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. Namely, the young +/b animal displays reticulocytosis of 3.1 +/- 1.0%, identical to b/b rat, compared with 0.8 +/- 0.4% in young normal animals. This conclusion was further supported by examination of beta-globin expression. The level of beta-globin mRNA in anemic and heterozygous reticulocytes is decreased, as estimated by dot blot hybridization, to 25% and 50% of normal level, respectively. Although inapparent phenotypically, b mutated allele disturbs early erythropoiesis and markedly decreases globin mRNA level in the heterozygous rat.

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.

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. rettgeri PAC-encoding gene, the E. coli pac is poorly expressed in yeast. The highest yield of P. rettgeri PAC was obtained with a multi-copy plasmid, resulting in of 1500units per liter. This yield is higher by an order of magnitude than that obtained in the best recombinant bacterial expression system. The recombinant P. rettgeri enzyme is only partially and selectively O-glycosylated. Only every sixth or seventh alpha-subunit is glycosylated, while the beta-subunit is not glycosylated at all. N-Glycosylation has not been detected.

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. Poly(I) x poly(C) and cycloheximide induction resulted in an increased RNA level, but this was not reflected in an increased production of biologically active interferon.

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 1.4-2 times higher expression; (iii) the hybrid PLtl promoter permits flexible gene expression because it can be utilized under either or both repression controls simultaneously; (iv) the PLtl promoter permits enhanced expression of genes encoding products with unknown properties. When compared with the strong promoter PL from phage lambda, results with the PLtl promoter in lacZ fusion constructs show higher levels of beta-galactosidase activity. We also constructed a plasmid vector, pPLtl7G, which contains the hybrid PLtl promoter with a polylinker sequence at its 3' end, which facilitates efficient fusions of foreign genes in any of the reading frame.

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.3.5.1.11) of Providencia rettgeri was determined. We aligned our P. rettgeri PA with other known Gram negative periplasmically located beta-lactam amidases. The analysis revealed a high homology with other Enterobacteric amidases (60%-65%), while with similar Pseudomonas sp. amidases the homology exceeded 25%. These homologies indicate their common ancestry.

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. Our study indicates that the presence of hsp70-like protein in the b/b rat RBC plasma membrane is consistent with a primary defect and is not a consequence of life long stress, i.e. hypoxia.

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. One third of the total amount of mRNA found in mutant cells is not in polysomes as compared to only 6 percent for the mRNA from normal lysates. These results suggest that a translational control mechanism is involved although the decreased globin synthesis in b/b anemia can not be fully accounted for by its operation.

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. Iron-dextran administration corrected the level and globin ratio in b/b RBC thus confirming that the iron deficiency is the primary defect in b/b rats.

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. Spleen erythroid cells are defective in differentiation and contain a decreased share of globin polypeptides in total protein cell extracts compared to the control. Spleen cells are also characterized by a drastic imbalance of alpha- to beta-globin resulting in the beta-globin chains surplus.

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. Iron-dextran, as expected, ameliorated the imbalance of alpha- and beta-globin chains in the b/b rat spleen. On the other hand, hemin, as we have hypothesized in the accompanying paper, leads to a surplus of beta-globin chains in the bone marrow, similar to the one detected in the b/b rat spleen. Therefore, an iron-rich microenvironment has a stimulatory effect, while a hemin-rich microenvironment has an inhibitory effect on erythropoiesis.

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. Savic, J. Bacteriol. 140:311-319, 1979; D. J. Savic, S. Romac, and S. D. Ehrlich, J. Bacteriol. 155:943-946, 1983). The data show a slight structural divergence of alpha 3 beta 3 and beta 5 alpha 5 elements, but they do not reveal new sequences within recombined IS3 elements that could influence the expression of nearby genes.

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. These cloned cDNAs will be useful probes of structure and function of rat globin genes. The deduced amino acid sequences extend the information about the variability of rat globin chains.

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 4.1 in the b/b rat reticulocytes is the same as in normal rat. When haemolytic anaemia was induced in normal rat by in vivo phenyhydrazine treatment the same phenomenon, i.e., the absence of protein 4.1a in the RBC membrane was observed. The increase of 4.1a isoform was monitored in RBCs during the recovery of normal rat after phenyhydrazine treatment. Hence, the portion of membrane protein 4.1a isoform is increasing during rat RBC aging. Likewise, when the RBC life span is prolonged (but not normalised) in the b/b rats by iron-dextran treatment protein 4.1a is present in small portion in the RBC membrane. All these data indicate that the lack of protein 4.1a isoform in the b/b rat is due to the presence of young RBCs in the circulation.

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.

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. These data indicate that 68-kDa protein is normally present in rat RBC cytosol, is exported via exosomes during reticulocyte maturation, and is increased in induced haemolytic anaemia.

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. Another regulatory element from this region, betaDRE, binds erythroid-specific and ubiquitous factors from REL cells. Although both the CCAAT box region and betaDRE element bind the same protein factors before and after induction of REL cells, we show by South-Western blot analysis that the concentrations of 150-kDa, 70-kDa and 60-kDa factors binding to the betaDRE are increased in DMSO-induced REL cells.