Efficient transfection of the archaebacterium Halobacterium halobium.

We developed an efficient polyethylene glycol-mediated spheroplast transfection method for the extremely halophilic archaebacterium Halobacterium halobium. The 59-kilobase-pair linear phage phi H DNA molecule routinely produced between 5 X 10(6) and 2 X 10(7) transfectants per microgram of DNA. Between 0.5 and 1% of spheroplasts were transfected per microgram of luminal diameter H DNA. Under our conditions, survival and regeneration of H. halobium spheroplasts were also quite efficient, suggesting that this method will be useful for introducing other DNAs into these bacteria.     

Polyadenylated RNA isolated from the archaebacterium Halobacterium halobium.

Polyadenylated [poly(A)+] RNA has been isolated from the halophilic archaebacterium Halobacterium halobium by binding, at 4 degrees C, to oligo(dT)-cellulose. H. halobium contains approximately 12 times more poly(A) per unit of RNA than does the methanogenic archaebacterium Methanococcus vannielii. The 3' poly(A) tracts in poly(A)+ RNA molecules are approximately twice as long (average length of 20 nucleotides) in H. halobium as in M. vannielii. In both archaebacterial species, poly(A)+ RNAs are unstable.     

Putative promoter region of rRNA operon from archaebacterium Halobacterium halobium.

The 100 bp sequence from the beginning of the 16S rRNA gene of archaebacterium Halobacterium halobium and the adjacent 800 bp upstream sequence were determined. Four long (80 bp) direct repeats were found in the region preceeding the structural gene of the 16S rRNA. These repeats are proposed to constitute the promoter region of the rRNA operon of H. halobium.     

Soluble succinate dehydrogenase from the halophilic archaebacterium, Halobacterium halobium

Succinate dehydrogenase activity was found in both the cytoplasmic and the membrane fractions from disrupted Halobacterium halobium cells. The cytoplasmic enzyme was found to be soluble in aqueous media and had an apparent molecular weight of 90,000. The enzyme activity of the cytoplasmic succinate dehydrogenase was salt dependent, with preference for KCl over KNO3. The Km values for succinate of the soluble and the membrane-bound succinate dehydrogenases from H. halobium were 2.3 +/- 0.3 and 0.7 +/- 0.1 mM, respectively. The soluble succinate dehydrogenase was obtained from two different strains of H. halobium and was obtained independently of the method used to disrupt the bacteria. Thus, the archaebacterium, H. halobium, contains a succinate dehydrogenase which differs from the succinate dehydrogenase in most eucaryotic and eubacterial cells, where the enzyme is tightly membrane-bound.     

tRNA binding capacities of ribosomal subunits from the archaebacterium Halobacterium halobium

tRNA saturation experiments were performed with ribosomal subunits from the extreme halophilic archaebacterium Halobacterium halobium. In the presence of poly(U) the 30S subunit could bind equally well one AcPhe-tRNAPhe, Phe-tRNAPhe, or deacylated tRNAPhe molecule, respectively. Binding experiments with a mixture of two differently labeled tRNA species revealed that all three kinds of tRNA bound to one and the same binding site on the 30S subunit. Poly(U) dependent binding to the 50S subunit was insignificant for AcPhe-tRNA and Phe-tRNA. In the absence of poly(U) both AcPhe-tRNAPhe and Phe-tRNAPhe showed no significant binding to either subunit, whereas the binding of deacylated tRNAPhe could not be clearly determined. These results are in good agreement with those obtained from ribosomal subunits of the eubacterium Escherichia coli.     

Transformation of the archaebacterium Halobacterium volcanii with genomic DNA.

We describe optimization of a transformation system for the halophilic archaebacterium Halobacterium volcanii. Transformation of spheroplasts in the presence of polyethylene glycol permits the uptake and expression of high-molecular-weight linear fragments of genomic DNA as well as plasmid or bacteriophage DNA. Transformations can be performed with either fresh or frozen cell preparations. Auxotrophic mutants were transformed to prototrophy with genomic DNA from wild-type cells with efficiencies of 5 x 10(4)/micrograms of DNA and frequencies of 8 x 10(-5) per regenerated spheroplast. The overall efficiency of transformation with genomic DNA implies that genetic recombination is an efficient process in H. volcanii.     

Four different b-type cytochromes in the halophilic archaebacterium, Halobacterium halobium

The halophilic archaebacterium, Halobacterium halobium has been found to contain four different b-type cytochromes. The four components were recognized by their potentiometric characteristics in situ in their functional environment in the membrane of H. halobium. Oxidation-reduction midpoint potentials of these four b-type cytochromes were determined to be +261, +160, +30, and -153 mV, respectively. We also demonstrate that the pathway involved in the transport of reducing equivalents from succinate to oxygen proceeds through the b-type cytochromes with oxidation-reduction midpoint potentials of +261 and +161 mV. The cytochrome with oxidation-reduction midpoint potential of -153 mV was not substrate reducible by NADH but was chemically reducible by dithionite. Antimycin inhibits reduction of b-type cytochrome in the succinate pathway, but has no effect on b-type cytochrome reduction when reducing equivalents are provided by NADH. The carbon monoxide difference spectrum of H. halobium membranes shows at least one carbon monoxide-binding b-type cytochrome, indicating a terminal oxidase. A scheme for electron transport in H.halobium involving the b-type cytochromes and terminal oxidase is suggested.     

Nucleosomelike structures associated with chromosomes of the archaebacterium Halobacterium salinarium.

Chromosomes of the halophilic archaebacterium Halobacterium salinarium were examined by electron microscopy after being spread onto water. The major part of the chromosomal DNA was associated with protein particles with diameter of 9.4 nm, arranged tandemly along the DNA fibers. Thus, the primary structure of the chromosome resembles that of eucaryote chromosomes.     

Cloning of the gene for ribosomal protein HS4 in the archaebacterium Halobacterium halobium

Using a synthetic oligonucleotide probe, a gene of the ribosomal protein HS4 from an archaebacterium Halobacterium halobium has been cloned and partly sequenced. The translation initiation region contains a sequence complementary to the 3' end of the 16S rRNA.     

DNA polymerases alpha beta and gamma in inherited diseases affecting DNA repair.

Fibroblasts derived from patients with diseases affecting DNA repair processes, such as Xeroderma Pigmentosum (classical and variant), Fanconi's anemia, Bloom's syndrome, Ataxia Telangiectasica, Progeria and Werner's syndrome, were assayed for the three DNA polymerases. The specific activities of these enzymes were found within the limits observed in normal human fibroblasts. Also the sedimentation properties of the three polymerases were unaltered.     

Stimulation of an alpha like DNA polymerase by v-myc related protein of Halobacterium halobium

Partial DNA sequencing of a genomic clone of the archaebacterium Halobacterium halobium, which hydridized with an avian v-myc probe, showed especially the presence, in the organism of one of the conserved regions through myb, myc and adenovirus E1a oncogenes. The archaebacterial deduced amino acid sequence displayed significant homology with the v-myc gene product. In accordance with the partial DNA sequencing which assured a sufficient homology to have similar epitopes, a protein having a molecular weight of 70,000 and possessing high antigenicity with a polyclonal antiserum against avian v-myc protein was isolated and purified from H. halobium extracts. The purified v-myc like protein stimulated in vitro DNA synthesis carried out by the alpha like DNA polymerase of H. halobium.     

Oxidation-reduction potentials and absorption spectra of two b-type cytochromes from the halophilic archaebacterium, Halobacterium halobium

The oxidation-reduction midpoint potentials were determined for two b-type cytochromes, which had been solubilized from the membrane of Halobacterium halobium and partially purified. The two b-type cytochromes have oxidation-reduction midpoint potentials of 175 and 7 mV, respectively. These b-type cytochromes could also be resolved by difference absorption spectroscopy, which revealed one b-type cytochrome with absorption maximum (alpha-peak) at 558 nm, reducible by ascorbate-tetramethyl-p-phenylenediamine, and the other with absorption maximum (alpha-peak) at 560 nm, reducible by dithionite. Different substrates such as succinate, NADH, and alpha-glycerophosphate were used to study the b-type cytochromes in situ when bound to the membrane in a functional state. Reducing equivalents from succinate and alpha-glycerophosphate appear to enter the respiratory chain at the 175 mV b-type cytochrome. Cytochrome a3 is spectrophotometrically shown to be present in the membrane of H. halobium.     

Immunocytochemical localization of chick DNA polymerases alpha and beta +

An immunofluorescent method using specific antibodies was employed to detect DNA polymerases alpha and beta in chick cells. With monoclonal antibodies produced by four independent hybridoma clones, most of the DNA polymerase alpha was shown to be present in nuclei of cultured chick embryonic cells. With a polyclonal, but highly specific, antibody against DNA polymerase beta, this enzyme was also shown to be present in nuclei. DNA polymerase alpha was detected in proliferating cells before cell contact and in lesser amount in resting cells after cell contact, indicating that its content is closely correlated with cell proliferation. On the other hand, similar amounts of DNA polymerase beta were detected in proliferating and resting cells. Furthermore, DNA polymerase beta was detected in nuclei of most cells, while DNA polymerase alpha was detected only in large round nuclei in seminiferous tubules of chick testis. DNA polymerase alpha is presumably present in cells that are capable of DNA replication, and during the cell cycle it seems to remain in the nuclei during the G1, S, and G2 phases, but to leave from condensed chromatin for the cytoplasm during the mitotic phase.     

Chemosensory responses of Halobacterium halobium.

Responses of Halobacterium halobium cells to chemical stimuli have been shown by a capillary technique. Cells were attacted by D-glucose and several amino acids and repelled by phenol. Certain chemicals, such as acetate, benzoate, indole, and NiSO4, that are known to act as repellents of Escherichia coli cells served as attractants for Halobacterium. In the presence of ethionine, sensitivity to attractants was reduced. Arsenate prevented the attraction by glucose without lowering the cellular adenosine 5'-triphosphate level. The ability for chemo-accumulation toward glucose and histidine was interfered with by the formation of photosensory systems. Light-induced motor responses and chemosensory behavior toward glucose and histidine became detectable in the late stationary growth phase only. The behavior toward acetate and indole was not connected to photobehavior in that way: both substances acted as attractants already in the late log phase. Inhibition of bacteriorhodopsin synthesis by L-nicotine allowed chemo-accumulation toward glucose and histidine already in the late logarithmic phase.     

The number, physical organization and transcription of ribosomal RNA cistrons in an archaebacterium: Halobacterium halobium.

Because it is now clear that archaebacteria may be as distinct from eubacteria as either group is from eukaryotic cells, and because a specifically archaebacterial ancestry has been proposed for the nuclear-cytoplasmic component of eukaryotic cells, we undertook to characterize, for the first time, the ribosomal RNA cistrons of an archaebacterium (Halobacterium halobium). We found these cistrons to be physically linked in the order 16S-23S-5S, and obtained evidence that they are also transcribed from a common promoter(s) in the order 5'-16S-23S-5S-3'. We showed that, although slightly larger immediate precursors of 16S and 23S are readily seen, no common precursor of both 16S and 23S can be easily detected in vivo. In all these respects the archaebacterium H. halobium is like a eubacterium and unlike the nuclear-cytoplasmic component of eukaryotic cells. We found, however, that it differs from eubacteria of comparable (large) genome size in having only one copy of the rRNA gene cluster per genome.     

DNA-processing activities associated with the purified alpha, beta 2, and alpha beta molecular forms of avian sarcoma virus RNA-dependent DNA polymerase.

The RNA-dependent DNA polymerase purified from B77 avian sarcoma virus exhibited two distinct DNA-processing activities. The alpha and beta 2 isoenzymes possessed an endodeoxyribonuclease activity capable of nicking simian virus 40 superhelical DNA, whereas the alpha beta isoenzyme performed as an untwisting topoisomerase. Both activities associated with the three molecular forms of the retroviral DNA polymerase were dependent on the presence of either Mn2+ or Mg2+ ions. From analysis of the denaturated DNA products, it is apparent that the alpha and beta 2 isoenzymes introduced two nicks, one per each strand in the superhelical simian virus 40 DNA molecules, whereas the alpha beta polymerase converted these supercoiled molecules to the relaxed covalently closed circular form. The notion that the DNA-processing activities are located on the DNA polymerase molecules was supported by the following: (i) the three isoenzymes were of a high purity; (ii) the activities cosedimented in glycerol gradients with the DNA polymerase activities of the alpha, beta 2, and alpha beta molecular forms; and (iii) immunoglobulin directed against the purified polymerase immunoprecipitated the DNA-processing activities. Chemical treatments of the DNA polymerase molecules (with pyridoxalphosphate, iodoacetamide, and sulfhydryl reagents), which inhibited the polymerase activity, also suppressed the endonucleolytic and topoisomerase activities, suggesting that cystein and amino groups play an important role in the active sites of the DNA-processing activities as well.