Biomedical subjects

Moreland D Gibbs

Publications and source records attributed to Moreland D Gibbs.

10 recordsLinked to original sources

PubMed2007

Degenerate oligonucleotide gene shuffling.

Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling. Shuffling techniques can be used on a collection of mutants of the same gene, or related families of genes can be shuffled to produce mutants encoding chimeric gene products. One difficulty with current shuffling procedures is the predominance of unshuffled ("parental") molecules in the pool of mutants. We describe a procedure for gene shuffling using degenerate primers that allows control of the relative levels of recombination between the genes that are shuffled and reduces the regeneration of unshuffled parental genes. This procedure has the advantage of avoiding the use of endonucleases for gene fragmentation before shuffling and allows the use of random mutagenesis of selected segments of the gene as part of the procedure. We illustrate the use of the technique with a diverse family of beta-xylanase genes that possess widely different G and C contents.

Amino Acid Sequence↗

PubMed2006

New high fidelity polymerases from Thermococcus species.

Two DNA polymerase genes have been isolated from Thermococcus strains, Thermococcus zilligii from New Zealand, and the other, Thermococcus 'GT', a fast-growing strain isolated from the Galapagos trench. Both genes were isolated by genomic walking PCR, a technique that does not require expression of the gene product. Phylogenetic analysis of SSU rDNA showed that the two strains were not closely related, as confirmed by an examination of the DNA polymerase sequences. Inteinless versions of each gene were generated by overlap-extension PCR and transferred into plasmid expression vectors. The proteins were produced in an Escherichia coli strain with additional copies of tRNAs corresponding to rarely used codons and purified by standard chromatographic procedures. Both enzymes were able to support PCR, but the Thermococcus 'GT' polymerase required higher concentrations of template than the enzyme from T. zilligii. Both enzymes showed 3' to 5' exonuclease activity, which was abolished in the case of T. zilligii by mutating the aspartic acid at position 141 and the glutamic acid at position 143 to alanine. Both enzymes showed a significant increase in fidelity of replication compared to the family A Thermus aquaticus DNA polymerase, in agreement with other results reported for family B polymerases with proof-reading ability.

Amino Acid Sequence↗

PubMed2005

Degenerate oligonucleotide gene shuffling (DOGS) and random drift mutagenesis (RNDM): two complementary techniques for enzyme evolution.

Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling. Many gene shuffling techniques result predominantly in the regeneration of unshuffled (parental) molecules. We describe a procedure for gene shuffling using degenerate primers that allows control of the relative levels of recombination between the genes that are shuffled, and reduces the regeneration of unshuffled parental genes. This shuffling procedure avoids the use of endonucleases for gene fragmentation prior to shuffling and allows the inclusion of random mutagenesis of selected portions of the chimeric genes as part of the procedure. We illustrate the use of the shuffling technique with a family of beta-xylanase genes that possess widely different G+C contents. In addition, we introduce a new method (RNDM) for rapid screening of mutants from libraries where no adaptive selection has been imposed on the cells. They are identified only by their retention of enzymatic activity. The combination of RNDM followed by DOGS allows a comprehensive exploration of a protein's functional sequence space.

Bacterial Proteins↗

PubMed2005

Distribution of genes encoding the microbial non-oxidative reversible hydroxyarylic acid decarboxylases/phenol carboxylases.

Bacterial non-oxidative, reversible multi subunit hydroxyarylic acid decarboxylases/phenol carboxylases are encoded by the three clustered genes, B, C, and D, of approximately 0.6, 1.4, and 0.2 kb, respectively. There are more than 160 homologues in the database with significant similarity to gene B (homology to ubiX) and C (ubiD) distributed in all three microbial domains, however, homologues to gene D, are not numerous ( approximately 15). The occurrence of the entire BCD gene cluster encoding for either identified or presumptive hydroxyarylic acid decarboxylase to date has been revealed in Sedimentibacter hydroxybenzoicus (unique genes arrangement CDB), Streptomyces sp. D7, Bacillus subtilis, B. licheniformis, E. coli O157:H7, Klebsiella pneumoniae, Enterobacter cloacae, Shigella dysenteriae, Salmonella enterica, S. paratyphi, S. typhimurium, S. bongori, and S. diarizonae. The corresponding genes from S. hydroxybenzoicus, B. subtilis, Streptomyces sp. D7, E. coli O157:H7, K. pneumoniae, and S. typhimurium were cloned and expressed in E. coli DH5alpha (void of analogous genes), and shown to code for proteins exhibiting non-oxidative hydroxyarylic acid decarboxylase activity.

Bacteria↗

PubMed2004

Isolation, characterization and expression of the hex1 gene from Trichoderma reesei.

Polymers of the HEX1 protein produce Woronin bodies in filamentous fungi. We have isolated and sequenced the hex1 gene and flanking regions from the industrially exploited fungus Trichoderma reesei. Multiple transcription start sites (TSS) and the 5' untranslated region (UTR) were identified by 5'RACE PCR. There are three hex1 transcript types, two of which originate from two TSSs at approximately -320 and -1335 from the start codon, which are separated by a 500-bp intron within the 5'UTR. The third transcript type results from alternative splicing of the intron within the coding sequence at the 3' end, which results in the inclusion or exclusion of an unconserved histidine-rich coding region. The three transcripts code for two forms of HEX1 protein. N-terminal sequencing of HEX1 separated by 2D gel electrophoresis confirms that there are two forms of HEX1 protein which are modified further by alternative cleavage of the N-terminus. The dominant form of HEX1 is coded by a cDNA with TSS at position -1335. Expression of hex1 on cellulase-inducing medium peaks strongly within 24 h of growth but the protein is expressed at a lower and more consistent level in medium containing glucose. This is the first investigation of expression of the hex1 gene encoding a protein unique to filamentous fungi.

Amino Acid Sequence↗

PubMed2004

Thermophilic bacterial DNA polymerases with reverse-transcriptase activity.

Conserved motifs found in known bacterial polI DNA polymerase sequences were identified, and degenerate PCR primers were designed for PCR amplification of an internal portion of polI genes from all bacterial divisions. We describe here a method that has allowed the rapid identification and isolation of 13 polI genes from a diverse selection of thermophilic bacteria and report on the biochemical characteristics of nine of the purified recombinant enzymes. Several enzymes showed significant reverse-transcriptase activity in the presence of Mg2+, particularly the polymerases from Bacillus caldolyticus EA1, Caldibacillus cellovorans CompA.2, and Clostridium stercorarium.

Bacillus↗

PubMed2004

A yeast intron as a translational terminator in a plasmid shuttle vector.

Plasmid shuttle vectors that contain both prokaryotic (Escherichia coli) and eukaryotic origins of replication are routinely used in molecular biology since E. coli is generally the organism of choice for manipulation of recombinant DNA. Initial transformation of the shuttle vector into E. coli allows production of microgram quantities of DNA suitable for transformation of low-transformation-efficiency hosts. A shuttle/expression vector for the yeast Kluyveromyces lactis, pCWK1, allows recombinant protein fused to the killer toxin signal sequence to be secreted to the medium. The heterologous genes are transcribed under the control of the K. lactis LAC4 promoter, which is tightly regulated in K. lactis. However, in E. coli the LAC4 promoter functions constitutively, and as a result, uncontrolled transcription and translation of genes that are toxic in E. coli can result in cell death, and subsequent failure to recover intact E. coli transformants. We have constructed and tested a modified shuttle vector that contains a K. lactis ribosomal intron that acts as a translational terminator in E. coli, preventing or reducing the expression of recombinant proteins and avoiding toxicity. When transcribed in K. lactis, the intron is spliced from the mRNA allowing the translation of intact full-length, active recombinant gene product.

Amino Acid Sequence↗

PubMed2002

Prospecting for novel lipase genes using PCR.

A PCR method suitable for the isolation of lipase genes directly from environmental DNA is described. The problems associated with the low levels of similarity between lipase genes were overcome by extensive analysis of conserved regions and careful primer design. Using this method, a lipase gene (oli-lipase) was isolated directly from environmental DNA. This lipase showed less than 20% similarity with other known lipases at the amino acid level. The study also revealed that distantly related members of the alpha/beta hydrolase superfamily share similar conserved motifs with the lipases, thus making these genes targets for gene prospecting by PCR.

Bacillus↗

PubMed2002

Production of recombinant bleaching enzymes from thermophilic microorganisms in fungal hosts.

Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host-vector expression system critical. We have developed two systems for the bulk production of bleaching enzymes from thermophiles. Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2mu-like plasmid pKD1 of Kluyveromyces drosophilarium. Our second system involves the filamentous fungus Trichoderma reesei. Fusion and nonfusion vectors have been constructed using the strong cellobiohydrolase 1 (cbh1) promoter. The KEX2 protease cleavage site and a 6 x HIS-tag have been incorporated to facilitate both cleavage and purification of the mature foreign proteins.

Amino Acid Sequence↗

PubMed2000

A novel thermostable multidomain 1,4-beta-xylanase from 'Caldibacillus cellulovorans' and effect of its xylan-binding domain on enzyme activity.

The nucleotide sequence of the complete xynA gene, encoding a novel multidomain xylanase XynA of 'Caldibacillus cellulovorans', was determined by genomic-walking PCR. The putative XynA comprises an N-terminal domain (D1), recently identified as a xylan-binding domain (XBD), homologous to non-catalytic thermostabilizing domains from other xylanases. D1 is followed by a xylanase catalytic domain (D2) homologous to family 10 glycosyl hydrolases. Downstream of this domain two cellulose-binding domains (CBD), D3 and D4, were found linked via proline-threonine (PT)-rich peptides. Both CBDs showed sequence similarity to family IIIb CBDs. Upstream of xynA an incomplete open reading frame was identified, encoding a putative C-terminal CBD homologous to family IIIb CBDs. Two expression plasmids encoding the N-terminal XBD plus the catalytic domain (XynAd1/2) and the xylanase catalytic domain alone (XynAd2) were constructed and the biochemical properties of the recombinant enzymes compared. The absence of the XBD resulted in a decrease in thermostability of the catalytic domain from 70 degrees C (XynAd1/2) to 60 degrees C (XynAd2). Substrate-specificity experiments and analysis of the main products released from xylan hydrolysis indicate that both recombinant enzymes act as endo-1, 4-beta-xylanases, but differ in their ability to cleave small xylooligosaccharides.

Bacillaceae↗