Chromosomal location and expression of the single-copy gene encoding high-mobility-group protein HMG-I/Y in Arabidopsis thaliana

A cDNA encoding the HMG-I/Y protein from Arabidopsis thaliana has been isolated and characterised by nucleotide sequencing. The 903 bp cDNA contains a 612 bp open reading frame encoding a protein of 204 amino acid residues showing homology to HMG-I/Y proteins from other plant species. The protein contains four copies of the AT-hook motif which is involved in binding A/T-rich DNA. Southern blotting showed that the HMG-I/Y gene was present in a single copy in the Arabidopsis genome. The gene was localised to the top of chromosome 1 by RFLP analysis of F₈ recombinant inbred lines. Northern blotting showed that the gene was expressed in all organs examined, with the highest expression in flowers and developing siliques.     

Studies on the tissue specificity of the high-mobility-group non-histone chromosomal proteins from calf.

The high-mobility-group (HMG) non-histone chromosomal proteins from calf thymus, liver, spleen and kidney were extracted, and fractionated by CM-Sephadex chromatography and trichloroacetic acid precipitation. The isolated proteins HMG 1, HMG 2 and HMG 17 from the tissues were compared by polyacrylamide-gel electrophoresis, isoelectric focusing and amino acid analysis. The results show that the three proteins are very similar in the tissues studied, implying a lack of tissue specificity.     

A method for the fractionation of the high-mobility-group non-histone chromosomal proteins

A method is given for the preparation of four non-histone chromosomal proteins, one of which, protein 14, hitherto has not been isolated. The method also enables the preparation of histone H1 in gram quantities. The four non-histone chromosomal proteins so prepared are all polar molecules over 50% of each being composed of acidic and basic amino acids. It is also shown that protein 14 can be prepared from calf thymus without prior isolation of chromatin.     

Comparison of the high-mobility-group chromosomal proteins in rainbow-trout (Salmo gairdnerii) liver and testis.

Chromatography and characterization of the proteins extracted by 5% (w/v) HClO4 from rainbow-trout (Salmo gairdnerii) liver and testis show that the two tissues present a characteristically different spectrum of high-mobility-group (HMG) proteins. A variant subfraction of HMG C is found in liver, but is not detectable in testis, where even the main fraction of HMG C is present in only very low quantity. A protein, F, which appears to be related to protein H6 has similarly been isolated only from liver and not from testis. Quantification of the HMG proteins in total 5%-HClO4 extracts of trout liver and testis nuclei shows that, in relation to DNA, levels of HMG T1 and T2, and D are more than 2-fold, and C, 20-fold higher in liver than in testis. However, these differences do not result merely from the sequential withdrawal of HMG proteins at the same time that histones are replaced by protamines in the developing spermatid, since in testis, at some stages of maturation, levels of H6 are almost 2-fold higher than in liver. The implications of these findings for the function of HMG proteins are discussed.     

Abundance of mRNAs encoding HMG1/HMG2 class high-mobility-group DNA-binding proteins are differentially regulated in cotyledons of Pharbitis nil

The abundance of an mRNA encoding an HMG1/2 protein from Pharbitis nil (HMG1) has been previously shown to be regulated by light and an endogenous rhythm in cotyledons. A second Pharbitis nil HMG cDNA (HMG2) was characterized. The sequence of HMG2 was 82% and 86% identical to HMG1 at the nucleotide and amino acid level, respectively. As with HMG1, HMG2 mRNA was detected in all vegetative tissues and was most abundant in roots. However, unlike HMG1, HMG2 mRNA abundance did not increase upon transfer of cotyledons to darkness and did not exhibit regulation by an endogenous circadian rhythm when maintained in continuous darkness over a 68 h period. Similarly, while the abundance of HMG1 mRNA during a dark period that induced photoperiodically controlled flowering was dramatically affected by brief light exposure (night break), this treatment had no effect on HMG2 mRNA abundance. Collectively, these data are consistent with a role of HMG1 in contributing to the circadian-regulated and/or dark-regulated gene expression with constitutive expression of HMG2 playing a housekeeping role in the general regulation of gene expression in Pharbitis nil cotyledons.     

The amino acid sequence of the chromosomal protein HMG-Y, its relation to HMG-I and possible domains for the preferential binding of the proteins to stretches of A-T base pairs

The primary structure of the human high mobility group (HMG) protein HMG-Y has been established except for a few amino acids in the N-terminal and the C-terminal part of the protein. It was found that the sequence was identical to that of HMG-I except for a run of eleven amino acids. Like HMG-I the protein was N-terminally blocked and the palindromic sequence Pro-Arg-Gly-Arg-Pro occurred twice as in HMG-I. The binding of peptides derived from HMG-I (after thermolysin cleavage) to poly (dA-dT).poly(dA-dT) suggested that there are at least two different binding domains in the protein and that binding is not dependent upon an intact protein.     

The single-copy gene encoding high-mobility-group protein HMG-I/Y from pea contains a single intron and is expressed in all organs

The coding and 3-downstream regions of the gene encoding the high mobility group protein HMG-I/Y from pea have been isolated, sequenced and characterised. A 795 bp pea genomic fragment containing the coding region of the pea HMG-I/Y gene with a single intron of 201 bp was isolated by PCR. The gene encodes a protein of 197 amino acid residues with four copies of the AT-hook DNA-binding motif encoded by exon 2. Southern blot analysis on genomic DNA revealed the presence of a single copy of the HMG-I/Y gene in the haploid genome. The pea HMG-I/Y gene is expressed in all organs of pea including roots, stems, leaves, flowers, tendrils and developing seeds, as determined by northern blot analysis.     

Comparative studies on microinjected high-mobility-group chromosomal proteins, HMG1 and HMG2

The nonhistone chromosomal proteins, HMG1 and HMG2, were iodinated and introduced into HeLa cells, bovine fibroblasts, or mouse 3T3 cells by erythrocyte-mediated microinjection. Autoradiographic analysis of injected cells fixed with glutaraldehyde consistently showed both molecules concentrated within nuclei. Fixation with methanol, on the other hand, resulted in some leakage of the microinjected proteins from the nuclei so that more autoradiographic grains appeared over the cytoplasm or outside the cells. Both injected and endogenous HMG1 and HMG2 partitioned unexpectedly upon fractionation of bovine fibroblasts, HeLa, or 3T3 cells, appearing in the cytoplasmic fractions. However, in calf thymus, HMG1 and HMG2 molecules appeared in the 0.35 M NaCl extract of isolated nuclei, as expected. These observations show that the binding of HMG1 and HMG2 to chromatin differs among cell types or that other tissue-specific components can influence their binding. Coinjection of [125I]HMG1 and [131I]HMG2 into HeLa cells revealed that the two molecules display virtually equivalent distributions upon cell fractionation, identical stability, identical intracellular distributions, and equal rates of equilibration between nuclei. In addition, HMG1 and HMG2 did not differ in their partitioning upon fractionation nor in their stability in growing vs. nongrowing 3T3 cells. Thus, we have not detected any significant differences in the intracellular behavior of HMG1 and HMG2 after microinjection into human, bovine, or murine cells.     

Stability of the maize chromosomal high-mobility-group proteins,HMGa and HMGb,in vivo

Chromosomal non-histone high-mobility-group (HMG) proteins represent essential components of eukaryotic chromatin and have also been isolated from a variety of plants. In maize, studies on structure and function of the two larger of the four major HMG proteins have recently been performed and are now extended by analysis of theirin vivo stability using pulse-chase experiments in a cell suspension culture. The half-life of the analyzed HMGa and HMGb proteins was found to be 65 h or more than 78 h, respectively.     

Kinetic analysis of high-mobility-group proteins HMG-1 and HMG-I/Y binding to cholesterol-tagged DNA on a supported lipid monolayer

High-mobility-group proteins HMG-1 and HMG-I/Y bind to multiple sites within a 268 bp A/T-rich enhancer element of the pea plastocyanin gene (PetE). Within a 31 bp region of the enhancer, the binding site for HMG-1 overlaps with the binding site for HMG-I/Y. The kinetics of binding and the affinities of HMG-1 and HMG-I/Y for the 31 bp DNA were determined using surface plasmon resonance. Due to very high non-specific interactions of the HMG proteins with a carboxymethyl–dextran matrix, a novel method using a cholesterol tag to anchor the DNA in a supported lipid monolayer on a thin gold film was devised. The phosphatidylcholine monolayer produced a surface that reduced background interactions to a minimum and permitted the measurement of highly reproducible protein–DNA interactions. The association rate constant (k_a) of HMG-I/Y with the 31 bp DNA was ~5-fold higher than the rate constant for HMG-1, whereas the dissociation constant (K_D) for HMG-I/Y (3.1 nM) was ~7-fold lower than that for HMG-1 (20.1 nM). This suggests that HMG-I/Y should bind preferentially at the overlapping binding site within this region of the PetE enhancer.     

C1 proteins: a class of high-mobility-group non-histone chromosomal proteins from the fruit fly Ceratitis capitata

1. CM-cellulose chromatography of a fraction soluble in 5% perchloric acid from Ceratitis capitata chromatin yields three proteins, C1a1, C1a2 and C1b, which have been purified to electrophoretical homogeneity. 2. C1a1, C1a2 and C1b analyse like high mobility group (HMG) non-histone chromosomal proteins, although they do not exactly correspond with those from vertebrates. It is proposed that C1 proteins, as well as Drosophila D1 [Rodríguez Alfageme et al. (1980) Chromosoma, 78, 1-31] are representative of a class of insect-specific HMG proteins. Tryptic fingerprints show that C1a1 and C1a2 are very similar, but C1b is a somewhat distinct protein. Circular dichroism studies have shown that these preparations do not appreciably fold on increasing ionic strength. 3. The interactions between DNA and C1 proteins have been studied. These proteins precipitate DNA in 0.15 M NaCl, 0.015 M sodium citrate and the precipitation curves are cooperative. Soluble complexes between C1 proteins and DNA could be prepared in low ionic strength media and their thermal denaturation profiles obtained. C1 proteins do not destabilize DNA under the conditions used to prepare the complexes but the three proteins stabilize DNA to a different degree. From these studies it has been concluded that the association constant of C1b to DNA is smaller than that of C1a1 and C1a2.     

Localization of mRNAs encoding human mitochondrial oxidative phosphorylation proteins

The mitochondrial oxidative phosphorylation (OXPHOS) proteins are encoded by both nuclear and mitochondrial DNA. The nuclear-encoded OXPHOS mRNAs have specific subcellular localizations, but little is known about which localize near mitochondria. Here, we compared mRNAs in mitochondria-bound polysome fractions with those in cytosolic, free polysome fractions. mRNAs encoding hydrophobic OXPHOS proteins, which insert into the inner membrane, were localized near mitochondria. Conversely, OXPHOS gene which mRNAs were predominantly localized in cytosol had less than one transmembrane domain. The RNA-binding protein Y-box binding protein-1 is localized at the mitochondrial outer membrane and bound to the OXPHOS mRNAs. Our findings offer new insight into mitochondrial co-translational import in human cells.     

Sequence of a cDNA encoding chicken high-mobility-group protein-2.

There are several members of the high-mobility-group (HMG) of DNA-binding proteins, including HMG-1, HMG-2, HMG-14 and HMG-17 [Johns: The HMG Chromosomal Proteins. Academic Press, London, 1982]. We report here sequences encoding the chicken HMG-2 protein of 207 amino acids (aa). This assignment is made on the basis of available data which indicate 89% homology of the chicken aa sequence to porcine HMG-2. This compares with 78-81% homology to the HMG-1 proteins of rat, hamster, human, porcine, and bovine origin.     

Partitioning and translation of mRNAs encoding soluble proteins on membrane-bound ribosomes

In eukaryotic cells, it is generally accepted that protein synthesis is compartmentalized; soluble proteins are synthesized on free ribosomes, whereas secretory and membrane proteins are synthesized on endoplasmic reticulum (ER)-bound ribosomes. The partitioning of mRNAs that accompanies such compartmentalization arises early in protein synthesis, when ribosomes engaged in the translation of mRNAs encoding signal-sequence-bearing proteins are targeted to the ER. In this report, we use multiple cell fractionation protocols, in combination with cDNA microarray, nuclease protection, and Northern blot analyses, to assess the distribution of mRNAs between free and ER-bound ribosomes. We find a broad representation of mRNAs encoding soluble proteins in the ER fraction, with a subset of such mRNAs displaying substantial ER partitioning. In addition, we present evidence that membrane-bound ribosomes engage in the translation of mRNAs encoding soluble proteins. Single-cell in situ hybridization analysis of the subcellular distribution of mRNAs encoding ER-localized and soluble proteins identify two overall patterns of mRNA distribution in the cell-endoplasmic reticular and cytosolic. However, both partitioning patterns include a distinct perinuclear component. These results identify previously unappreciated roles for membrane-bound ribosomes in the subcellular compartmentalization of protein synthesis and indicate possible functions for the perinuclear membrane domain in mRNA sorting in the cell.