ISSN:0008-4166    

DOI:10.1139/m89-001

出版商:NRC Research Press    

年代:1989

数据来源: NRC

摘要:

By using affinity chromatography methods, we have purified elongation factor Tu (EF-Tu) proteins from a host of archaebacteria covering all known divisions in the archaebacterial tree except halophiles, and from such distantly related eubacteria asThermotoga maritimaandEscherichia coli. Polyclonal antibodies were raised against the Tu proteins ofSulfolobus solfataricus,Thermoproteus tenax,Thermococcus celer,Pyrococcus wosei,Archaeoglobus fulgidus,Methanococcus thermolitotrophicus,Thermoplasma acidophilum, andThermotogaand used to probe the immunochemical relatedness of elongation factors both within and across kingdom boundaries. A selection of the results, presented here, indicates that (i) every archaebacterial EF-Tu is closer (immunochemically) to every other archaebacterial EF-Tu than to the functionally analogous proteins of eubacteria and eukaryotes, with only one possible exception concerning die recognition of eukaryotic (EF-1α) factors byThermococcusEF-Tu antibodies, and (ii) within the archaebacteria there appears to be a correlation between EF-Tu immunochemical similarities and the phylogenetic relatedness of the organisms inferred from other (sequence) criteria. On the whole, immunochemical similarity data argue against the proposal that the archaebacterial taxon should be split and redistributed between two superkingdoms.Key words: phylogeny, archaebacteria, elongation factor Tu antibodies, eubacteria, eukaryotes.

ISSN:0008-4166    

DOI:10.1139/m89-002

出版商:NRC Research Press    

年代:1989

数据来源: NRC

摘要:

Using data from a partial protein sequence analysis of ribosomal proteins derived from the archaebacteriumMethanococcus vannielii, oligonucleotide probes were synthesized. The probes enabled us to localize several ribosomal protein genes and to determine their nucleotide sequences. The amino acid sequences that were deduced from the genes correspond to proteins L12 and L10 from therifoperon, according to the genome organization inEscherichia coli, and to proteins L23 and L2, which have comparable locations, as in theEscherichia coliS10 operon. Various degrees of similarity were found when the four proteins were compared with the corresponding ribosomal proteins of prokaryotic or eukaryotic organisms. The highest sequence homology was found in counterparts from other archaebacteria, such asHalobacterium marismortui,Halobacterium halobium, orSulfolobus. In general, theM.vannieliiprotein sequences were more related to the eukaryotic kingdom than to the Gram-positive or Gram-negative eubacteria. On the other hand, the organization of the ribosomal protein genes clearly follows the operon structure of theEscherichia coligenome and is different from the monocistronic eukaryotic gene arrangements. The protein coding regions were not interrupted by introns. Furthermore, the Shine–Dalgarno type sequences of methanogenic bacteria are homologous with those of eubacteria, and also their terminator regions are similar.Key words: archaebacteria, ribosomal proteins, evolution, gene organization,Methanococcus vannielii.

ISSN:0008-4166    

DOI:10.1139/m89-003

出版商:NRC Research Press    

年代:1989

数据来源: NRC

摘要:

The goal of our research is to produce an ordered set of cosmid clones for each of several species of halobacteria for use in physical and genetic mapping. These maps will answer questions about genome evolution and about gene organization and regulation in this archaebacterial lineage. Progress in cloning and mapping the genome ofHalobacterium volcaniiDS2 (synonymHaloferax volcaniiDS2) is reported. Overlapping cosmids are recognized by a strategy which makes use of the distinctive restriction fragments around relatively rare restriction sites. Each site recognized by the infrequently cutting restriction enzymes is a landmark from which to identify different regions of the genome. The main advantage of this strategy is that only a small overlap (10–20%) between cosmid clones is required, resulting in a correspondingly small number of cosmid clones to be analyzed. The certainty of overlap is high, and computation is simple. The final 5–10% of each genome is cloned, linked, and identified by chromosome walking methods. Hybridization of cloned homologous or heterologous genes and of stable RNAs to the minimal cosmid set localizes these genes on the physical map. Additional genes have been and will be cloned by complementation of auxotrophic mutants, or as determinants of resistance to antibiotics.Key words: landmark strategy, genome mapping, archaebacteria, cosmid.

ISSN:0008-4166    

DOI:10.1139/m89-004

出版商:NRC Research Press    

年代:1989

数据来源: NRC

摘要:

To identify an archaebacterial promoter sequence, nuclease protection studies with the purified RNA polymerase ofMethanococcus vannieliiwere performed. The enzyme binds specifically both at protein-encoding (hisAand methyl CoM reductase, component C) and tRNA–rRNA genes. The binding region of the RNA polymerase extends from 30 base pairs (bp) upstream (−30) to 20 bp downstream (+20) from thein vivotranscription start site. This finding indicates that the archaebacterial enzyme recognizes promoters without transacting traascription factors. The DNA segment protected from nuclease digestion by bound RNA polymerase contains an octanucleotide sequence centered at −25, which is conserved between the protein-encoding and the stable RNA genes. According to the specific binding of the enzyme to only DNA-fragments harbouring this motif, we propose the sequence TTTATATA as the major recognition signal of theMethanococcusRNA polymerase. Comparison of this motif with published archaebacterial DNA sequences revealed the presence of homologous sequences at the same location upstream of 36 genes. We therefore consider the overall consensusas a general element of promoters in archaebacteria. In spite of the specific binding of the enzyme, most preparations of theMethanococcus vannieliiRNA polymerase are unable to initiate transcription at the correct sitesin vitro. Here we present first evidence for the possible existence of a transcription factor conferring the ability to the enzyme to initiate and terminate transcription specificallyin vitro.Key words: promoter, footprint, TATA box, RNA polymerase, transcription.

ISSN:0008-4166    

DOI:10.1139/m89-005

出版商:NRC Research Press    

年代:1989

数据来源: NRC

摘要:

Anin vitroassay system has been developed for theHalobacterium volcaniitRNA intron endonuclease usingin vitrogenerated precursor RNAs. A partially purified enzyme preparation is capable of precise and accurate excision of the intron from the halobacterial tRNATrpprecursor. The cleavage reaction produces products having 5′ hydroxyl and 2′,3′ cyclic phosphate termini. Processing of precursor molecules containing deletions within the exon regions indicates that the halobacterial endonuclease does not require intact mature tRNA structure in the substrate; this is in contrast to the eukaryotic endonuclease enzyme that has an absolute requirement for these structures. The large halobacterial tRNATrpintron does not appear to be a primary site for recognition by the endonuclease, however, its removal affects cleavage efficiency. Through a comparison of the structural and sequence features of the halobacterial substrates and the precursors of other archaebacterial intron-containing precursors, a common element is proposed for the recognition of substrates by intron endonuclease.Key words: archaebacteria, intron, tRNA, evolution, manipulation.

ISSN:0008-4166    

DOI:10.1139/m89-006

出版商:NRC Research Press    

年代:1989

数据来源: NRC

摘要:

A cladistic analysis of 553 5S rRNA sequences has revealed a Ur-5S rRNA, the ancestor of all present-day 5S rRNA molecules. Previously stated characteristic differences between the eubacterial and eukaryotic molecules, namely, the length base-pairing schemes of helices D, can be used as a marker for the various archaebacterial branches. One model comprisesThermococcus,Thermoplasma, methanobacteria, and halobacteria; a second comprises theSulfolobales; and a third is represented only by the single organism Octopus Spring species 1. A relaxed selection pressure on helix E with subsequent deletions is observed inMethanobacteriales,Methanococcales, and eubacteria. The secondary structures are supported by enzymatic digestion and chemical modification studies of the 5S rRNAs. Reconstitution of eubacterial 50S ribosomal subunits with 5S rRNA fromHalobacteriumandThermoplasmahas revealed 100% incorporation, while eukaryotic 5S rRNAs yielded a 50% incorporation. Relevant positions of the small-subunit rRNA are selected to answer the question of the monophyly of archaebacteria. Eight positions account for monophyly, eight for an ancestry of eubacteria with halophile methanogens and eukaryotes with eocytes (paraphyly of archaebacteria), and two for an ancestry of eubacteria with eocytes. A refinement of the neighborliness method of S. Sattath and A. Tversky resulted in a monophyly of archaebacteria when all positions are treated equally and in a paraphyly when tranversions are weighted twice over transitions.Key words: archaebacteria, ribosomal RNA, evolution, cladistic analysis.

ISSN:0008-4166    

DOI:10.1139/m89-007

出版商:NRC Research Press    

年代:1989

数据来源: NRC

摘要:

A comparative analysis of cotranscribed gene clusters comprising the structural genesmcrA,mcrB,mcrC,mcrD, andmcrG was carried out in three species of methanogens.mcrA,mcrB, andmcrG are the structural genes for the three subunits of methyl coenzyme M reductase, while the two other genes encode polypeptides of unknown functions. The degree of conservation of themcrgene products among different species of methanogens varies. No correlation was found between the conservation of the G + C contents of the homologous genes and of the amino acid sequences of their products among the different bacteria. The comparison of RNA polymerase core subunit genes ofMethanobacterium thermoautotrophicumas evolutionary markers with their equivalents inEscherichia coli,Saccharomyces cerevisiae, andDrosophila melanogastershowed that homologous polypeptide domains are encoded by different numbers of genes suggesting gene fusion of adjacent genes in the course of evolution. The archaebacterial subunits exhibit much stronger homology with their eukaryotic than with their eubacterial equivalents on the polypeptide sequence level. All the analyzed genes are preceded by ribosome binding sites of eubacterial type. In addition to known putative promoter sequences, conserved structural elements of the DNA were detected surrounding the transcription initiation sites of themcrgenes.Key words: archaebacteria, methanogens, gene structure, RNA polymerase, promoter.

ISSN:0008-4166    

DOI:10.1139/m89-008

出版商:NRC Research Press    

年代:1989

数据来源: NRC

ISSN:0008-4166    

DOI:10.1139/m89-009

出版商:NRC Research Press    

年代:1989

数据来源: NRC

摘要:

Several phenotypic variants ofHalobacterium halobiumarise spontaneously at extremely high frequencies (up to 1%) and are readily identified by inspection of bacterial colonies. Two mutant types, those lacking the buoyant gas vesicles or the photosynthetic purple membrane, have been studied in detail by phenotypic and molecular genetic analysis. In the wild-type NRC-1 strain, thebopgene, encoding the purple membrane protein bacterio-opsin, is found on the bacterial chromosome, while the gas vesicle protein genes,gvpAandgvpC, are present on pNRC100, a multicopy plasmid of ~150 kilobase pairs. ThegvpAandgvpCgenes are on a single transcription unit, while the majorbopmRNA is monocistronic. Essentially all of the purple membrane deficient mutants contain insertion sequence (IS) elements into or upstream of thebopgene. Two elements, ISH 1 and ISH 2, account for most (80–90%) of the purple membrane mutations, but at least three other elements, ISH 23, ISH 26, and ISH S1, have also been implicated. The gas vesicle mutants are more heterogeneous, with many displaying partial phenotypes. Three major classes of gas vesicle mutants are distinguishable: class I and class III mutants are the result of large deletions in pNCR100; however, while class I mutants are partially gas vesicle deficient and contain a correspondingly reduced number ofgvpACoperon copies, class III mutants contain no detectable copies of the gas vesicle genes and are essentially completely gas vesicle deficient. Class II mutants, like the purple membrane mutants, contain IS elements into or upstream of the gas vesicle genes. At least five different IS elements are involved: ISH 2, ISH 3, ISH 4, ISH 6, and ISH 8.Key words: insertion sequence, transposable element, plasmid,Halobacterium halobium, bacteriorhodopsin, gas vesicle.

ISSN:0008-4166    

DOI:10.1139/m89-010

出版商:NRC Research Press    

年代:1989

数据来源: NRC