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A fast and economical approach, referred to as cDNA clone tagging, was adapted to identify and isolate zebrafish cDNA clones. The basic approach was to partially sequence the coding region of size selected cDNA clones and the partial sequences were then used as tags for identifying the clones through homology search. To benefit maximally from the tagging approach, two cDNA libraries, derived from...
As a preliminary step in an analysis of Hox gene expression and radial body plan specification in sea urchin development, we amplified partial homeobox sequences in H. purpurescens by PCR using degenerate primers. The primers, HoxE and HoxF (), spanned a highly conserved region of 82 nucleotides encompassing amino acids 21-47 of the homeodomain. Seven Hox-type homeobox sequences and two non-Hox homeobox...
In order to investigate the role of bacterioferritin (Bfr) in the biomineralization of magnetite by microorganisms, we have cloned and sequenced the bfr genes from M. magnetotacticum. The organism has two bfr genes that overlap by one nucleotide. Both encode putative protein products of 18kDa, the expected size for Bfr subunits, and show a strong similarity to other Bfr subunit proteins. By scanning...
Proteasomes are large, multisubunit particles that act as the proteolytic machinery for most regulated intracellular protein breakdown in eukaryotic cells. The core proteinase of this complex, known as the 20S proteasome, is a hollow barrel-shaped structure made up of four stacked rings of seven subunits each, with the outer two rings each being made up of seven distinct α-type subunits, and the two...
A protein homologous to the Escherichia coli FtsY which in turn has characteristics in common with the α-subunit of the eukaryotic signal recognition particle receptor (SRα) in the membrane of the endoplasmic reticulum, was identified in Mycoplasma hominis and its encoding DNA sequenced. The aa similarity to E. coli FtsY and B. subtilis FtsY was 38% and 51%, respectively. The protein was synthesized...
The role of ecdysteroids in modulating exoskeletal growth during the moult cycle of Crustacea has been well described. However, little is known about the action of ecdysteroids at the level of gene transcription and regulation in Crustacea. This paper reports the cloning of an ecdysteroid responsive gene, HHR3, a potential Manduca sexta MHR3 homologue in the American lobster, Homarus americanus. Levels...
Two full-length cDNAs, gbr-2A and gbr-2B, encoding inhibitory amino acid receptor subunits have been amplified and cloned from Caenorhabditis elegans mRNA. The 5 732bp of the two cDNAs, encoding 237 amino acids, are identical. The 3 758bp of the gbr-2B cDNA are present within the 3 untranslated region of the gbr-2A clone. As a result, the two cDNAs are predicted to encode subunits which share...
Hepatitis C virus (HCV) has a positive-strand RNA genome that codes for a polyprotein precursor, which is processed co- and post-translationally by cellular and viral proteinases into three structural and at least six non-structural (NS) proteins. The NS5A protein, expressed in mammalian cells, exists in two phosphorylated forms of 56-kDa and 58-kDa. In this study, we provide evidence for a stable...
A mutation strategy which utilises phage display technology and the Escherichia coli mutator strains, mutD5-FIT and XL1-RED, was applied to a Hepatitis B (HepB) specific single-chain Fv (scFv) to incorporate random mutations throughout the gene. Messenger RNA from a hybridoma producing antibodies against HepB was isolated, reverse transcribed and used as template for the production of scFv. Following...
The ubiquitin-mediated degradation of cellular proteins requires the sequential activity of E1, E2 and, in some cases, E3 enzymes. Using the yeast two-hybrid system, we have cloned 1.0- and 2.5-kb cDNAs encoding the identical murine E2, or ubiquitin conjugating enzyme by virtue of its interaction with the E2A transcription factor. This cDNA encodes the 158-amino-acid protein, mUBC9, which has considerable...
The role of the polymeric receptor (pIgR) is to transport polymeric IgA across various mucosal epithelial layers. Although several mammalian pIgR cDNAs, including mouse, have been cloned, genomic structure has only been partially analyzed in the human, and neither its 5 -upstream region nor its transcriptional start site is known. We report the isolation and characterization of the murine pIgR gene...
Carrot cells cultured in vitro in a medium supplemented with 2,4-D (2,4-dichlorophenoxyacetic acid) proliferate as unorganized cell clusters. Upon removal of 2,4-D from the culture medium, these cells undergo somatic embryo formation through globular, heart, and torpedo stages. Since the proximal -255bp upstream region of the rolC gene of the Ri plasmid confers somatic embryogenesis-related activation...
Clostridium difficile is a causative agent in antibiotically induced diarrhea and pseudomembraneous colitis. The ability of strains of C. difficile to cause disease depends upon the presence of two toxin genes and their corresponding proteins, designated toxin A and toxin B. Previous studies conducted in this laboratory indicated that toxigenic strains of C. difficile possess both toxin genes, whereas...
The full length cDNA sequences of rat and mouse filensin are presented, as well as the structure of the rat filensin gene. This gene spanned 31kb and included seven introns. The first six introns were conserved in position and phase with those found in the intermediate filament (IF) protein genes of the type II (type II keratin), type III (vimentin) and type V (lamin). The last intron of the filensin...
In fish, cathepsin D, an aspartyl protease, is believed to mediate the processing of yolk proteins in the oocyte. Cathepsin D, therefore, is vital for the production of a viable egg. This study set out to isolate and sequence the cDNA encoding cathepsin D, and to determine the developmental expression of the message in the ovary and subsequently during embryogenesis in the rainbow trout, Oncorhynchus...
The gene encoding murine tetranectin (Tna) and its 5 -flanking region was isolated and cloned from a EMBL3 SP6/T7 genomic library. The compiled nucleotide sequence was determined by sequencing, revealing a conserved Tna structure in man and mouse. Mapping of the transcription start point (tsp) suggests that murine Tna has more than one of these. In addition, no consensus TATA-box was found uptream...
CneMDR1, a gene encoding a protein related to several eukaryotic multidrug resistance (MDR) proteins, was identified, cloned, and characterized from a clinical isolate of Cryptococcus neoformans (Cn) (strain M1-106). Polymerase chain reaction (PCR) amplification of a DNA region encompassing conserved motifs of other MDR-like proteins was initially used to identify and clone CneMDR1. Analysis of the...
The lrp gene of the extreme thermophilic archaeon Sulfolofus solfataricus, encoding a homologue of the eubacterial global leucine-responsive regulatory protein, was identified by DNA sequencing and sequence comparisons on a 6.9-kb genomic fragment cloned into Escherichia coli. The S. solfataricus Lrp subunit is a 155-aa polypeptide that bears between 24.5 and 29% sequence identity with eubacterial...
Chimeric genes containing portions of the mouse myelin proteolipid protein (PLP) gene fused to the lacZ reporter gene were used to detect the effect of PLP intron 1 sequences on cell type-specific expression. A transfected fusion gene containing PLP intron 1 sequences was expressed in an oligodendrocyte cell line but not in a liver cell line, consistent with endogenous PLP gene expression. However,...
The `translocon associated protein' is a tetrameric complex residing in the translocation sites, in which nascent polypeptides pass through the endoplasmic reticulum membrane. The β subunit of this complex is a single spanning membrane protein, as deduced from cDNAs deriving from canine or human epithelial tissues. We have isolated and analysed a cDNA clone of the β subunit from chick nervous tissue,...
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