Immunological evidence for the existence of H1-like histone in yeast

In view of the controversies about the existence of histone H1 in yeast we have reinvestigated the problem by studying yeast proteins extracted with perchloric acid and salt. Perchloric-acid-extracted proteins from whole cells contain only two fractions which comigrate with 'authentic' yeast high-mobility-group proteins (HMG) in both SDS and acid urea gels. These extracts show a considerable cross-reaction with anti-(calf thymus HMG) antiserum and do not react with antiserum to mouse liver H1. The isolation of 'authentic' yeast HMG by the standard salt/trichloroacetic acid procedure gives two types of preparations containing different numbers of protein bands. The poorer preparation reacts only with the anti-HMG antiserum whereas the richer preparation also gives considerable cross-reaction with the anti-H1 antiserum. Immunoblotting analysis performed on the salt-extracted proteins reveals the presence of three protein bands giving positive immunoreaction with the anti-H1 antiserum. The immunoreactive bands have electrophoretic mobilities close to that of the marker calf thymus H1 and similar to the mobilities of the presumptive yeast H1 fractions found by other authors.     

Segregation of nucleosomes in replicated mouse alpha-globin gene

DNA of mouse erythroleukemia cells grown in vitro was labeled with bromodeoxyuridine during cycloheximide-inhibited protein synthesis. Isolated nuclei were digested with micrococcal nuclease to obtain monosomes and monosomal dsDNA. The protection of the heavy and of the light strands of the newly replicated DNA was studied by dot hybridization with the coding and with its complementary noncoding strand of the alpha-globin gene. The results show that both sides of the replication fork contain protected sequences of the gene, thus supporting a bilateral (dispersive) mode of nucleosome segregation during DNA replication.     

Prevention by N-acetylcysteine of benzo[a]pyrene clastogenicity and DNA adducts in rats

The daily i.t. administration of benzo[a]pyrene (BP) to Sprague-Dawley rats, for 3 consecutive days, did not cause any toxicity or clastogenicity in bone marrow cells, as evaluated by monitoring the ratio of polychromatic to normochromatic erythrocytes and the frequency of micronucleated polychromatic erythrocytes. However, BP produced a considerable enhancement of binucleated and micronucleated pulmonary alveolar macrophages, as well as a significant increase in polymorphonucleates recovered by bronchoalveolar lavage. These effects were prevented by administering the thiol N-acetylcysteine (NAC) by gavage 5 h before each BP instillation. In addition, the i.t. treatment with BP resulted in the formation of BP diolepoxide (BPDE)-DNA adducts in lungs and liver, as assessed by synchronous fluorescence spectrophotometry, with fluorescence peaks of similar magnitude in the 2 tissues. Pretreatment with NAC by gavage completely prevented BPDE adducts to liver DNA and significantly decreased those to lung DNA.     

A rapid coagglutination test for the detection and serogrouping of Legionellae

The applicability of coagglutination for the rapid detection and serogrouping of Legionellae has been investigated. The coagglutination reaction is carried out with the aid of self-made preparations of protein A containing staphylococci, sensitized with specific antibodies against the antigens of L. pneumophila (serogroups I to 6), L. bozemanii and L. micdadei. Preliminary heating of Legionella suspensions at 100% C for 15 min was used to prevent cross coagglutination reactions and ensure greater safety of laboratory personnel during the performance of the test. The results obtained demonstrate a high specificity of coagglutination. With the aid of the coagglutination reactions it has been shown that L. pneumophila strains isolated in Bulgaria belong to serogroup I. The coagglutination method is characterized by its rapidity, simplicity and feasibility. It is a useful and convenient means for the rapid detection and serogrouping of Legionellae.     

A sensitive,simultaneous analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase efficiencies: Graphical determination of the CO2/O2 specificity factor

A simple approach to determine CO₂/O₂ specificity factor () of ribulose 1,5-bisphosphate carboxylase/oxygenase is described. The assay measures the amount of CO₂ fixation at varying [CO₂]/[O₂] ratios after complete consumption of ribulose 1,5-bisphosphate (RuBP). Carbon dioxide fixation catalyzed by the carboxylase was monitored by directly measuring the moles of ¹⁴CO₂ incorporated into 3-phosphoglycerate (PGA). This measurement at different [CO₂]/[O₂] ratios is used to determine graphically by several different linear plots the total RuBP consumed by the two activities and the CO₂/O₂ specificity factor. The assay can be used to measure the amounts of products of the carboxylase and oxygenase reactions and to determine the concentration of the substrate RuBP converted to an endpoint amount of PGA and phosphoglycolate. The assay was found to be suitable for all [CO₂]/[O₂] ratios examined, ranging from 14 to 215 micromolar CO₂ (provided as 1–16 mM NaHCO₃) and 614 micromolar O₂ provided as 50% O₂. The procedure described is extremely rapid and sensitive. Specificity factors for enzymes of highly divergent values are in good agreement with previously published data.Abbreviations HEPPS N-(2-hydroxyethyl)piperazine-N-(3-propanesulfonic acid) - L large subunit of rubisco - PGA 3-phosphoglyceric acid - rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP d-ribulose 1,5-bisphosphate - S small subunit of rubisco - XuBP d-xylulose 1,5-bisphosphate     

Uroporphyrinogen-I-synthetase in erythrocytes in acute intermittent porphyria

The primary genetic defect in acute intermittent porphyria is a decreased uroporphyrinogen I-synthetase [EC.4.3.1.8] activity. As a beginning of a genealogical study of the known families with members suffering from this disease in the People's Republic of Bulgaria, the red cell uroporphyrinogen I-synthetase was determined in 3 families by the method of Mandel et al [8]. Except for the three propositi, an enzyme deficiency was established in 3 latent carriers of the pathological gene, two of whom had normal values of the urinary epsilon-aminolevulinic acid and porphobilinogen. The determination of red cell uroporphyrinogen I-synthetase proved to be a valuable parameter for revealing the latent AIP.     

An antigen located in the kinetochore region in metaphase and on polar microtubule ends in the midbody region in anaphase,characterised using a monoclonal antibody

We describe a new component of the kinetochore region of Chinese hamster ovary cells, which was characterised using a monoclonal antibody (mAb). This antigen was localised on the kinetochore regions of purified metaphase chromosomes, but in anaphase it was instead located on the polar microtubules in the midbody region, where they terminate in the stembody. It was not detectable in prophase or interphase cells by immunofluorescence, but was present in the interphase nucleus as shown by immunoblotting after SDS-polyacrylamide gel electrophoresis. The mAb recognised two polypeptides of M_r 140 000 and 155 000. The localisation of this antigen in metaphase on the kinetochore region, where the plus ends of the kinetochore microtubules are temporarily stabilised when they attach, and later in the stembody and midbody where the plus ends of the polar microtubules are stabilised in anaphase and telophase, suggests that it could play a role in stabilising the plus ends of microtubules and thus in the control of microtubule dynamics during mitosis.     

Fractionation of rat-liver-chromatin nonhistone proteins into two groups with different metabolic rates.

In the pH interval 10.5-11.8, 70% of the nonhistone proteins normally present in rat liver chromatin were dissociated. The rest remained complexed with DNA even at pH 13. Dodecylsulfate-polyacrylamide gel electrophoresis revealed that the majority of the high-molecular-weight nonhistone proteins together with a few characteristic fractions with molecular weights of 40 000-60 000 remained in the alkali-resistant group. L-[14C]Leucine pulse-labelling experiments showed that the specific radioactivity of the alkali-labile nonhistone proteins was 2-3 times higher than that of the alkali-resistant nonhistone proteins, which, in turn, had the same specific radioactivity as that of the histones. The same held true for chromatin from regenerating rat liver. In the course of a 21-day chase the specific radioactivity of the alkali-labile nonhistone proteins gradually decreased and finally became 3 times lower than that of the alkali-resistant nonhistone proteins. On the contrary, the ratio of the specific radioactivities of the alkali-resistant nonhistone proteins and of the histones to the specific radioactivity of DNA remained constant during the chase. A conclusion can be drawn that a fraction of liver nonhistone proteins exists which is alkali-resistant and is conserved in chromatin like histones.     

Differential DNase I Sensitivity of the Two Complementary Nucleosomal DNA Strands in Cycloheximide-Treated Ehrlich Ascites Tumor Cells

Abstract

The accessibility of the two complementary DNA strands in newly replicated chromatin of Ehrlich ascites tumor (EAT) cells grown under conditions of cycloheximide-inhibrted protein synthesis was studied by analysis of the DNase I digestion of isolated nuclei. Bulk DNA was labeled with ¹⁴C-thymidine and the newly synthesized strands - with bromodeoxyu ridine and ³H-thymidine. The DNase I digests were fractionated in two successive CsCl density gradient centrifugations to obtain a dense fraction containing 15–20% newly replica ted DNA Analysis of the distribution of ¹⁴C-labeled parental DNA fragments complementary to the ³H-nascent strand has shown that the ¹⁴C-labeled fragments prevail in the region of 30–50 nucleotides. Simulation experiments using the rate constants for DNase I attack show that this result may be explained by an enhanced accessibility at the nucleosomal 5′-end region of the parental strands, where the H2a-H2b dimer interacts with DNA. This asymmetry seems tobe induced by interactions in the chromatin.