 20 degrees C (from 45 degrees C to about 65 degrees C). 2. There are 6.0 amino acids/nucleotide in HMG1-bound DNA and 5.0 in HMGI-bound DNA which suggests that each HMB1 moleculae would cover about 20 base pairs of DNA and each HMG2 molecule would cover about 25 base pairs. 3. The alpha-helical content of these two non-histone proteins in the complexes, estimated from the CD value at 220 nm, is about one third to one half that of total proteins in calf thymus chromatin. 4. DNA conformation is distorted only slightly by the binding of protein HMG1 or HMG2. 5. Neither the melting nor the CD properties of HMG1-DNA or HMG2-DNA complexes differ substantially whether they are prepared by NaCl-gradient dialysis in urea or by direct mixing of protein and DNA at 0.15 M NaCl, followed by dialysis against the same buffer i.e. 0.25 mM EDTA (pH 8.0).