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Genomic clones of ECI-6 (endothelial cell inducible), the porcine IκBα gene that encodes a cytoplasmic inhibitor of the transcription factor NF-κB, were isolated, spanning the entire transcribed region plus 2.1 and 0.35 kb of 5′- and 3′-flanking sequences, respectively. The gene contains five introns ranging in size from 0.6 to 0.1 kb. Four of the introns are located in the coding regions for four...
A novel protein tyrosine kinase (PTK) was previously identified by us from the rat insulin-producing cell line, RINm5F, by polymerase chain reaction (PCR). By using this PCR fragment to screen a cDNA library from the mouse insulin-producing cell line βTC-1, a cDNA clone of about 2.0 kb was obtained which encodes the entire amino acid (aa) sequence of the corresponding PTK. The deduced aa sequence...
We report on the construction and use of two eukaryotic expression vectors which add well-characterized epitope tags to the N termini of proteins. The utility of these vectors is demonstrated for detecting the expression of a variety of proteins. As the addition of these epitope tags can in some cases obviate the need to generate specific antisera to each individual protein, these vectors provide...
The cloning and sequencing of the murine Mxil gene encoding Mxil, one of Max-associated proteins, is described. Murine and human sequences showed 87.9% nucleotide (nt) and 90.3% amino acid (aa) sequence homology, whereas murine and zebra fish sequences showed 67.2% nt and 67.8% aa sequence homology.
The murine macrophage inflammatory protein 1β mRNA (MIP-1β) is rapidly and transiently induced in macrophages by lipopolysacharride (LPS), serum or cycloheximide. Functional studies of the MIP-1β proximal promoter indicate that it is cell-specific, and serum- and LPS-responsive in macrophages. A 76-bp proximal promoter sequence (−51 to −127 bp) confers cell-specific and LPS-inducible activity when...
A subtraction cDNA library was constructed from control hybrid NG108-15 (mouse neuroblastoma x rat glioma) cells and NG108-15 cells which had been treated for 48 h with the δ-opioid agonist d-Ala 2 -d-Leu 5 enkephalin (DADLE) to down-regulate the δ-opioid receptor on these cells. Among the clones isolated from this library was NGD16-4, a 2768-bp clone encoding a putative 64-kDa protein...
Full-length genomic clones encoding the α- and β-subunits of the pyrophosphate-dependent phosphofructokinase (PFP) from the castor plant have been isolated and sequenced. The gene (PFPα) encoding PFPα is approx. 5.8 kb in length and contains 19 exons, which collectively encode a protein of 617 amino acids (aa) having a deduced M r of 67 360. PFPβ is approx. 4.6-kb long and contains 16 exons...
A 2935-bp cDNA clone encoding glutamine synthetase (GS) was isolated from a cDNA library prepared from four-blastomere Paracentrotus lividus sea urchin embryos. The sequence consists of a 75-bp 5′ untranslated region (5′-UTR) followed by a 1095-bp coding region corresponding to a 365-amino-acid (aa) protein, a 1747-bp 3′-UTR and a terminal 18-bp poly(A) tail. The encoded protein shows about 66% identical...
A simple procedure for in vitro site-directed mutagenesis (SDM) was developed using two oligodeoxyribonucleotide (oligo) primers, one serving as selection primer and the other for mutagenesis in the target DNA (mutagenic primer). The mutant clones can be selected by reversion mutations of dual amber (am) to dual Gln codons in the cat gene, resulting in a chloramphenicol-resistant phenotype in sup...
The mutation spectrum attributable to an antimutator DNA polymerase in a eukaryotic cell was examined. Drug-resistant thymidine kinase (tk) mutants derived from both a wild-type (wt) strain, KOS and an antimutator DNA polymerase (pol) mutant, PAA r 5, of herpes simplex virus (HSV), were isolated, and the mutated tk genes were characterized at the sequence level. While both transition and frameshift...
The cDNAs encoding the porcine 65- and 67-kDa glutamic acid decar☐ylases (GAD 65 and GAD 67 , respectively) were cloned by the PCR method. The 2246-nucleotide (nt) GAD 65 cDNA contained an open reading frame (ORF) coding for a protein of 585 amino acids (aa), and the 3262-nt GAD 67 cDNA contained an ORF coding for a protein of 594 aa. A remarkable conservation was shown...
The mouse vitamin D receptor (VDR)-encoding cDNA was cloned and the coding regions were sequenced. Comparison of the amino-acid sequence to that of humans and rats revealed high homology in the DNA- and ligand-binding domains. Divergence appeared in the internal region between the two domains.
Genomic and cDNA clones of a mcol gene encoding mini-collagen (MCOL), a nematocyst capsule protein, have been isolated from a reef-building coral, Acropora donei (Anthozoa). The gene and its flanking regions, comprising 5382 bp and covering three exons and two introns, were sequenced. Exons 2 and 3 together have an open reading frame which can encode a MCOL of 176 amino acids (aa). The coral MCOL...
A human cDNA specifying a member of the Tis11 early response gene family was cloned and sequenced. The human gene differs from its mouse homologue by encoding an additional 97 amino acids at its C-terminal end. The sequence has transactivation-like motifs, an unusual Cys-Ser-Ala-rich motif and displays sequence similarity at the extreme C-terminal end with another Tis11 family member, ERF-1.
Thioredoxins (TXN) are small proteins with various biological functions, such as redox regulation, found in many species including bacteria, plants and animals. We previously reported the isolation of the TXN-encoding cDNAs from human and mouse. In order to elucidate the functions of the mammalian TXN system, we planned to generate Txn knockout mice, and cloned the genomic DNA fragments using the...
A cDNA encoding a novel mammalian member of the Clp/HSP104 family was isolated from a mouse macrophage-like cell line (J774.1) cDNA library by suppression of the growth defect of a Saccharomyces cerevisiae trkl trk2 double mutant. The full-length version of this cDNA, termed SKD3, encodes a putative 76-kDa protein of 677 amino acids (aa). The deduced aa sequence of the SKD3 polypeptide contains four...
An integrated hepatitis B virus (HBV) DNA has been identified in a human neuroblastoma, cloned and sequenced. The integrated HBV DNA is not rearranged, although a 480-bp fragment is deleted. The integrated viral DNA covers the C gene fragment, the region of Pre-S and S, and the 3′-truncated X gene.
Genes involved in deoxysugar metabolism, encoding thymidine diphospho (TDP)-glucose 4,6-dehydratase (gdh) and a putative TDP-4-keto-6-deoxyglucose 3,5-epimerase (kde), were cloned from the erythromycin (Er)-producingSaccharopolyspora erythraea by means of an oligodeoxynucleotide corresponding to a segment of the purified Gdh protein. Determination of the nucleotide sequence established that kde lies...
Sequence analysis of a fragment of Azospirillum brasilense DNA revealed the presence of a ribA homologue, of which the 3 portion encodes a putative GTP cyclohydrolase II. The 5 portion (approx. half of the ORF) does not show homology to any other sequence from the databases.
The energy-dependent efflux of erythromycin (Er) in staphylococci is due to the presence of msrA, which encodes an ATP-binding protein. MsrA is related to the multi-component ATP-binding cassette (ABC) transporters which characteristically also contain membrane-spanning domains. Since MsrA functions in a heterologous host in the absence of other plasmid-encoded products, the requirement for a transmembrane...
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