Biomedical subjects

Gregory W Warr

Publications and source records attributed to Gregory W Warr.

26 records · Page 2Linked to original sources

PubMed2004

Evolution of transcriptional control of the IgH locus: characterization, expression, and function of TF12/HEB homologs of the catfish.

The transcriptional enhancer (Emu3') of the IgH locus of the channel catfish, Ictalurus punctatus, differs from enhancers of the mammalian IgH locus in terms of its position, structure, and function. Transcription factors binding to multiple octamer motifs and a single muE5 motif (an E-box site, consensus CANNTG) interact for its function. E-box binding transcription factors of the class I basic helix-loop-helix family were cloned from a catfish B cell cDNA library in this study, and homologs of TF12/HEB were identified as the most highly represented E-proteins. Two alternatively spliced forms of catfish TF12 (termed CFEB1 and -2) were identified and contained regions homologous to the basic helix-loop-helix and activation domains of other vertebrate E-proteins. CFEB message is widely expressed, with CFEB1 message predominating over that of CFEB2. Both CFEB1 and -2 strongly activated transcription from a muE5-dependent artificial promoter. In catfish B cells, CFEB1 and -2 also activated transcription from the core region of the catfish IgH enhancer (Emu3') in a manner dependent on the presence of the muE5 site. Both CFEB1 and -2 bound the muE5 motif, and formed both homo- and heterodimers. CFEB1 and -2 were weakly active or inactive (in a promoter-dependent fashion) in mammalian B-lineage cells. Although E-proteins have been highly conserved in vertebrate evolution, the present results indicate that, at the phylogenetic level of a teleost fish, the TF12/HEB homolog differs from that of mammals in terms of 1) its high level of expression and 2) the presence of isoforms generated by alternative RNA processing.

Alternative Splicing↗

PubMed2004

Diversity of metallothioneins in the American oyster, Crassostrea virginica, revealed by transcriptomic and proteomic approaches.

Metallothioneins are typically low relative molecular mass (6000-7000), sulfhydryl-rich metal-binding proteins with characteristic repeating cysteine motifs (Cys-X-Cys or Cys-X(n)-Cys) and a prolate ellipsoid shape containing single alpha- and beta-domains. While functionally diverse, they play important roles in the homeostasis, detoxification and stress response of metals. The originally reported metallothionein of the American oyster, Crassostrea virginica showed the canonical molluscan alphabeta-domain structure. Oyster metallothioneins have been characterized as cDNA and as expressed proteins, and here it is shown that the previously reported metallothionein is a prototypical member of a subfamily (designated as CvMT-I) of alphabeta-domain metallothioneins. A second extensive subfamily of oyster metallothioneins (designated as CvMT-II) has apparently arisen from (a) a stop mutation that truncates the protein after the alpha-domain, and (b) a subsequent series of duplication and recombination events that have led to the development of metallothionein isoforms containing one to four alpha-domains and that lack a beta-domain. Analysis of metallothioneins revealed that certain CvMT-I isoforms showed preferential association either with cadmium or with copper and zinc, even after exposure to cadmium. These data extend our knowledge of the evolutionary diversification of metallothioneins, and indicate differences in metal-binding preferences between isoforms within the same family.

Animals↗

PubMed2002

The IgH locus of the channel catfish, Ictalurus punctatus, contains multiple constant region gene sequences: different genes encode heavy chains of membrane and secreted IgD.

The delta-chain of catfish IgD was initially characterized as a unique chimeric molecule containing a rearranged VDJ spliced to C micro 1, seven C domain-encoding exons (delta1-delta7), and a transmembrane tail. The presence of cDNA forms showing splicing of delta7 to an exon encoding a secretory tail was interpreted to indicate that membrane (deltam) and secreted (deltas) forms were likely expressed from a single gene by alternative RNA processing. Subsequent cloning and sequence analyses have unexpectedly revealed the presence of three delta C region genes, each linked to a micro gene or pseudogene. The first (IGHD1) is located 1.6 kb 3' of the functional C micro (IGHM1). The second (IGHD3) is positioned immediately downstream of a pseudo C micro (IGHM3P), approximately 725 kb 5' of IGHM1. These two delta genes are highly similar in sequence and each contains a tandem duplication of delta2-delta3-delta4. However, IGHD1 has a terminal exon encoding the transmembrane region, whereas IGHD3 has a single terminal exon encoding a secreted tail. The occurrence of IGHD3 immediately downstream of a micro pseudogene indicates that the putative deltas product may not be expressed as a chimeric micro delta molecule. Western blots and protein sequencing data indicate that an IGHD3-encoded protein is expressed in catfish serum. Thus, catfish deltam transcripts appear to originate from IGHD1, whereas deltas transcripts originate from IGHD3 rather than, as previously inferred, from a single expressed delta gene. The third delta (IGHD2) is associated with a pseudo C micro (IGHM2P); its presence is inferred by Southern blot analyses.

Amino Acid Sequence↗

PubMed2002

Oct2 transcription factor of a teleost fish: activation domains and function from an enhancer.

Oct2 transcription factors of the catfish (Ictalurus punctatus) are expressed as alternatively spliced alpha and beta isoforms. Functional analysis revealed an N-terminal glutamine (Q)-rich transactivation domain common to both isoforms of catfish Oct2. A C-terminal proline, serine, threonine (PST)-rich activation domain was identified exclusively in the beta isoform. Activation domains of fish and mammalian Oct2 showed cell type- and species-specific activity correlated with their biochemical composition (Q-rich vs PST-rich). In contrast the activation domains of the aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator of fish and mammals showed no correlation of activity with biochemical composition or species of origin. Although isolated catfish Oct2 activation domains were unable to drive transcription from a site 1.9kb distal to the promoter, Oct2beta activated transcription from both an IgH enhancer and an array of octamer motifs at this distal position. The properties of catfish Oct2 activation domains differ depending on whether they are studied in isolation or as components of the intact transcription factor.

Alternative Splicing↗

PubMed2002

Potential indicators of stress response identified by expressed sequence tag analysis of hemocytes and embryos from the American oyster, Crassostrea virginica.

A pilot program was initiated to identify genes from the American oyster, Crassostrea virginica, that are potentially involved in the stress response for use as bioindicators of exposure to environmental pollutants and to toxic and infectious agents. A PCR-based method was used to construct cDNA libraries from pooled embryos and the hemocytes of a single individual. A total of 998 randomly selected clones (expressed sequence tags, ESTs) were sequenced. Approximately 40% of the ESTs are novel sequences. Several potential biomarkers identified include an antimicrobial peptide, recognition molecules (lectin receptors), proteinases and proteinase inhibitors, and a novel metallothionein. Diversity analysis shows that 363 and 286 unique genes were identified from the hemocyte and embryo libraries, respectively, indicating that full-scale EST collection is a valuable approach for the discovery of new genes of potential significance in the molluscan stress response.

Journal Article↗

PubMed2002

Crustins, homologues of an 11.5-kDa antibacterial peptide, from two species of penaeid shrimp, Litopenaeus vannamei and Litopenaeus setiferus.

The response of crustaceans to pathogens is believed to depend solely on innate, nonadaptive immune mechanisms, including phagocytosis, encapsulation, clotting, and a variety of soluble antimicrobial activities. Arthropod antimicrobial peptides, while characterized primarily from insects, also have been isolated from crustaceans. Expressed sequence tag analysis of hemocyte complementary DNA libraries from 2 species of shrimp, Litopenaeus vannamei and Litopenaeus setiferus, revealed transcripts with strong sequence similarity to an 11.5-kDa antibacterial peptide (crustin Cm1) found in Carcinus maenas. Crustins were also observed to contain motifs common to proteinase inhibitors. Analysis of these cDNA libraries yielded at least 3 different isoforms of this peptide in L. vannamei (crustin Lv1-Lv3) and 3 in L. setiferus (crustin Ls1-Ls3). Further analysis of a second L. vannamei cDNA library revealed the presence of 3 more possible isoforms (crustin Lv4-Lv6), which differed from those seen in the first L. vannamei cDNA library. Genomic Southern blot analysis revealed a complex family of crustin-related sequences. However, full-length crustin appears to be encoded by a much more restricted subset of sequences within this family.

Journal Article↗

PubMed2002

Enhanced bacterial disease resistance of transgenic channel catfish Ictalurus punctatus possessing cecropin genes.

The cecropin B gene from the moth Hyalophora cecropia, driven by the cytomegalovirus promoter, was transferred to the channel catfish Ictalurus punctatus. Transgenic individuals (P1) were mated to produce individuals (F1) that exhibited enhanced disease resistance and survival when challenged with pathogenic bacteria. During the epizootic of Flavobacterium columnare in an earthen pond, the percentage of transgenic individuals containing preprocecropin B construct that survived (100%) was significantly greater (P <0.005) than that of nontransgenic controls (27.3%). Also, when challenged in tanks with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish, the percentage of transgenic individuals containing catfish IG leader cecropin B construct that survived (40.7%) was significantly greater (P <0.01) than that of nontransgenic controls (14.8%). There were no pleiotropic effects of the transgenes, and growth rates of the transgenic and nontransgenic siblings were not different (P > 0.05). Inheritance of the transgene by the F1 generation, 20.2% to 30.7% was typical of that in studies with transgenic channel catfish.

Journal Article↗

PubMed2002

Cloning of the IgM heavy chain of the bottlenose dolphin (Tursiops truncatus), and initial analysis of VH gene usage.

Clones encoding the dolphin IgM heavy (micro) chain gene were isolated from a cDNA library of peripheral blood leukocytes. Genomic Southern blot analyses showed that the dolphin IGHM gene is most likely present in a single copy, and its sequence shows greatest similarity to those of the IGHM gene of the sheep, pig and cow, evolutionarily related artiodactyls. The transmembrane (TM) form of the IGHM chain was isolated by 3' RACE. While showing similarities to the TM regions of other mammalian IGHM chains, the highly conserved Ser residue of the CART motif is substituted with a Gly in the dolphin. In contrast to the pig and cow, which utilize only a single VH family, the dolphin expresses at least two distinct VH families, belonging to the mammalian VH clans I and III. At least two JH genes were identified in the dolphin. Some CDR3 regions of the dolphin VH are long (up to 21 amino acids), and contain multiple Cys residues, hypothesized to stabilize the CDR3 structure through disulfide bond formation.

Amino Acid Sequence↗