Romanowsky dyes and Romanowsky-Giemsa effect

Summary A reproducible Romanowsky-Giemsa staining (RGS) can be carried out with standardized staining solutions containing the two dyes azure B (AB) and eosin Y (EY). After staining, cell nuclei have a purple coloration generated by DNA-AB-EY complexes. The microspectra of cell nuclei have a sharp and intense absorption band at 18 100 cm^–1 (552 nm), the so called Romanowsky band (RB), which is due to the EY chromophore of the dye complexes. Other absorption bands can be assigned to the DNA-bound AB cations.Artificial DNA-AB-EY complexes can be prepared outside the cell by subsequent staining of DNA with AB and EY. In the first step of our staining experiments we prepared thin films of blue DNA-AB complexes on microslides with 1:1 composition: each anionic phosphodiester residue of the nucleic acid was occupied by one AB cation. Microspectrophotometric investigations of the dye preparations demonstrated that, besides monomers and dimers, mainly higher AB aggregates are bound to DNA by electrostatic and hydrophobic interactions. These DNA-AB complexes are insoluble in water. Therefore it was possible to stain the DNA-AB films with aqueous EY solutions and also to prepare insoluble DNA-AB-EY films in the second step of the staining experiments. After the reaction with EY, thin sites within the dye preparations were purple. The microspectra of the purple spots show a strong Romanowsky band at 18 100 cm^–1. Using a special technique it was possible to estimate the composition of the purple dye complexes. The ratio of the two dyes was approximately EY:AB1:3. The EY anions are mainly bound by hydrophobic interaction to the AB framework of the electrical neutral DNA-AB complexes. The EY absorption is red shifted by the interaction of EY with the AB framework of DNA-AB-EY. We suppose that this red shift is caused by a dielectric polarization of the bound EY dianions.The DNA chains in the DNA-AB complexes can mechanically be aligned in a preferred direction k. Highly orientated dye complexes prepared on microslides were birefringent and dichroic. The orientation is maintained during subsequent staining with aqueous EY solutions. In this way we also prepared highly orientated purple DNA-AB-EY complexes on microslides. The light absorption of both types of dye complexes was studied by means of a microspectrophotometer equipped with a polarizer and an analyser. The sites of best orientation within the dye preparations were selected under crossed nicols according to the quality of birefringence. Subsequently, the absorption spectra of the highly orientated dye complexes were measured with plane polarized light. We found that the transition moments, m _AB, of the bound AB cations in DNA-AB and DNA-AB-EY are orientated almost perpendicular to k, i.e. m _ABk. On the contrary, the transition moments, m _EY, of the bound EY anions in DNA-AB-EY are polarized parallel to k, i.e. m _EY k. The transition moments m _AB and m _EY lay in the direction of the long axes of the AB and EY chromophores. For that reason, in both DNA-AB and DNA-AB-EY the long molecular axes of the AB cations are orientated approximately perpendicular to the DNA chains, while the long molecular axes of the EY chromophores are polarized in the direction of the DNA chains. Therefore, in DNA-AB-EY the long axes of AB and EY are perpendicular to each other, m _ABm _EY. This molecular arrangement fully agrees with our quantitative measurements and with the theory of the absorption of plane polarized light by orientated dye complexes, which has been developed and discussed in detail.     

Evolutionary conservation of the chromosomal configuration and regulation of amylase genes among eight species of the Drosophila melanogaster species subgroup

Nuclear DNA was extracted from each of the eight species comprising the Drosophila melanogaster species subgroup. Southern hybridization of this DNA by using a molecular probe specific for the alpha-amylase coding region showed that the duplicated structure of the amylase locus, first found in D. melanogaster, is conserved among all species of the melanogaster subgroup. Evidence is also presented for the concerted evolution of the duplicated genes within each species. In addition, it is shown that the glucose repression of amylase gene expression, which has been extensively studied in D. melanogaster, is not confined to this species but occurs in all eight members of the species subgroup. Thus, both the duplicated gene structure and the glucose repression of Drosophila amylase gene activity are stable over extended periods of evolutionary time.      

Modelluntersuchungen zur Struktur des purpurnen Farbstoffkomplexes der Giemsa-Färbung

Summary Nuclei of Giemsa stained cells show a purple coloration, which is generated by a complex of DNA, azure B (AB) and eosin Y (EY). The structure of this complex is unknown. Its absorption spectrum shows a sharp and strong band at 18 100 cm^–1 (552 nm), the so called Romanowsky band (RB). It is possible to produce the complex outside of the cell, but it is cubersome to handle. Easier to handle is a purple complex composed of chondroitin sulfate (CHS), AB and EY, which also shows a sharp and strong RB at 18100 cm^–1 in the absorption spectrum. This CHS-AB-EY complex is a model for the DNA-AB-EY complex of Giemsa stained cell nuclei. We tried to investigate its structure.In the first step of the staining procedure CHS binds AB cations forming a stable CHS-AB complex. In the case of saturation each anionic SO ₄ ^– - and COO^–-binding site of CHS is occupied by one dye cation and the complex has 1:1 composition. It has a strong and broad absorption band with its maximum at ca. 18000 cm^–1 (556 nm). In the second step the CHS-AB complex additionally binds EY dianions forming the purple CHS-AB-EY complex with its RB at 18100 cm^–1. This band can be clearly distinguished from the broad absorption of the bound AB cations. RB is generated by the EY chromophore, whose absorption is shifted to longer wavelength by the interaction with the CHS-AB framework.The CHS chains of the CHS-AB and CHS-AB-EY complexes can be mechanically aligned in a preferred direction k. Fine films of highly orientated complexes were prepared with a special technique and studied with a microspectrophotometer equipped with a polarizer and an analyzer. They are birefringent and dichroic-the more birefringent, the better the mechanical orientation. The sites of best orientation within the film were selected according to the quality of the birefringence. We measured the absorption of these regions with linearly polarized light. By setting the polarizer (e _p parallel () or perpendicular () to k, we found that the transition moment m _AB of the long wave-length absorption of AB in the CHS-AB and the CHS-AB-EY complexes is polarized almost perpendicular to the preferred direction k, m _AB k. But the transition moment m _EY of EY in CHS-AB-EY is polarized parallel to k, m _EY k. The transition moments m _AB and m _EY lay in the molecular plane in the direction of the long axes of the AB and EY chromophores, respectively. Therefore, in both CHS-AB and CHS-AB-EY the long axes of the AB molecules are approximately perpendicular to the CHS chain; but in CHS-AB-EY the long axes of the EY chromophore are parallel to the chain of the biopolymer. This structure is somewhat surprising. In the CHS-AB-EY dye complex the chromophores of AB and EY are not parallel but approximately perpendicular to each other.     

Growth of Phytomonas serpens in a Defined Medium; Nutritional Requirements

ABSTRACT. Three strains of Phytomonas serpens two from tomatoes, Lycopersicon esculentum one from the insect Phtia picta (Hemiptera, Coreidae), were cultivated in a chemically defined medium developed from a defined medium for cultivating insect flagellates. Besides organic growth factors required by other insect trypanosomatids this flagellate requires, serine and inositol. Glutamine stimulates growth, and, surprisingly, does not require heme.     

Membrane Skeletal Proteins and Their Integral Membrane Protein Anchors are Targets for Tyrosine and Threonine Kinases in Euglena

ABSTRACT. Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [³²P]-orthophosphate or γ-[³²P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.     

Sequence variation in the outer-surface-protein genes of Borrelia burgdorferi

Borrelia burgdorferi is a spirochete pathogen transmitted among warm- blooded hosts by ixodid ticks. Frequency-dependent selection for variant outer-surface proteins might be expected to arise in this species, since rare variants are more likely to avoid immune surveillance in previously infected hosts. We sequenced the OspA and OspB genes of nine North American strains and compared them with nine strains previously described. For each gene, the mean number of synonymous substitutions per synonymous site and the mean number of nonsynonymous substitutions per nonsynonymous site show only a twofold excess of silent mutations. Synonymous rates vary widely along the OspB protein. Some regions show a significant excess of silent substitutions, while divergence in other regions is constrained by biased base composition or selection. The presence, in antigenically important regions of the protein, of significant variation among strains, as well as evidence for recombination among strains, should be considered in attempts to develop vaccines against this disease.      

Biosafety investigations in an r-DNA production plant

Employees of a biotechnological production plant for recombinant interferon-2a have been participating in a 1-year study on occupational hygiene and the resulting biosafety aspects. Most of the employees have been employed in the plant for more than 6 years. Weekly stool samples were analysed for tetracycline (used as selection marker)-resistant coliforms as well as for rDNA (IFN gene) (interferon gene) and for the production organism. Various analytical methods, including the polymerase chain reaction, were applied to show that neither rDNA nor the production organism could be found in any of the stool samples and that there was no change or trend in the gut flora with respect to tetracycline resistance. In addition it could be shown that the tetracycline-resistance gene, as well as the rDNA, are completely inactivated in the course of the production process and thus no further recombination can take place. Blood samples were taken to show that none of the employees had anti-product antibodies. Correspondence to: E. K. Weibel     

Characterisation of a 34 kD protein from potato cyst nematodes, using monoclonal antibodies with potential for species diagnosis

Two monoclonal antibodies, which differentially recognise the two species of potato cyst nematodes (PCN), Globodera pallida and G. rostochiensis, are described. They have been shown to have potential for quantification of these two species, recognising proteins of the same molecular weight (34 kD) in both species. Further investigation showed these proteins to have isoelectric points at pH values of 5.7 in G. pallida and 5.9 in G. rostochiensis, in common with the proteins used by Fleming & Marks (1983) to differentiate the species of PCN. They are likely to be structurally very similar, with the same physiological function (and therefore similar concentrations) in the two species. In cross-reactivity tests with a wide range of soil nematode species, the antibodies reacted strongly only with species of the genus Globodera, and thereby confirmed their potential as the basis of a quantitative immunoassay likely to be useful in management of PCN populations.