Clinical Studies and Physiological Properties of Hopkins-2 Haemoglobin

HAEMOGLOBIN Hopkins-2 (Ho-2) was discovered in a family in which haemoglobin S was also present¹. Independent segregation of the two abnormal haemoglobins provided the first convincing evidence that two genetic loci are concerned with synthesis of the haemoglobin molecule^1,2. Subsequently, evidence was obtained that the abnormality in haemoglobin Ho-2 is in the alpha chain³, whereas the lesion in haemoglobin S is in the beta chain of globin. We have reported the structural abnormality in Ho-2 and we now present a revised pedigree and the clinical status of carriers; we attempt to relate these findings to functional abnormalities exhibited by the haemoglobin.     

Duplicated α-Chain Genes in Hopkins-2 Haemoglobin of Man and Evidence for Unequal Crossing Over between Them

SYNTHESIS of the α and β-chains for haemoglobin is dictated by independent genetic loci. The first evidence for this notion came from Smith and Torbert's observation that inheritance of haemoglobin Hopkins-2 (Ho-2) was independent from that of haemoglobin S¹ and that Ho-2 was an α-chain variant². We wish to report the amino-acid replacements involved. These structural changes establish the presence of at least two α-chain genes in man. Some physical and physiological properties of the abnormal haemoglobin and the clinical status of carriers, have been reported in another article³.     

Identification of Mouse Haemoglobin Messenger RNA

RETICULOCYTE polyribosomes contain 9S RNA with many of the properties expected for the haemoglobin messenger RNA (mRNA)^1–12. Proof that this RNA is the haemoglobin (Hb) mRNA, however, can be obtained only by showing that it directs the synthesis of globin chains. Laycock and Hunt¹³ added an RNA isolated from rabbit reticulocytes to an E. coli cell-free preparation and observed the synthesis of material, with the properties of globin in the presence of N-acetylvalyl tRNA. We added the mouse reticulocyte 9S RNA to a rabbit reticulocyte cell-free system and have shown that material is synthesized which co-chromatographs with mouse globin β-chains¹⁴. We now present evidence that the material synthesized under the direction of the mouse 9S RNA is indeed mouse haemoglobin β-chains.     

Evidence for Various Types of Synthesis of Human γ Chains of Haemoglobin in Acquired Haematological Disorders

AT first, the γ chain of human haemoglobin seemed to be a constant chemical species¹, but Schroeder et al. showed that two or possibly four of the γ chain loci which control the synthesis of the two types of chains, ^Aγ and ^Bγ, are active in new born infants². The two chains differ only at position 136, where ^Aγ has alanine and ^Gγ has glycine. Thus although they are inseparable by chromatography and electrophoresis, their relative concentrations can be estimated from the ratio of these two amino-acids in the third and last peptide (γ^CB3) produced by hydrolysis of the chains by cyanogen bromide. Schroeder et al. studied the variations in the levels of these two chains during the normal ontogenic development and in several cases of hereditary persistence of foetal haemoglobin (HPHF) and β thalassaemias^3–5. We have studied the γ chains synthesized in different types of acquired anaemia with increased amounts of foetal haemoglobin, of which haemoglobin F constitutes between 1 and 15%. Such anaemias include refractory anaemias with excess myeloblasts (RAEM), acquired sideroblastic idiopathic anaemias (ASIA), idiopathic aplastic anaemias and some varieties of leukaemias, especially juvenile myeloid leukaemias (JML).     

Separation of α- and β-Globin Messenger RNAs

THE 10S RNA fraction of reticulocytes from various species contains the haemoglobin messenger RNA^1–4. When this 10S RNA fraction is added to a cell-free system derived from reticulocytes or Krebs II ascites cells, it directs the synthesis of α and β chains of haemoglobin^5–8. The α and β messenger RNA molecules contained in this fraction, however, have not yet been separated and identified. When reticulocyte. RNA of mouse is subjected to electrophoresis on 6% polyacrylamide gels, the 10S fraction contains two major bands and three minor bands⁹, suggesting that the major lOS RNA bands contain the messenger RNAs for the α- and β-globin chains.     

Possible Cytoplasmic Precursor of Haemoglobin Messenger RNA

THE “rapidly labelled” RNA of immature erythroblasts includes a rather homogeneous high specific activity RNA in the 9S region of linear sucrose gradients, but when the same RNA is assayed for ability to stimulate protein synthesis in a cell-free system, the peak of activity is found just trailing the 18S ribosomal RNA^1,2. Evidence has been assembled to support the contention that the 9S species of RNA is the haemoglobin messenger RNA³. While investigating the 9S RNA of chicken erythroblasts, we have found conditions in which a well defined rapidly labelled RNA peak could be observed in the 9S and/or the 17S region of the gradient. The concentration of pulse labelled RNA in the 17S region has been reported in diverse systems⁴ and may be a general phenomenon. It is particularly striking in the erythroblast system in which background ribosomal RNA synthesis is at a minimum.     

2,3-Diphosphoglycerate Content of Human Arterial and Venous Blood

THE glycolytic intermediate, 2,3-diphosphoglycerate, is an intracellular regulator of the oxygen affinity of haemoglobin^1,2. At high altitudes there is a direct relationship between the decreased oxygen affinity of haemoglobin and the increased concentration of diphosphoglycerate in the blood³. This was explained by Benesch et al.⁴ and Chanutin et al.⁵, who found that the binding of diphosphoglycerate to haemoglobin reduces the oxygen affinity and by our finding that the concentration of diphosphoglycerate increases when the red cells are incubated under low oxygen tension^6,7, thereby releasing oxygen from haemoglobin. For the same reason, the oxygen tension is reduced during the circulation of blood from the pulmonary alveoli to the tissues; the decreased level of the diphosphoglycerate facilitates the binding of oxygen to haemoglobin in the pulmonary alveoli and the increased level of the diphosphoglycerate in the blood of the capillaries decreases the affinity of haemoglobin for oxygen. We have measured the amount of 2,3-diphosphoglycerate and other glycolytic intermediates in arterial and venous blood to test this supposition.     

Differences in shoot Na+ accumulation between two tomato species are due to differences in ion affinity of HKT1;2

HKT1 has been shown to be essential in Na⁺ homeostasis in plants. In this paper, we report the analysis of Na⁺ accumulation in different plant organs of two tomato species with contrasting salt tolerances: Solanum lycopersicum and Solanum pennellii. Furthermore, we relate these differences in Na⁺ accumulation between the two species to the differences in HKT1;2 transport kinetics and HKT1;2 expression. S. lycopersicum showed “Na⁺ excluder” behaviour, whereas S. pennellii showed “Na⁺ includer” behaviour. SlHKT1;2 expression, in contrast to SpHKT1;2 expression showed a significant effect of NaCl treatment, especially stems had a high increase in SlHKT1;2 expression. SlHKT1;2 promoter-GUS reporter gene analysis showed that SlHKT1;2 is expressed in the vasculature surrounding the roots and shoots of transformed Arabidopsis plants. In this paper, we present HKT1;2 protein sequences of both tomato species and provide evidence that both SlHKT1;2 and SpHKT1;2 are Na⁺ transporters. Our kinetic studies showed that SpHKT1;2, in comparison with SlHKT1;2, had a lower affinity for Na⁺. This low affinity of SpHKT1;2 correlated with higher xylem Na⁺ and higher accumulation of Na⁺ in stems and leaves of S. pennellii. Our findings demonstrate the importance of the understanding of transport characteristics of HKT1;2 transporters to improve the understanding of Na⁺ homeostasis in plants.     

The xeroderma pigmentosum group E gene product DDB2 activates nucleotide excision repair by regulating the level of p21Waf1/Cip1

The xeroderma pigmentosum group E gene product DDB2, a protein involved in nucleotide excision repair (NER), associates with the E3 ubiquitin ligase complex Cul4A-DDB1. But the precise role of these interactions in the NER activity of DDB2 is unclear. Several models, including DDB2-mediated ubiquitination of histones in UV-irradiated cells, have been proposed. But those models lack clear genetic evidence. Here we show that DDB2 participates in NER by regulating the cellular levels of p21^Waf1/Cip1. We show that DDB2 enhances nuclear accumulation of DDB1, which binds to a modified form of p53 containing phosphorylation at Ser18 (p53^S18P) and targets it for degradation in low-dose-UV-irradiated cells. DDB2^−/− mouse embryonic fibroblasts (MEFs), unlike wild-type MEFs, are deficient in the proteolysis of p53^S18P. Accumulation of p53^S18P in DDB2^−/− MEFs causes higher expression p21^Waf1/Cip1. We show that the increased expression of p21^Waf1/Cip1 is the cause NER deficiency in DDB2^−/− cells because deletion or knockdown of p21^Waf1/Cip1 reverses their NER-deficient phenotype. p21^Waf1/Cip1 was shown to bind PCNA, which is required for both DNA replication and NER. Moreover, an increased level of p21^Waf1/Cip1 was shown to inhibit NER both in vitro and in vivo. Our results provide genetic evidence linking the regulation of p21^Waf1/Cip1 to the NER activity of DDB2.     

Chemical rescue of a site-modified ligand to a [4Fe-4S] cluster in PsaC, a bacterial-like dicluster ferredoxin bound to Photosystem I

Chemical rescue of site-modified amino acids using externally supplied organic molecules represents a powerful method to investigate structure-function relationships in proteins. Here we provide definitive evidence that aryl and alkyl thiolates, reagents typically used for in vitro iron-sulfur cluster reconstitutions, serve as rescue ligands to a site-specifically modified [4Fe-4S]^1+,2+ cluster in PsaC, a bacterial dicluster ferredoxin-like subunit of Photosystem I. PsaC binds two low-potential [4Fe-4S]^1+,2+ clusters termed F_A and F_B. In the C13G/C33S variant of PsaC, glycine has replaced cysteine at position 13 creating a protein that is missing one of the ligating amino acids to iron-sulfur cluster F_B. Using a variety of analytical techniques, including non-heme iron and acid-labile sulfur assays, and EPR, resonance Raman, and Mössbauer spectroscopies, we showed that the C13G/C33S variant of PsaC binds two [4Fe-4S]^1+,2+ clusters, despite the absence of one of the biological ligands. ¹⁹F NMR spectroscopy indicated that the external thiolate replaces cysteine 13 as a substitute ligand to the F_B cluster. The finding that site-modified [4Fe-4S]^1+,2+ clusters can be chemically rescued with external thiolates opens new opportunities for modulating their properties in proteins. In particular, it provides a mechanism to attach an additional electron transfer cofactor to the protein via a bound, external ligand.     

An evolutionary approach uncovers a diverse response of tRNA 2-thiolation to elevated temperatures in yeast

Chemical modifications of transfer RNA (tRNA) molecules are evolutionarily well conserved and critical for translation and tRNA structure. Little is known how these nucleoside modifications respond to physiological stress. Using mass spectrometry and complementary methods, we defined tRNA modification levels in six yeast species in response to elevated temperatures. We show that 2-thiolation of uridine at position 34 (s²U₃₄) is impaired at temperatures exceeding 30°C in the commonly used Saccharomyces cerevisiae laboratory strains S288C and W303, and in Saccharomyces bayanus. Upon stress relief, thiolation levels recover and we find no evidence that modified tRNA or s²U₃₄ nucleosides are actively removed. Our results suggest that loss of 2-thiolation follows accumulation of newly synthesized tRNA that lack s²U₃₄ modification due to temperature sensitivity of the URM1 pathway in S. cerevisiae and S. bayanus. Furthermore, our analysis of the tRNA modification pattern in selected yeast species revealed two alternative phenotypes. Most strains moderately increase their tRNA modification levels in response to heat, possibly constituting a common adaptation to high temperatures. However, an overall reduction of nucleoside modifications was observed exclusively in S288C. This surprising finding emphasizes the importance of studies that utilize the power of evolutionary biology, and highlights the need for future systematic studies on tRNA modifications in additional model organisms.     

The measurement of the intrinsic alkaline Bohr effect of various human haemoglobins by isoelectric focusing

We have used isoelectric focusing to measure the differences between the pI values of various normal and mutant human haemoglobins when completely deoxygenated and when fully liganded with CO. It was assumed that the ΔpI(deox.–ox.) values might correspond quantitatively to the intrinsic alkaline Bohr effect, as most of the anionic cofactors of the haemoglobin molecule are `stripped' off during the electrophoretic process. In haemoglobins known to exhibit a normal Bohr coefficient (ΔlogP₅₀/ΔpH) in solutions, the ΔpI(deox.–ox.) values are lower the higher their respective pI(ox.) values. This indicates that for any particular haemoglobin the ΔpI(deox.–ox.) value accounts for the difference in surface charges at the pH of its pI value. This was confirmed by measuring, by the direct-titration technique, the difference in pH of deoxy and fully liganded haemoglobin A₀ (α₂β₂) solutions in conditions approximating those of the isoelectric focusing, i.e. at 5°C and very low concentration of KCl. The variation of the ΔpH(deox.–ox.) curve as a function of pH (ox.) was similar to the isoelectric-focusing curve relating the variation of ΔpI(deox.–ox.) versus pI(ox.) in various haemoglobins with Bohr factor identical with that of haemoglobin A₀. In haemoglobin A₀ the ΔpI(deox.–ox.) value is 0.17 pH unit, which corresponds to a difference of 1.20 positive charges between the oxy and deoxy states of the tetrameric haemoglobin. This value compares favourably with the values of the intrinsic Bohr effect estimated in back-titration experiments. The ΔpI(deox.–ox.) values of mutant or chemically modified haemoglobins carrying an abnormality at the N- or C-terminus of the α-chains are decreased by 30% compared with the ΔpI value measured in haemoglobin A₀. When the C-terminus of the β-chains is altered, as in Hb Nancy (α₂β^{Tyr-145→Asp}₂), we observed a 70% decrease in the ΔpI value compared with that measured in haemoglobin A₀. These values are in close agreement with the estimated respective roles of the two major Bohr groups, Val-1α and His-146β, at the origin of the intrinsic alkaline Bohr effect [Kilmartin, Fogg, Luzzana & Rossi-Bernardi (1973) J. Biol. Chem. 248, 7039–7043; Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema (1980) J. Mol. Biol. 138, 649–670]. In other mutant haemoglobins it is demonstrated also that the ΔpI(deox.–ox.) value may be decreased or even suppressed when the substitution affects residues involved in the stability of the tetramer. These results support the interpretation proposed by Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema [(1980), J. Mol. Biol. 138, 649–670] for the mechanism of the alkaline Bohr effect, and also indicate that the transition between the two quaternary configurations is a prerequisite for the full expression of the alkaline Bohr effect.     

The role of membrane-bound magnesium in the permeability of ghosts to K+

Mg²⁺ is required for restoring a low cation permeability of erythrocyte membranes after osmotic lysis. These ions promote binding of haemoglobin to the ghost membrane. Because the optimal binding occurs at a pH where the K⁺ retention by ghosts is maximal, the possibility exists that Mg²⁺ exerts an indirect effect by facilitating the adsorption of haemoglobin, which may be essential for the maintenance of a low permeability. In order to investigate this possibility, a systematic study was done of the effects of pH and hypotonic washing on the retention of haemoglobin, Mg²⁺ and K⁺ by human erythrocyte ghosts.Between pH 5.5 and 7.5, the retention of haemoglobin paralleled that of K⁺ and Mg²⁺ on resealing immediately after lysis. Such a parallelism was not observed if ghosts were both washed with fresh haemolytic media and resuspended in a medium of lower osmolarity before reversal.No correspondence was found between ghost K⁺ content and membrane-bound haemoglobin, indicating that the latter is not involved in the maintenance of a low permeability. By contrast, Mg²⁺ binding was altered by pH in the same way as ghost K⁺ and a strict relationship between membrane-bound Mg²⁺ and K⁺ retention was obtained.The results suggest that K⁺ permeability is mainly determined by the amount of Mg²⁺ associated with the ghost membrane.     

Establishment and characterization of three new cell lines from the embryonic tissue of Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae)

The establishment of new insect cell lines plays important roles in the researches of insect pathology, insect toxicology, insecticide screening and activity assay, etc. Using embryos of Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae) as materials, this study describes the establishment of three cell lines designated as QAU-Ho-E-3 (Ho-3), QAU-Ho-E-4 (Ho-4), and QAU-Ho-E-6 (Ho-6), respectively. Currently, the three cell lines have been passaged more than 50 times in the TNM-FH insect cell medium containing 10% fetal bovine serum (FBS). All of them showed adherent growth. The majority of Ho-3 cells are spindle-shaped, with a size of 24.35?±?5.29?×?11.56?±?1.67 μm. The Ho-4 cells were either spindle-shaped or oblong, with a size of 38.07?±?8.57?×?17.62?±?2.48 μm. The Ho-6 cells were primarily round in shape with a diameter of 14.54?±?1.96 μm. The Ho-3 and Ho-4 cell lines contained 20 chromosomes (i.e., diploid, 2n?=?20) at passages 14 and 45. The Ho-6 cell line contained 20 chromosomes (i.e., diploid, 2n?=?20) at passage 14 but 40 chromosomes (i.e., polyploidy, 4n?=?40) at passage 45. The results of random amplified polymorphic DNA (RAPD) analysis showed that the RAPD fingerprint of the three cell lines was consistent with that of H. oblita eggs, but clearly different from that of BTI-Tn5B1-4 and Sf-9 cells, demonstrating that the three cell lines Ho-3, Ho-4, and Ho-6 are H. oblita cell lines. The results of the growth curve test showed that the population doubling times of Ho-3, Ho-4, and Ho-6 were 101.1, 105.2, and 83.6 h, respectively. The viral infection assay indicated that these H. oblita cell lines were not permissive to infection by Autographa californica multiple nucleopolyhedrovirus (AcMNPV) or Bombyx mori nucleopolyhedrovirus (BmNPV).     

Molecular cytogenetic evidence for a high level of chromosome pairing among different genomes in Triticum aestivum–Thinopyrum intermedium hybrids

Intergeneric hybrids (ABDJJ^sS genomes) were made between Triticum aestivum cv. Chinese Spring (CS) and Thinopyrum intermedium. Genomic in situ hybridization (GISH) using genomic DNA probes from Pseudoroegneria libanotica (Hackel) D.R. Dewey (genome S, 2n = 14) was used to study chromosome pairing among J, J^s, S and wheat ABD genomes in the hybrids. It was shown that in the hexaploid (ABDJJ^sS) hybrids, high pairing occurred among wheat chromosomes and among Thinopyrum chromosomes. A closer relationship was observed among the three genomes of Th. intermedium than among the three genomes of T. aestivum. It was further discerned that S genome chromosomes paired with J- and J^s-genome chromosomes at a high frequency. The frequency of heterologous pairing between S and J or S and J^s chromosomes was higher than those between J and J^s chromosomes, indicating that the S-genome was more closely related with these two genomes. Our results provided direct molecular cytogenetic evidence for the hypothesis that S-genome chromosomes are genetically similar to the J-genome chromosomes and, therefore, genetic exchange between these genomes is possible. The discovery of a close relationship among S, J and J^s genomes provides valuable markers for molecular cytogenetic analyses using S-genomic DNA probes in monitoring the transfer of useful traits from Thinopyrum species into wheat. Received: 23 August 2000 / Accepted: 5 September 2000     

Pleiotropic role of the Sco1/SenC family copper chaperone in the physiology of Streptomyces

Antibiotic production and cell differentiation in Streptomyces is stimulated by micromolar levels of Cu²⁺. Here, we knocked out the Sco1/SenC family copper chaperone (ScoC) encoded in the conserved gene cluster ‘sco’ (the S treptomycesco pper utilization) in Streptomyces coelicolor A3(2) and S. griseus. It is known that the Sco1/SenC family incorporates Cu²⁺ into the active centre of cytochrome oxidase (cox). The knockout caused a marked delay in antibiotic production and aerial mycelium formation on solid medium, temporal pH decline in glucose‐containing liquid medium, and significant reduction of cox activity in S. coelicolor. The scoC mutant produced two‐ to threefold higher cellular mass of the wild type exhibiting a marked cox activity in liquid medium supplied with 10 µM CuSO₄, suggesting that ScoC is involved in not only the construction but also the deactivation of cox. The scoC mutant was defective in the monoamine oxidase activity responsible for cell aggregation and sedimentation. These features were similarly observed with regard to the scoC mutant of S. griseus. The scoC mutant of S. griseus was also defective in the extracellular activity oxidizing N,N′‐dimethyl‐p‐phenylenediamine sulfate. Addition of 10 µM CuSO₄ repressed the activity of the conserved promoter preceding scoA and caused phenylalanine auxotrophy in some Streptomyces spp. probably because of the repression of pheA; pheA encodes prephenate dehydratase, which is located at the 3′ terminus of the putative operon structure. Overall, the evidence indicates that Sco is crucial for the utilization of copper under a low‐copper condition and for the activation of the multiple Cu²⁺‐containing oxidases that play divergent roles in the complex physiology of Streptomyces.     

Antigenic Difference between Informofers and Protein bound to Polyribosomal mRNA from Rat Liver

NUCLEAR RNA with DNA-like base composition (heterogeneous nuclear RNA, Hn-RNA) is complexed with globular protein particles called informofers^1–4. When mRNA is liberated from polysomes by EDTA or incubation with puromycin, it is isolated as a ribonucleoprotein complex (mRNP)^5–8. Polyacrylamide gel electrophoresis has been used to study whether the informofer protein and the protein of polysomal mRNP are completely or partly identical^9–11. The protein bound to haemoglobin mRNA is different from the informofer protein, but in rat liver and sheep thyroid polysomes a protein was found with characteristics similar to the informofer protein^6,12,13. We have used a new immunological procedure to show that the proteins of the two ribonucleoprotein complexes in the rat liver are immunologically different.