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C B Dugan

Publications and source records attributed to C B Dugan.

13 recordsLinked to original sources
PubMed1996

Integration of foreign DNA in an intergenic region of the archaeon Methanosarcina mazei without effect on transcription of adjacent genes.

Transformation systems for methanogenic archaea are scarce, none has been reported for the genus Methanosarcina, and plasmids useful as vectors for cloning foreign DNA into methanogens that stably replicate as extrachromosomal elements are not available. We developed an integration vector for transformation of a member of the genus Methanosarcina, i.e. Methanosarcina mazei, using a segment (Int alpha; 1015 bp) which encompasses the intergenic region (431 bp) between the stress (heat-shock) genes grpE and dnaK. This segment also includes the 3' end (270 bp) of the grpE protein-coding region and the 5' end (314 bp) of the dnaK protein-coding region. Int alpha has an EcoRI site, useful for cloning, situated in the 3' direction beyond the grpE transcription termination region, and far upstream from the dnaK promoter. This location of the site, and the monocistronic mode of transcription of grpE and dnaK in M. mazei, suggested to us that a foreign insert in the site would not affect transcription of either flanking gene. A puromycin-resistance cassette (pac cassette) was inserted in the EcoRI site of Int alpha already inserted in pUC18, to obtain a vector which integrated the pac cassette in the chromosome between grpE and dnaK. The pac gene was transcribed and the transformants acquired puromycin resistance. Constitutive and heat-shock-induced transcription of grpE and dnaK in the transformants was the same as in wild-type cells. The two vectors found with transforming ability differed in the orientation of the pac cassette but both had M. mazei's DNA on each flank of the cassette, with the same orientation as that of the homologous segments in the chromosome.

Bacterial Proteins↗
PubMed1994

Identification of a grpE heat-shock gene homolog in the archaeon Methanosarcina mazei.

A grpE heat-shock gene was found by sequencing in the genome of the methanogenic archaeon Methanosarcina mazei S-6. It is the first example of grpE from the phylogenetic domain Archaea. Since the other seven sequenced homologs are from the domain Bacteria, it may be concluded that grpE appeared early in evolution, before the two domains separated. The archaeal grpE is located in the dnaK locus, 431 base-pairs upstream of dnaK, which is followed downstream by the dnaJ gene. The organization of these three genes is known for Bacillus subtilis, Clostridium acetobutylicum, Borrelia burgdorferi and Mycobacterium tuberculosis. The archaeal locus organization, grpE-dnaK-dnaJ, is similar to that of the former three bacteria, but different from that of M. tuberculosis. This, and sequence homologies, suggest that the M. tuberculosis GrpE belongs, together with the Streptomyces coelicolor homolog, to a subgroup of the GrpE proteins. The M. mazei grpE gene encodes a protein of 209 amino acid residues. The deduced amino acid sequence shows 28.2 to 34.6% identities, and 50.3 to 58.9 similarities (identities plus conservative substitutions) with the other six complete GrpE sequences available. These percentages fall within the range observed for the other GrpEs. Two regions in the second and fourth quarters of the GrpE molecule show higher homology, particularly in three stretches of nine, six and nine amino acid residues, respectively. The archaeal gene uses all codons but three, whereas the bacterial homologs lack higher numbers of codons. The M. mazei grpE responded to heat-shock by increasing transcription, in a manner similar to that of the nearby heat-shock gene dnaK.

Amino Acid Sequence↗
PubMed1993

An archaeal trkA homolog near dnaK and dnaJ.

The first trkA gene homolog in the phylogenetic domain Archaea is reported. The gene is located near the dnaK-dnaJ gene cluster in the genome of Methanosarcina mazei S-6, and encodes a protein homologous to the only other TrkA known, i.e., that of the bacterium Escherichia coli, involved in K+ transport. This finding supports an essential, evolutionarily early, and conserved role for this gene in cell survival and adaptation.

Amino Acid Sequence↗
PubMed1993

dnaJ in Archaea.

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Amino Acid Sequence↗
PubMed1991

A dnaK homolog in the archaebacterium Methanosarcina mazei S6.

A fragment of genomic DNA cloned from the methanogenic archaebacterium, Methanosarcina mazei strain S6, was found to contain an 1857-bp open reading frame (ORF). A sequence matching the consensus ribosome-binding sequence determined for other methanogens was found upstream from the ORF. The amino acid (aa) sequence encoded by the ORF was compared with reference sequences and was found to be related to six DnaK sequences determined for five species of eubacteria (none exist for archaebacteria). The M. mazei S6 aa sequence was over 61% identical and over 77% similar (identities plus conservative substitutions) to the closest four reference sequences, which were all DnaKs. The gene described here is therefore proposed to be the first member of the dnaK family sequenced from the archaebacterial kingdom (Archaea). This finding confirms that DnaK proteins are highly conserved, occurring not only in eubacteria (Bacteria) and eukaryotes (Eucaria), but also in archaebacteria (Archaea).

Amino Acid Sequence↗
PubMed1987

Antigenic mosaic of Methanogenium spp.: analysis with poly- and monoclonal antibody probes.

Eight well-characterized Methanogenium strains, including the six described type strains, were analyzed with poly- and monoclonal antibody probes to examine the antigenic mosaic of the genus. The pattern of cross-reactions showed that the mosaic is complex and varies with the strains; thus, these organisms have developed a considerable antigenic diversity, which is expressed in their envelopes. Every strain shared at least one determinant with at least one other strain, demonstrating the antigenic cohesiveness of the group. This finding, together with the fact that most strains displayed a distinctive antigenic fingerprint (notwithstanding the limited number of probes available), emphasizes the potential of antibodies for rapid identification of new isolates and for direct elucidation of Methanogenium strains in microbial mixtures.

Antibodies↗
PubMed1981

Phenotypic diversification of a cultured tumor line as a function of substratum.

We have found that a murine hepatoma displays a considerable phenotypic diversification in culture, which depends upon the substratum utilized, and is manifested by the formation of multicellular structures of differing geometry: Monolayer on glass and plastic, thick multilayer pads on Gelfilm, and spheroids on agar and agarose. These multicellular morphological phenotypes were assayed without disruption to ascertain their antigenicity in vitro and their tumorigenicity in vivo and to obtain quantitative information on the effect of the spatial arrangement of the hepatoma cells upon the ability of each multicellular structure to interact, as a whole, with molecules and cells in its surroundings. The antigenicity of the multicellular structures was determined with calibrated probes and a methodology that measures the total antigenicity, as well as antigenicity per unit of surface area. Antigenicity was found to differ in the following decreasing order: Monolayer on plastic greater than spheroids on agarose greater than spheroids on agar greater than multilayer on Gelfilm. At least part of these antigenic variants arise from different degrees of masking of the structures' surface determinants by a trypsin-sensitive material. The multicellular phenotypes also differed in tumorigenicity. When assayed in syngeneic hosts under comparable conditions, agar-grown spheroids produced the fewest tumors, whereas Gelfilm-grown multilayers produced the most. These two independent sets of data show that the various geometries that a tumor tissue is induced to acquire by the culture substratum are accompanied by a distinctive combination of surface and biological properties.

Agar↗