Isolation of DNA-free RNA, DNA, and proteins by cesium trifluoroacetate centrifugation.

The ability to simultaneously isolate intact DNA-free RNA, genomic DNA, and proteins from a biological specimen can be useful in cloning genes and analyzing gene expression. Equilibrium density gradient centrifugation with CsCl is a useful tool for fractionating, quantitatively separating, and characterizing RNA, DNA, and the total quota of proteins, respectively, based on differences in their buoyant densities. In the present study we have reexamined the rarely used cesium salt, cesium trifluoroacetate, for the same purpose.

Expression and characterization of human interferon-beta1 in the methylotrophic yeast Pichia pastoris.

We describe the heterologous expression of a human interferon-beta1 in the methylotrophic yeast Pichia pastoris. Biologically active recombinant human interferon-beta1 (rHuIFN-beta1) was secreted from shake-flask-grown P. pastoris cells into the medium using the Saccharomyces cerevisiae alpha-mating factor prepro-leader sequence at the level of (1-3) x 10(5) i.

Induced synthesis of albumin-like protein in damaged rat reticulocytes.

In this study, we present evidence that red blood cell (RBC) membrane p68 in the Belgrade (b/b) rat is similar if not identical to rat serum albumin (RSA). Structural homology between RSA and the RBC p68 has been determined by a variety of biochemical and immunological criteria. This albumin-like protein is a normal constituent of rat RBC and it is partially exported by exosomes during erythroid differentiation.

The rat beta (b miny)-globin promoter: nuclear protein factors and erythroid-specific induction of transcription.

We show that the rat adult beta (b miny)-globin gene is transcriptionally active. The - 100-bp promoter region contains control elements that are important for the induction of transcription in rat erythroleukemia (REL) cells. By using DNaseI footprinting and gel mobility shift assays, we have shown that the CCAAT box, a regulatory element from an analyzed promoter region, binds NF-Y and GATA-1 transcription factors.

Differences in rat rbc cytosol induced after in vivo phenylhydrazine treatment.

Analysis of rat red blood cell (RBC) cytosol protein fraction after in vivo phenylhydrazine treatment revealed increased amounts of 68-kDa protein. This protein is present in trace amounts in normal rat RBC cytosol. We also present data that 68-kDa protein from RBC cytosol has an identical isoelectric point, partial proteolytic map and immunological determinants as a protein of the same molecular mass from rat exosomes.

DNA region responsible for transcriptional regulation of the Escherichia coli penicillin amidase (pac) gene by CRP and PAA.

Transcriptional regulation of Escherichia coli ATCC11105 penicillin amidase gene (pac) by cAMP receptor protein (CRP) and phenylacetic acid (PAA) was studied by using operon fusions with divergent reporter gene (lacZ, and phoA) constructs. A 150 bp DNA segment essential for the regulation of pac gene transcription by CRP and PAA was defined.

Lack of protein 4.1a in red blood cells of the hereditarily anemic belgrade laboratory (b/b) rat.

We have demonstrated that the red blood cell (RBC) membrane of the hereditarily anemic Belgrade laboratory (b/b) rat contains protein 4. 1b isoform, only. The evidence are given that the synthesis of protein 4.

Synthesis and secretion of Providencia rettgeri and Escherichia coli heterodimeric penicillin amidases in Saccharomyces cerevisiae.

The Providencia rettgeri and Escherichia coli pac genes encoding heterodimeric penicillin G amidases (PAC) were successfully expressed in Saccharomyces cerevisiae. Furthermore, these recombinant enzymes are secreted from the yeast cell into the medium which is in contrast to bacterial hosts, where the enzymes are retained in the periplasm. Contrary to the P.

Alpha- and beta-globins of the anemic Belgrade laboratory rat. II. The effect of hemin and iron-dextran treatment.

We have studied the changes in bone marrow and spleen globin chain levels after in vivo hemin and iron-dextran treatment of hereditarily anemic Belgrade laboratory (b/b) rats. The increase of globin chains was detected in the bone marrow and in the spleen when b/b animals were treated with either iron or hemin. The analysis of changes in alpha- and beta-globin chain ratios revealed the distinctive role of these molecules in regulating globin chain status.

Alpha- and beta-globins of the anemic Belgrade laboratory rat. I. Status of alpha- and beta-globins in bone marrow and spleen.

In this study we have demonstrated that the bone marrow of the anemic Belgrade laboratory (b/b) rat, as the primary site of erythropoiesis, has a decreased globin polypeptide synthesis in total protein cell extracts. Therefore, it is the source of red blood cells containing decreased amounts of globin mRNAs and polypeptides. In spite of the fact that the b/b rat shows a splenomegaly, the spleen is not capable of compensating for the anemic state.

Evidence for HSP70-like protein in the RBC membrane of the hereditarily anemic Belgrade laboratory (b/b) rat.

We have demonstrated that in normal and b/b rat red blood cells (RBCs) hsp70-like protein (heat shock protein 70-like) is localized in the cytosol and it is exported via exosomes during in vivo reticulocytes maturation. As we have presumed, in the mutant (b/b) rat, hsp70-like protein transfers from cytosol to the RBC membrane. In the normal rat RBCs this happens when those cells are submitted to heat stress conditions.

Molecular evidence for increased hematopoietic proliferation in the spleen of the b/b laboratory rat.

The splenomegaly and the appearance of a significant number of CFU-E (erythroid colony-forming units) and BFU-E1 (erythroid burst-forming units) in the Belgrade laboratory rat (b/b) spleen prompted us to analyse further the molecular evidence for increased hematopoietic proliferation in the b/b spleen. Messenger RNAs (mRNAs) specific for globins, proteins for iron transport and deposition and the band 3 protein were used in rat erythropoietic tissues as markers for proliferation and erythroid differentiation. In the b/b spleen, all mRNAs analysed display an erythroid-specific pattern of expression.

The disbalance of alpha- and beta-globins in anemic Belgrade rat red blood cells.

The Belgrade Laboratory (b/b) rat has an autosomal mutation which in homozygous state induces severe anemia. This study was based on solubilization of total rat globin chains and their separation into alpha- and beta-globins using a 20% SDS polyacrylamide gel. These analyses demonstrated that the disbalance of alpha/beta globins in b/b red blood cells (RBC) is due to decreased level of alpha-globins.

The penicillin amidase of Arthrobacter viscosus (ATCC 15294).

The nucleotide (nt) sequence of the gene encoding penicillin G amidase (PA) of Arthrobacter viscosus strain ATCC 15,294 was determined. The sequence contained an open reading frame of 2406 nt with a G+C content of 37%. The deduced amino-acid sequence shows significant homology with other so far identified beta-lactam amidases of Gram- bacteria.

Constitutive interferon expression from retroviral vector.

A genomic fragment with the human beta-interferon gene was cloned into a pL3-4, a defective Moloney murine leukemia virus (M-MuLV) vector. Here we show that clones selected after viral infection of mouse NIH 3T3 cells constitutively produced 128 IU/ml of human beta-interferon. Constitutive synthesis of retroviral RNA was confirmed by dot blot hybridization of RNA isolated from two of the selected clones.

The "b" mutated gene in heterozygous Belgrade anemic rat.

The Belgrade b/b rat has an autosomal recessive mutation which in homozygous state induces severe anemia. So far, this mutation has been considered a recessive one and the heterozygous animals (+/b) as phenotypically normal. In this study, we showed that at the hematologic level, the heterozygous animals acquire some of the anemic characteristics as well.

The primary structure of Providencia rettgeri penicillin G amidase gene and its relationship to other gram negative amidases.

The nucleotide sequence of Penicillin G amidase (PA,E.C.3.

Hybrid PLtl promoter with dual regulation control.

The newly constructed PLtl hybrid promoter is composed of the operator and promoter sequences of tac and the -35 to -135 region of the phage lambda PL promoter, which contains the AT-rich block and the OL2 and OL3 segments. Transcriptional properties of PLtl were examined and compared with tac and lambda PL as reference promoters. The hybrid PLtl exhibits different and improved properties over tac promoter in four ways: (i) when repressed, the repression is almost complete; (ii) after complete induction, the hybrid PLtl promoter shows a 1.

Nucleotide sequence analysis of the inversion termini located within IS3 elements alpha 3 beta 3 and beta 5 alpha 5 of Escherichia coli K-12.

This paper presents the first detailed structural analysis of termini of an inversion mediated by recombination between Escherichia coli native IS elements. The complete nucleotide sequence of the inversion termini in the lactose region of Escherichia coli K-12 confirms our previous suggestion that the inversion occurred by homologous recombination between alpha 3 beta 3 and beta 5 alpha 5 IS3 elements (D. J.

Characterization of two rat globin cDNA clones.

The rat globin gene system is suitable for studying a coordinated regulation of seven genes from two gene families. A rat reticulocyte cDNA globin library has been constructed and two clones analyzed in detail. pBRrg 5 contains alpha while pBRrg X contains beta type sequence.

Rat b/b anemia: translation of normal and anemic globin mRNA in wheat-germ cell-free system.

Globin mRNAs were isolated from circulating reticulocytes both from rats carrying a homozygous, recessive mutation causing a severe thalassemia-like syndrome and from normal rats. After first identifying the rat globin chains as alpha or beta chains, the translational products primed by both polysomal and nonpolysomal mRNAs in wheat germ 30000 x g supernatant were analyzed: the ratio of alpha to beta globin mRNAs found in polysomes isolated from mutant rats is identical to the ratio of their products synthesized in vivo while the ratio of these mRNAs is quite different in the nonpolysomal fraction, the latter being enriched in alpha globin mRNA. No difference is found in the ratio of alpha and beta globin mRNAs in the polysomal and nonpolysomal RNA isolated from normal rats, both being identical to the ratio of their products synthesized in vivo.