Structure of the rat alpha 2-macroglobulin-coding gene

Rat alpha 2-macroglobulin (alpha 2M) is an acute-phase protein, i.e., produced upon tissue inflammation. Genomic DNA clones covering the entire sequence of the alpha 2M gene were isolated and characterized by restriction mapping. Southern blotting and (partial) DNA sequencing. The rat alpha 2M gene is approx. 50 kb in length and consists of 36 exons ranging in size from 21 to 229 bp. Two functional domains, a bait region and a thiol ester site, are encoded by the exon 18 and 24, respectively. Several possible regulatory signals such as a TPA-inducible enhancer core, an identifier sequence, purine-pyrimidine alternative stretches and viral enhancer core sequences were identified. Several genomic DNA clones which cross-hybridized with the alpha 2M cDNA probe were also identified. Sequence analysis showed that they possessed sequences identical to a part of the rat alpha 1-inhibitor III cDNA and that they had a strikingly similar exon organization to the alpha 2M gene.     

a1 protein alters the DNA binding specificity of alpha 2 repressor

The alpha 2 protein of S. cerevisiae, the product of the MAT alpha 2 gene, represses a set of cell-type-specific genes (the a-specific genes) by binding to an operator sequence upstream of each gene. We demonstrate that a second yeast regulatory protein, a1, the product of the MATa1 gene, can alter the binding specificity of alpha 2 so that it no longer recognizes the a-specific gene operator, but instead acquires the ability to recognize a different operator sequence found upstream of haploid-specific genes. Thus, under the influence of a1, alpha 2 can repress haploid-specific genes. An alpha cell expresses alpha 2 but not a1, so that alpha 2 turns off only the a-specific genes. An a/alpha cell makes both a1 and alpha 2, in a ratio that ensures that alpha 2 is distributed between two distinct binding modes: the alpha 2 binding mode and the a1-alpha 2 binding mode. Thus in an a/alpha cell, alpha 2 represses two distinct classes of genes.     

Partial hepatectomy and mediators of inflammation decrease the expression of liver alpha 2-HS glycoprotein gene in rats

Liver mRNA levels of two acute phase reactant (APR) proteins, alpha 2-HS glycoprotein (a major negative APR) and alpha 1-acid glycoprotein (a major positive APR) were measured in male rats at different times after the administration of turpentine, of tumor necrosis factor, or following partial hepatectomy. In every case, a marked decrease in mRNA levels of alpha 2-HS glycoprotein was observed which reached a maximum at 24 h. A concomitant increase of alpha 1-acid glycoprotein mRNA levels was observed under the same conditions. These results indicate that the decreased levels of alpha 2-HS glycoprotein induced by the acute-phase response following inflammatory mediators and partial hepatectomy are due to a down-regulation of the gene expression of this protein in rat liver.     

Characterization of the DNA-binding activity of GCR1: in vivo evidence for two GCR1-binding sites in the upstream activating sequence of TPI of Saccharomyces cerevisiae.

GCR1 gene function is required for high-level glycolytic gene expression in Saccharomyces cerevisiae. Recently, we suggested that the CTTCC sequence motif found in front of many genes encoding glycolytic enzymes lay at the core of the GCR1-binding site. Here we mapped the DNA-binding domain of GCR1 to the carboxy-terminal 154 amino acids of the polypeptide. DNase I protection studies showed that a hybrid MBP-GCR1 fusion protein protected a region of the upstream activating sequence of TPI (UASTPI), which harbored the CTTCC sequence motif, and suggested that the fusion protein might also interact with a region of the UAS that contained the related sequence CATCC. A series of in vivo G methylation protection experiments of the native TPI promoter were carried out with wild-type and gcr1 deletion mutant strains. The G doublets that correspond to the C doublets in each site were protected in the wild-type strain but not in the gcr1 mutant strain. These data demonstrate that the UAS of TPI contains two GCR1-binding sites which are occupied in vivo. Furthermore, adjacent RAP1/GRF1/TUF- and REB1/GRF2/QBP/Y-binding sites in UASTPI were occupied in the backgrounds of both strains. In addition, DNA band-shift assays were used to show that the MBP-GCR1 fusion protein was able to form nucleoprotein complexes with oligonucleotides that contained CTTCC sequence elements found in front of other glycolytic genes, namely, PGK, ENO1, PYK, and ADH1, all of which are dependent on GCR1 gene function for full expression. However, we were unable to detect specific interactions with CTTCC sequence elements found in front of the translational component genes TEF1, TEF2, and CRY1. Taken together, these experiments have allowed us to propose a consensus GCR1-binding site which is 5'-(T/A)N(T/C)N(G/A)NC(T/A)TCC(T/A)N(T/A)(T/A)(T/G)-3'.     

Induction of several acute-phase protein genes by heavy metals: a new class of metal-responsive genes.

Acute-phase reactants, metallothioneins, and heat-shock proteins are the products of three families of genes that respond to glucocorticoids and cytokines. Metallothioneins and heat-shock proteins, however, are also stimulated by heavy metals, whereas very little is known about the effect of heavy metals on acute-phase-reactant genes. We have studied the effect of heavy metals (Hg, Cd, Pb, Cu, Ni, and Zn) and Mg on the acute-phase reactants alpha 1-acid glycoprotein, C-reactive protein, alpha 1-antitrypsin and alpha 1-antichymotrypsin. alpha 1-Acid glycoprotein and C-reactive protein mRNA levels were increased severalfold in livers of heavy-metal-treated Balb/c mice. The strongest induction was mediated by Hg, followed in order of response by Cd greater than Pb greater than Cu greater than Ni greater than Zn greater than Mg. None of the metals affected the mRNA levels of albumin, alpha 1-antitrypsin, and alpha 1-antichymotrypsin. Furthermore, failure to repress albumin, a negative acute-phase reactant, indicated that the induction of these genes was not due to a metal-mediated inflammatory response. The metals also induced alpha 1-acid glycoprotein and C-reactive protein in adrenalectomized animals, indicating that induction by the heavy metals is not mediated by the glucocorticoid induction pathway. Sequence analysis has revealed a region of homology to metal-responsive elements in the alpha 1-acid glycoprotein and C-reactive protein promoters. Additionally, an alpha 1-acid glycoprotein expression vector, pAGP(-595)CAT, responded to Hg and Cd when transfected into human HepG2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the contact sites of a factor that interacts with motif I (alpha CE1) of the chicken alpha A-crystallin lens-specific enhancer.

A lens-specific enhancer, an 84bp element between base pairs -162 and -79, of the chicken alpha A-crystallin gene is composed of two motifs, alpha CE1 (-162 and -134) and alpha CE2 (-119 and -99). Previous studies showed that a nuclear factor which binds to alpha CE1, termed alpha CEF1, is present at high levels in lens cells. Methylation interference analysis identified an inverted repeat of 5bp separated by 4bp, 5'-CTGGTTCCCACCAG-3', between positions -153 and -140 as an alpha CEF1-binding site. Gel mobility shift assays using synthetic oligonucleotides with site-directed mutations revealed that the alpha CEF1-binding consensus sequence is 5'-C(T/A)GGN6CC(A/T)G-3'. Comparison of this binding motif with regulatory sequences of diverse crystallin genes from diverse species suggests that alpha CE1 may be a ubiquitous crystallin gene enhancer.     

Drosophila contains three genes that encode distinct isoforms of protein phosphatase 1

The sequences of two Drosophila and one rabbit protein phosphatase (PP) 1 catalytic subunits were determined from their cDNA. The sequence of Drosophila PP1 alpha 1 was deduced from a 2.2-kb cDNA purified from an embryonic cDNA library, while that for Drosophila PP1 beta was obtained from overlapping clones isolated from both a head cDNA library and an eye imaginal disc cDNA library. The gene for Drosophila PP1 alpha 1 is at 96A2-5 on chromosome 3 and encodes a protein of 327 amino acids with a calculated molecular mass of 37.3 kDa. The gene for Drosophila PP1 beta is localized at 9C1-2 on the X chromosome and encodes a protein of 330 amino acids with a predicted molecular mass of 37.8 kDa. PP1 alpha 1 shows 96% amino acid sequence identity to PP1 alpha 2 (302 amino acids), an isoform whose gene is located in the 87B6-12 region of chromosome 3 [Dombrádi, V., Axton, J. M., Glover, D.M. Cohen, P.T.W. (1989) Eur. J. Biochem. 183, 603-610]. PP1 beta shows 85% identity to PP1 alpha 1 and PP1 alpha 2 over the 302 homologous amino acids. These results demonstrate that at least three genes are present in Drosophila that encode different isoforms of PP1. Drosophila PP1 alpha 1 and PP1 beta show 89% amino acid sequence identity to rabbit PP1 alpha (330 amino acids) [Cohen, P.T.W. (1988) FEBS Lett. 232, 17-23] and PP1 beta (327 amino acids), respectively, demonstrating that the structures of both isoforms are among the most conserved proteins known throughout the evolution of the animal kingdom. The presence of characteristic structural differences between PP1 alpha and PP1 beta, which have been preserved from insects to mammals, implies that the alpha and beta isoforms may have distinct biological functions.     

Microtubule depolymerization inhibits the regulation of alpha 1-acid glycoprotein mRNA by hepatocyte stimulating factor

Hepatocyte stimulating factor (HSF, a polypeptide cytokine) is a major regulatory hormone responsible for hepatic acute-phase reactant (APR) induction following acute systemic injury. The mechanisms by which HSF regulates APR synthesis in the liver are unknown. Microtubules are involved in a number of polypeptide hormone-mediated events which can be modified, either positively or negatively, by microtubule depolymerizing agents. In this study we have used colchicine (a microtubule depolymerizing drug) to assess whether or not HSF-mediated changes in rat hepatic alpha 1-acid glycoprotein (AGP) or albumin mRNA levels require an intact microtubule cytoskeletal system. Cultured rat hepatocytes were pretreated for 30 min with either colchicine (10(-6) M), or the inactive isomer lumicolchicine (10(-6) M), or fresh medium. Following pretreatment, purified murine macrophage HSF (10 units/ml) was added and the cells were incubated for an additional 12 h. Colchicine, but not lumicolchicine, significantly inhibited the HSF-dependent regulation of mRNA for the positive APR, AGP, but had no effect on the mRNA levels of albumin, a negative APR. Furthermore, removal of colchicine from previously inhibited cultures allowed HSF to restimulate AGP mRNA expression. These data suggest that microtubules may play a regulatory role in controlling the expression of the genes for positive acute-phase proteins and may explain the temporal differences found in vivo between positive and negative APR expression.     

Bacteriophage T4 gene 44 DNA polymerase accessory protein. Sequences of gene 44 and its protein product

Bacteriophage T4 gene 44 protein is a DNA polymerase accessory protein which is required for T4 DNA replication. We have isolated the gene for 44 protein from a previously constructed lambda-T4 hybrid phage (Wilson, G. G., Tanyashin, V. I., and Murray, N. E. (1977) Mol. Gen. Genet. 156, 203-214). We report here the nucleotide sequence of gene 44 and about 60 nucleotides 5' upstream from its coding region, which is immediately adjacent to gene 45. We have also purified 44 protein from T4-infected cells and submitted it to extensive protein chemistry characterization. Thus, considerable portions of the protein sequence predicted from the DNA sequence were confirmed by direct protein sequencing of peptides or by matching amino acid compositions of purified peptides. A total of 84% of the predicted amino acids was confirmed by the protein data. These studies indicate that gene 44 codes for a polypeptide containing 319 amino acids, with a calculated Mr = 35,371. The coding region of gene 44 is preceded by a potential regulatory region containing sequences homologous to the Escherichia coli (-10) RNA polymerase binding region and to a conserved sequence at -25 to -30 found in other T4 middle genes. In addition, there are sequence similarities in the translation initiation regions of genes 44, 45, and rIIB, all of which are subject to regulation by regA protein.     

Nucleotide sequence and characterization of the sfs1 gene: sfs1 is involved in CRP*-dependent mal gene expression in Escherichia coli.

We have cloned at least 12 different Escherichia coli genes which enable strain MK2001 to use maltose. The genes were designated sfs1 through sfs12 (sugar fermentation stimulation). Previously, one (sfs7) of them was mapped at 65 min on the E. coli chromosome and identified as nlp, which has high homology to repressor protein (Ner) of Mu phage, which contains a putative DNA binding region (Y.-L. Choi, T. Nishida, M. Kawamukai, R. Utsumi, H. Sakai, and T. Komano, J. Bacteriol. 171:5222-5225, 1989). In this study, another gene (sfs1) located at 3.5 min was newly found and analyzed. The nucleotide sequence of sfs1 encoded a protein of 234 amino acids (molecular mass, 26,227 Da) which also has a putative DNA binding domain. Overexpression of the sfs1 gene in MK2001 resulted in a 10-fold increase of amylomaltase, which was still dependent on MalT. These results suggest that Sfs1 could be a new regulatory factor involved in maltose metabolism.