Qualitative and quantitative immunofluorescence studies of chloroplast ferredoxin

Antibodies were raised in rabbits against 2Fe–2S ferredoxin from N. tabacum L. The antibodies showed partial cross-reactivity in the double diffusion test with ferredoxins from Spinacia oleracea L., Petunia inflata Fries., P. axillaris Lam., Phaseolus vulgaris L., Chlamydomonas remhardii Dang. A complete cross-reaction was observed with ferredoxins from five other Nicotiana species, thus with this test it was impossible to discriminate between these ferredoxins. Therefore the following test was performed. Heterologous ferredoxin (i.e., ferredoxin other than from N. tabacum) was coupled covalently to Sepharose beads. Rabbit anti-N. tabacum-serum was then pre-incubated with this ferredoxin which resulted in complete abolition of cross-reactivity with free heterologous ferredoxin. However, the serum retained antibody activity against specific antigenic determinants of N. tabacum ferredoxin. When this serum was tested against ferredoxin purified from the hybrid: N. tabacum ()xN. glutinosa () it gave a positive reaction. The relative content of maternal N. tabacum ferredoxin in the hybrid was estimated by using a fluorescent derivative of this specific antibody and estimating the cross-reactivity compared with that of artificial mixtures of pure N. tabacum and N. glutinosa ferredoxins. The hybrid contained 50% of maternal ferredoxin. This technique was also applied to ferredoxins of other species of Nicotiana and to the ferredoxin from the hybrid N. clevelandii ()xN. glutinosa (). We conclude that it provides a good test system for the study of the expression of chloroplast ferredoxin in Nicotiana hybrids in general.Abbreviations PBS phosphate buffered saline - FITC fluorescein isothiocyanate - S.E.M. standard error of means     

An unusual phenotypic expression of Hb-Leiden

After a boy of Mexican-American descent became jaundiced during treatment of a serious urinary tract infection with an oxidant drug, an extensive hematological examination was made. The important finding was the presence of Hb-Leiden to the extent of less than 3% or about a tenth of the usual percentage. Although inclusion bodies are present in the erythrocytes at all times, his hematological parameters have remained normal. The genetic basis for the unusually small amount of Hb-Leiden in the propositus may be due to the Hb-Leiden gene in an anti-Lepore configuration, that is, and Hb-Leiden gene in cis to the normal β and δ genes.     

Heterozygosity for the IVS-I-5 (G-->C) mutation with a G-->A change at codon 18 (Val-->Met; Hb Baden) in cis and a T-->G mutation at codon 126 (Val-->Gly; Hb Dhonburi) in trans resulting in a thalassemia intermedia.

We have analyzed the hemoglobins of a young German patient with beta-thalassemia intermedia and of his immediate family and included in these studies an evaluation of possible nucleotide changes in the beta-globin genes through sequencing of amplified DNA. One chromosome of the propositus and one of his father's carried the GTG-->GGG mutation at codon 126 leading to the synthesis of Hb Dhonburi or alpha 2 beta (2)126(H4)Val-->Gly; this variant is slightly unstable and is associated with mild thalassemic features. His second chromosome and one of his mother's had the common IVS-I-5 (G-->C) mutation that leads to a rather severe beta(+)-thalassemia and the GTG-->ATG mutation at codon 18, resulting in the replacement of a valine residue by a methionine residue. This newly discovered beta-chain variant, named Hb Baden, was present for only 2-3% in both the patient and his mother. This low amount results from a decreased splicing of RNA at the donor splice-site of the first intron that is nearly completely deactivated by the IVS-I-5 (G-->C) thalassemic mutation. The chromosome with the codon 18 (GTG-->ATG) and the IVS-I-5 (G-->C) mutations has thus far been found only in this German family; analysis of 51 chromosomes from patients with the IVS-I-5 (G-->C) mutation living in different countries failed to detect the codon 18 (GTG-->ATG) change.     

Synthesis of poly(3-hydroxyalkanoates) by mutant and recombinant Pseudomonas strains

We have studied the accumulation kinetics and physical characteristics of the poly(3-hydroxyalkanoates) (PHAs) formed by several Pseudomonas strains, mutants and recombinants. Although PHA synthesis generally begins only after an essential nutrient such as N, P, S or Mg becomes limiting, we have identified at least one strain (P. putida KT2442) that begins producing PHA during the exponential growth phase. This PHA is chemically and physically identical to that produced by P. oleovorans GPol, the strain in which we first identified PHA. Analysis of the PHA formed by a mutant strain defective in PHA degradation (P. oleovorans GPo500) revealed that the molecular mass (M_w), the monomer composition and thermal characteristics were similar to that of the PHA of the wild-type parent strain P. oleovorans GPo1. The pha locus of P. oleovorans encodes enzymes that are involved in PHA biosynthesis and degradation. It has been subcloned to study the two PHA polymerases separately in a PHA^– mutant (GPp104) derived from P. putida KT2442. The recombinant strains accumulated lower PHA levels than the wild-type strains, and the M_w of these polymers were lower than those produced by the wild-type P. oleovorans and parent strain. The monomer composition of the two PHAs formed by the two PHA polymerases differed, indicating that the PHA polymerases have different substrate specificities for the incorporation of 3-hydroxyoctanoate and 3-hydroxyhexanoate monomers into PHA. Despite these differences, the PHAs formed were essentially indistinguishable from wild-type PHAs with respect to their thermal characteristics.Correspondence to: B. Witholt     

Proteolysis and modulation of the activity of the cell division inhibitor SulA in Escherichia coli lon mutants.

Intracellular accumulation of the inducible cell division inhibitor SulA is modulated by proteases that ensure its degradation, namely, the Lon protease and another ATP-dependent protease(s). Lon- cells are UV sensitive because SulA is stable. We asked whether these ATP-dependent proteases are more active when lon cells are grown at high temperature or in synthetic medium since these conditions decrease the UV sensitivity of lon cells. We found that these growth conditions have no direct effect on Lon-independent degradation of SulA. They may, instead, decrease the SulA-FtsZ interaction.     

An X-ray determination of the molecular interactions in hemoglobin C: a disease characterized by intraerythrocytic crystals.

The X-ray structure of cyanomet human hemoglobin C has been solved and refined, R ～27%. The molecular packing can be represented in two dimensions by two sets of parallel strands, one set in the $\text{b}$ direction and the other in the $\text{c}$ direction. Taken together the two sets of strands interconnect the molecules into square nets or layers where each molecule contacts its four nearest neighbors. Molecules in one layer are displaced in $\text{a}$ and $\text{b}$ so that they fit into the “holes” of the square arrays of the adjacent layers (normal to $\text{a}$) resulting in a pseudo body-centered cubic packing. This packing can account for the hemoglobin crystallization in and fragility of the erythrocytes. The aberrant β6A3 Lys residue is in a position to influence the crystal formation.     

On the chromatographic heterogeneity of human fetal hemoglobin.

Minor fetal hemoglobins in red cell hemolysates of newborn and adults with elevated levels of Hb F have been separated and quantitated by Biorex 70 column chromatography. In addition to Hb F1, other minor hemoglobin zones eluting before F1, pre-F1, and after F1, post-f1 have been observed. The relative amounts of the two pre-F1 zones and F1 are higher in the red cells of adults with 97--100% Hb F (homozygous hereditary persistence of fetal hemoglobin, homozygous deltabeta-thalassemia and homozygous beta0-thalassemia) than in the red cells of an adult with homozygous beta+-thalassemia with 66% Hb F, a child with a trisomy-D-13 having 38% Hb F, and in two newborn. Hb F was glycosylated in vitro with [14C]glucose or [14C] glucose 6-phosphate, and was acetylated using chicken reticulocyte lysate or a crude acetyltransferase preparation isolated from the same lysate with [14C]acetyl-CoA as substrate. Chromatographic analyses indicated that the Hb F1 zone can be formed both by glycosylation and acetylation of Hb F, and that pre-F1 zones can be products of the reaction of Hb F with phosphorylated glycolytic intermediates. Biosynthesis of minor hemoglobins in reticulocytes was studied with [14C]leucine in the presence and absence of cycloheximide and by pulse-chase. The resulting data indicate that Hb F1 synthesis is dependent upon Hb F synthesis and that the posttranslational modification may take place at an early stage in Hb F synthesis.     

Suppression of tif-mediated induction of SOS functions in Escherichia coli by an altered dnaB protein.

The tif-1 mutation in the Escherichia coli recA gene is known to cause induction of the various "SOS" functions at high temperature, including massive synthesis of the recA protein, lethal filamentation, elevated mutagenesis, and, in lambda lysogens, induction of prophage. It is shown here that the deoxyribonucleic acid initiation mutation dnaB252 suppresses all these manifestations of tif expression. Induction of lambda by ultraviolet irradiation, however, is not affected by the dnaB252 mutation. No similar suppression of tif is observed with other dnaB mutations affecting deoxyribonucleic acid elongation or with other deoxyribonucleic acid initiation mutations at the dnaA and dnaC loci. The fact that an alteration of the dnaB protein specifically suppresses tif-mediated SOS induction implies a role of the replication apparatus in this process, as has been suggested for ultraviolet induction. The induction of lambda is known to proceed via repressor cleavage, presumably promoted by an activated (protease) form of the recA protein. Since lambda induction is normal after ultraviolet irradiation of the tif-1 dnaB252(lambda) strain, tif-mediated induction in this strain may be blocked in a tif-specific step leading to activation of the recA (tif) protein. It is possible that the recA (tif) mutant protein may be directly involved in the replication complex in processes leading to this activation.     

Characterization of ferredoxins on a nanomole scale

Two method are described for the characterization of ferredoxins. First, mapping of tryptic peptides from 2 to 3 nmol of carboxymethylated ferredoxin by two-dimensional thin-layer electrophoresis and chromatography. Second, gel electrophoresis of tryptic digests of apoferredoxins. The latter method discriminates between ferredoxins of closely related species.