In vitro translation.

The two most frequently used systems for in vitro translation are the rabbit reticulocyte system and the wheat germ extract. These systems are useful for mRNAs isolated from cells, tissues, and capped or uncapped mRNA produced in vitro by transcription of cDNA. In a combined system, mRNA can be transcribed and translated in a single reaction. In addition these systems can be used for translation reactions with biotinylated amino acids; this allows capture of the newly synthesized protein using streptavidin immobilized on agarose.

Conservation of primary structure in prokaryotic hydrogenases.

All prokaryotic (NiFe)-hydrogenases so far studied at the primary sequence level appear to have evolved from a common ancestral sequence. Highly conserved cysteinyl and histidinyl residues indicate regions likely to be essential for enzyme activity, ligand and co-factor binding. There is a very highly conserved sequence over 100 basepairs (bp) in length within the intergenic region upstream of the methyl-viologen hydrogenase encoding genes in several different strains of Methanobacterium thermoautotrophicum, indicating that a sequence of this length is needed to direct and regulate the expression of these genes.

Conservation of hydrogenase and polyferredoxin structures in the hyperthermophilic archaebacterium Methanothermus fervidus.

A 3.3-kilobase-pair region of the Methanothermus fervidus genome encoding part of the small subunit and all of the large subunit of the methyl viologen-reducing hydrogenase and a polyferredoxin was cloned and sequenced. The sequence of this hyperthermophilic hydrogenase conforms to the consensus sequence established for procaryotic [NiFe] hydrogenases. Although the M. fervidus polyferredoxin is the same size as the Methanobacterium thermoautotrophicum ferredoxin, containing six tandemly arranged bacterial ferredoxinlike domains, these two proteins are predicted to be only 64% identical in their primary sequences.

A hydrogenase-linked gene in Methanobacterium thermoautotrophicum strain delta H encodes a polyferredoxin.

The genes mvhDGA, which encode the subunit polypeptides of the methyl viologen-reducing hydrogenase in Methanobacterium thermoautotrophicum strain delta H, have been cloned and sequenced. These genes, together with a fourth open reading frame designated mvhB, are tightly linked and appear to form an operon that is transcribed starting 42 base pairs upstream of mvhD. The organization and sequences of the mvhG and mvhA genes indicate a common evolutionary ancestry with genes encoding the small and large subunits of hydrogenases in eubacterial species. The product of the mvhB gene is predicted to contain six tandomly repeated bacterial-ferredoxin-like domains and, therefore, is predicted to be a polyferredoxin that could contain as many as 48 iron atoms in 12 Fe4S4 clusters.

An archaebacterial promoter sequence assigned by RNA polymerase binding experiments.

To identify an archaebacterial promoter sequence, nuclease protection studies with the purified RNA polymerase of Methanococcus vannielii were performed. The enzyme binds specifically both at protein-encoding (hisA and 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 the in vivo transcription start site. This finding indicates that the archaebacterial enzyme recognizes promoters without transacting transcription 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 the Methanococcus RNA 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 consensus TTTATAATA as a general element of promoters in archaebacteria. In spite of the specific binding of the enzyme, most preparations of the Methanococcus vannielii RNA polymerase are unable to initiate transcription at the correct sites in 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 specifically in vitro.

An archaebacterial RNA polymerase binding site and transcription initiation of the hisA gene in Methanococcus vannielii.

Transcription initiation of the hisA gene in vivo in the archaebacterium Methanococcus vannielii, as determined by nuclease S1 and primer extension analyses, occurs 73 base pairs (bp) upstream of the translation initiation site. Binding of M. vannielii RNA polymerase protects 43 bp of DNA, from 35 bp upstream (-35) to 8 bp downstream (+8) of the hisA mRNA initiation site, from digestion by DNase I and exonuclease III. An A + T rich region, with a sequence which conforms to the consensus sequence for promoters of stable RNA-encoding genes in methanogens, is found at the same location (-25) upstream of the polypeptide-encoding hisA gene. It appears therefore that a TATA-like sequence is also an element of promoters which direct transcription of polypeptide-encoding genes in this archaebacterium.

Structure and organization of the hisA gene of the thermophilic archaebacterium Methanococcus thermolithotrophicus.

A restriction fragment of Methanococcus thermolithotrophicus genomic DNA was cloned into pUC8 to produce plasmid pET9301, which complements mutations in the hisA gene of Escherichia coli. Sequencing the DNA (2,155 base pairs) cloned from this thermophilic methanogen demonstrated that the M. thermolithotrophicus hisA gene is located within a cluster of open reading frames (ORFs) and is 68 and 69% homologous at the nucleotide level to the hisA genes of the mesophilic methanococci M. voltae and M. vannielii, respectively. The ORF (ORF 206) immediately 5' to the hisA gene of M. thermolithotrophicus is partially deleted in the genomes of the two mesophilic species, whereas ORF 114, which is 5' to ORF 206, is conserved in all three species.

Conservation of primary structure in the hisI gene of the archaebacterium, Methanococcus vannielii, the eubacterium Escherichia coli, and the eucaryote Saccharomyces cerevisiae.

A 2.7 kilobase pair (Kb) fragment of DNA, which complements mutations in the hisI locus of Escherichia coli, has been cloned and sequenced from the genome of the methanogenic archaebacterium Methanococcus vannielii. The cloned DNA directs the synthesis of three polypeptides, with molecular weights of 71,000, 29,000 and 15,600 in minicells of E. coli. Subcloning and mutagenesis demonstrates that hisI complementation results from the activity of the 15,600 molecular weight polypeptide. The primary structure of this archaebacterial gene and its gene product have been compared with the functionally equivalent gene and protein from the eubacterium E. coli (hisI) (Chiariotti et al. 1986) and from the eucaryote Saccharomyces cerevisiae (his4A) (Donahue et al. 1982). The DNA sequences of the archaebacterial and eubacterial genes are 40% homologous, the archaebacterial and eucaryotic DNA sequences are 47% homologous and, as previously reported (Bruni et al. 1986) the eubacterial and eucaryotic DNA sequences are 45% homologous. In E. coli the hisI locus is part of a bifunctional gene (hisI/E) within the single his operon. In S. cerevisiae the his4A locus is part of a multifunctional gene (his4) which encodes a protein with at least four enzymatic activities. The his genes of S. cerevisiae do not form an operon and are not physically linked. The M. vannielii hisI gene does not appear to be part of a multifunctional DNA sequence and, although it does appear to be within an operon, the open reading frames (ORFs) 5' and 3' to the M. vannielii hisI gene are not related to any published his sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and sequence divergence of two archaebacterial genes.

The DNA sequences of a region that includes the hisA gene of two related methanogenic archaebacteria, Methanococcus voltae and Methanococcus vannielii, have been compared. Both organisms show a similar genome organization in this region, displaying three open reading frames (ORFs) separated by regions of very high A + T content. Two of the ORFs, including ORFHisA, show significant DNA sequence homology. As might be expected for organisms having a genome that is A + T-rich, there is a high preference for A and U as the third base in codons. Although the regions upstream of the structural genes contain prokaryotic-like promoter sequences, it is not known whether they are recognized as promoters in these archaebacterial cells. A ribosome binding site, G-G-T-G, is located 6 base pairs preceding the ATG translation initiation sequence of both hisA genes. The sequences upstream of the two hisA genes show only limited sequence homology. The M. voltae intergenic region contains four tandemly arranged repetitions of an 11-base-pair sequence, whereas the M. vannielii sequence contains both direct and inverted repetitive sequences. Based on the degree of hisA sequence homology, we conclude that M. voltae and M. vannielii are less closely related taxonomically than are members of the enteric group of eubacteria.

Chloramphenicol acetyltransferase should not provide methanogens with resistance to chloramphenicol.

Growth of the four methanogens investigated was inhibited by chloramphenicol-3-acetate; therefore, introduction of chloramphenicol acetyltransferase-encoding genes should not confer chloramphenicol resistance on these methanogens. Reduction of the aryl nitro group of chloramphenicol produced a compound which did not inhibit the growth of these methanogens.