Gene
We cloned two rabbit GRO homologue cDNAs from a lipopolysaccharide-stimulated rabbit alveolar macrophage (AM) cDNA library. One cDNA contains the complete coding sequence for a new mature GRO protein, RabGRO, which shares 68, 78 and 70% amino-acid identity with human GRO-α, -β and -γ, respectively. The other cDNA contains previously unreported sequence encoding a second GRO protein, rabbit permeability...
Previous studies have indicated that there may be uncleavable TaqI sites (TCGA) within the mouse myogenic gene,Myo-D1 . Fragments of DNA bearing most of the presumed insensitive TaqI sites have been reproduced using PCR. The presence of each of the originally uncleavable TaqI sites has been confirmed and each TaqI site has been shown to be sensitive to TaqI hydrolysis in PCR-synthesised genomic DNA...
We have constructed rare restriction-site (NotI, SacII and ClaI) chromosome 3 (Chr 3)-specific linking libraries in a plasmid-based vector by mass transfer of a λ phage human Chr-3-specific library (LA03NS01-ATCC57717) into pUC18. Total plasmid DNA isolated from the plasmid-based Chr-3-specific library was digested with either ClaI, NotI or SacII. Linear molecules were separated from undigested circles...
The vast majority of proteins in the nematode cuticle are collagens. Cuticular collagen-encoding genes (col) have been described for the animal parasites Ascaris suum and Haemonchus contortus and for the free-living Caenorhabditis elegans. The proteins encoded by all these genes seem to have the same basic structure, indicating that there is a conserved subfamily of cuticular col in these nematodes...
A 2.91-kb fragment of the Escherichia coli chromosome containing the speC gene, encoding biosynthetic ornithine decarboxylase (ODC) was sequenced. The speC gene is encoded by a 2133-bp ORF; the deduced amino-acid sequence contains 711 residues whose predicted molecular mass is 79 505 Da.
A gene (minK) that encodes a minimal potassium channel has been cloned recently. We describe in this paper a human minK sequence which differs from the original sequence with a single A→G at position 112. This resulted in a change from a Ser codon (AGT) to a Gly codon (GGT) and created a new MspAI restriction site. Of the 32 alleles from 16 subjects studied, 25 had this newly discovered sequence...
Using oligodeoxyribonucleotide primers and the polymerase chain reaction, we cloned and sequenced a 3.2-kb region which contains the gyrB gene, encoding the β-subunit of gyrase from Mycoplasma capricolum. We localized gyrB opposite from the chromosomal replication origin (ori) of M. capricolum by genomic Southern hybridization.
As a first step towards developing a DNA transformation method for the ciliated protozoan Euplotes crassus we determined the minimum inhibitory concentration (MIC) for cell division in the presence of cycloheximide (Chx) for several cell lines and the range of Chx sensitivity for 106 different progeny cell lines derived by mating two lines. All of the cell lines are highly sensitive to Chx. Progeny...
Rod photoreceptor cyclic GMP-phosphodiesterase (cGMP-PDE) is one of the key enzymes of the visual phototransduction cascade in the vertebrate retina. The holoenzyme is a heterotetrameric complex, consisting of two large catalytic subunits, α (88 kDa) and β (84 kDa), and two identical inhibitory subunits, γ (11 kDa). Here we present the complete nucleotide sequence of the gene (cGMP-PDEγ) encoding...
In order to obtain the 5 ends of the three mouse calmodulin (CaM) cDNAs, we modified the standard 5 RACE (rapid amplification of cDNA ends) method to use degenerate synthetic oligodeoxyribonucleotides to prime cDNA synthesis of all three CaM mRNAs. In this modified method, the degenerate primers were annealed to mRNAs in an incubation step prior to the reverse transcription reaction. Separating...
Here we describe the cloning of a full-length cDNA encoding a neutrophil chemoattractant, peptide ENA-78, from human platelets. The cDNA encodes a predicted sequence of 114 amino acids and contains the Cys motif C-X-C found in other members of the α-chemokine family which also includes interleukin 8 (IL-8). ENA-78 has a high degree of sequence identity with other platelet-derived chemokines which...
A 2433-bp, high-copy-number expression vector, pUK, was constructed from pUC19 and pKK223-3. The genes encoding chicken adenylate kinase (AK) and phospholipase A 2 , cloned into pUK, were highly expressed in vitro using the coupled transcription/translation system of Zubay. High-level in vivo expression in Escherichia coli was also demonstrated for the AK gene cloned in this vector. The...
We have developed a facile procedure for rapid PCR-based site-directed mutagenesis of double-stranded DNA. Increasing the initial template concentration and decreasing the PCR cycles to 5-10 allows us to reduce the rate of undesired second-site mutations and dramatically increase the time savings. Following PCR, DpnI treatment is used to select against parental DNA molecules. The DpnI (target sequence...
Two-component regulatory systems play important roles in the adaptive responses of many bacteria to environmental changes. The sensor proteins of these systems are highly conserved near their C-termini. We exploited this feature to isolate a gene encoding a putative sensor component from the obligate intracellular rickettsial parasite Coxiella burnetii (Cb). Using degenerate primers and the polymerase...
Using an Escherichia coli expression system, pGEX-2T, that expresses foreign sequences as fusion proteins with a glutathione S-transferase (GST) carrier, we have produced several recombinant human salivary cystatin SN (reCsnSN) variants. These include a N-terminal-truncated form (aa 17-121), a C-terminal-truncated form (aa 1-102) and two deletion mutants (Δ12-16 and Δ56-60). A large amount of the...
Staphylococcal protein A (SPA) is ranked as a versatile probe in immunoassays because of its immunoglobulin G (IgG)-binding capability. However, poor binding of SPA to the IgG of some laboratory animals and its inability to bind human IgG3 restricts its universal utility. In the present study, DNA encoding the four IgG-binding domains of SPA (E, D, A and B) or the B domain alone has been fused, in...
Two pUC-derived vectors containing the promoterless xylE gene (encoding catechol 2,3-dioxygenase) of Pseudomonas putida mt-2 were constructed. The t o transcriptional terminator of phage λ was placed downstream from the stop codon of xylE. The new vectors, pXT1 and pXT2, contain xylE and the t o terminator within a cloning cassette which can be excised with several endonucleases...
A gene (pfhsp86) encoding a member of the heat-shock protein 90 (HSP90) family has been isolated from Plasmodium falciparum (Pf). The pfhsp86 coding region comprises two exons separated by an 0.8-kb intron with consensus splice junction sequences. The transcript itself is 3.4-kb long and includes a 0.65-kb 5 -untranslated region (UTR) and a 0.54-kb 3 -UTR. Upstream of the transcription start point...
We have cloned and expressed in vaccinia virus a cDNA encoding an ubiquitous 501-amino-acid (aa) phosphoprotein that corresponds to protein IEF SSP 9502 (79 400 Da, pI 4.5) in the master 2-D-gel keratinocyte protein database [Celis et al., Electrophoresis 14 (1993) 1091-1198]. The deduced aa sequence contains 9 Trp residues, some of which are localized in repeats and that characterise the protein...
We cloned the rat Gadd45 (growth arrest and DNA damage inducible) cDNA and examined its mRNA induction by γ-ray irradiation in the rat brain. The rat Gadd45 cDNA sequence was highly homologous to the previously published human and hamster cDNAs, and was partially similar to the 28S rRNA gene. The mRNA encoding rat GADD45 was induced in the brain after γ-ray irradiation. This finding indicates that...