Domestic yak (Bos grunniens) metallothionein-I (MT-I) and metallothionein-III (MT-III) cDNA 3′-end full-length sequences (331 and 378 bp; GenBank accession MT-I no. AY758557, MT-III no. DQ492300) from the total RNA of liver and brain tissues were amplified and cloned by reverse transcription polymerase chain reaction (RT-PCR) and 3′-rapid amplification of cDNA ends (RACE) using the primers YMT-I SP and M13 primers M4, and YMT-III SP and M13 primers M4, respectively, which included the MT-I (183 bp) coding sequence, MT-III (207 bp) cDNA coding sequences, and also the tailing signal AATAAA and Poly(A) at the 3′-ends of the MT-I and MT-III, respectively. The analyses showed that the yak cDNA sequence coding for MT-I protein was composed of 61 amino acids, including 20 cysteines having a conserved tripeptide structure, for example C–X–C, C–C–X–C–C, C–X–X–C and so on. The yak cDNA sequence coding for MT-III protein was composed of 68 amino acids, including 19 cysteines having both MT-III-specific conserved tripeptides and the same conserved tripeptides as MT-I, including T, CPCP, GEGAEA and so on. They were all comparatively conservative in molecular evolution. These structures indicated that MT-III had the same physiological functions regarding heavy metals (detoxification and so on) as MT-I, and also different specific functions from MT-I, including inhibition of neuron growth.

Ligand-gated chloride channels (LGCCs) are key components of the nervous system of parasitic nematodes and important targets for anthelmintics. Here, we describe the isolation and characterization of a novel member of the LGCC gene family (HcLGCC1) from the parasitic nematode Haemonchus contortus. Sequence analysis revealed that the channel subunit encoded by HcLGCC1 is anion selective and a member of a group of channels characterized as having two Cys-loops in the N-terminal ligand-binding domain†. Although the overall function of HcLGCC1 is presently unknown, the gene may play a key role in the early developmental stages of the parasite. Further investigations into the function of LGCCs, such as HcLGCC1, in parasitic nematodes should have implications for the discovery of new anthelmintic targets.

We performed genome-wide chemical mutagenesis of C57BL/6J mice using N-ethyl-N-nitrosourea (ENU). Electroretinographic screening of the third generation offspring revealed two G3 individuals from one G1 family with a normal a-wave but lacking the b-wave that we named nob4. The mutation was transmitted with a recessive mode of inheritance and mapped to chromosome 11 in a region containing the Grm6 gene, which encodes a metabotropic glutamate receptor protein, mGluR6. Sequencing confirmed a single nucleotide substitution from T to C in the Grm6 gene. The mutation is predicted to result in substitution of Pro for Ser at position 185 within the extracellular, ligand-binding domain and oocytes expressing the homologous mutation in mGluR6 did not display robust glutamate-induced currents. Retinal mRNA levels for Grm6 were not significantly reduced, but no immunoreactivity for mGluR6 protein was found. Histological and fundus evaluations of nob4 showed normal retinal morphology. In contrast, the mutation has severe consequences for visual function. In nob4 mice, fewer retinal ganglion cells (RGCs) responded to the onset (ON) of a bright full field stimulus. When ON responses could be evoked, their onset was significantly delayed. Visual acuity and contrast sensitivity, measured with optomotor responses, were reduced under both photopic and scotopic conditions. This mutant will be useful because its phenotype is similar to that of human patients with congenital stationary night blindness and will provide a tool for understanding retinal circuitry and the role of ganglion cell encoding of visual information.

A cDNA library was constructed from muscle larvae of Trichinella pseudospiralis. A cDNA clone, designated as Tp8 contained a cDNA transcript of 1326 bp length with a single open reading frame, which encoded 303 amino acid residues (34187 Da, estimated molecular mass). The predicted amino acid sequence of the clone had an identity of approximately 60% to the Rcd1 (Required cell differentiation 1) -like proteins among a wide range of organisms. Real-time quantitative polymerase chain reaction results showed that the transcription level of Tp8 gene reached the highest value in adult worms, and that the transcription level in muscle larvae before stichosome formation was higher than in muscle larvae after stichosome formation. The recombinant Tp8 protein migrated at 37 kDa and reacted to antibody against T. pseudospiralis excretory-secretory (E-S) products and sera from mice infected with T. pseudospiralis. An antibody against the Tp8 recombinant protein could stain proteins migrating at approximately 34 kDa (which is the expected size from the sequence) on Western blotting of E-S products from muscle larvae. An immunocytochemical study showed that the Tp8 protein was present within the stichocyte of muscle larvae and adults worms.

The bovine interferon-γ gene cloned from total RNA of peripheral blood lymphocytes stimulated with concanavalin A (ConA), using the reverse transcriptase-polymerase chain reaction (RT-PCR), was cloned into pGEM T-easy vector and sequenced. The mature peptide without signal peptide was then subcloned and constructed into the expression plasmids pET28a/BoIFN-γ and pPICZα/BoIFN-γ, respectively. The former was highly expressed, induced by isopropyl β-d-thiogalactoside (IPTG), in Escherichia coli BL21, with 30% of bacterial proteins in inclusion bodies. The latter was expressed in Pichia pastoris GS115 with methanol induction. The expressed products were secreted directly into cultural supernatant at concentrations of 1.0 g/l. The same recombinant proteins had antiviral activities 10 times higher in an MDBK (Madan-Darby bovine kidney)/VSV (Vesicular stomatitis virus) cell line than in a CEF (chicken embryo fibroblast)/VSV) cell line, and the antiviral activities expressed in P. pastoris were higher than those expressed in E. coli in the same cell line.

We performed genome-wide mutagenesis of C57BL/6J mice using the mutagen N-ethyl-N-nitrosourea (ENU) and screened the third generation (G3) offspring for visual system alterations using electroretinography and fundus photography. Several mice in one pedigree showed characteristics of retinal degeneration when tested at 12–14 weeks of age: no recordable electroretinogram (ERG), attenuation of retinal vessels, and speckled pigmentation of the fundus. Histological studies showed that the retinas undergo a photoreceptor degeneration with apoptotic loss of outer nuclear layer nuclei but visual acuity measured using the optomotor response under photopic conditions persists in spite of considerable photoreceptor loss. The Noerg-1 mutation showed an autosomal dominant pattern of inheritance in progeny. Studies in early postnatal mice showed degeneration to occur after formation of partially functional rods. The Noerg-1 mutation was mapped genetically to chromosome 6 by crossing C57BL/6J mutants with DBA/2J or BALB/cJ mice to produce an N2 generation and then determining the ERG phenotypes and the genotypes of the N2 offspring at multiple loci using SSLP and SNP markers. Fine mapping was accomplished with a set of closely spaced markers. A nonrecombinant region from 112.8 Mb to 115.1 Mb was identified, encompassing the rhodopsin (Rho) coding region. A single nucleotide transition from G to A was found in the Rho gene that is predicted to result in a substitution of Tyr for Cys at position 110, in an intradiscal loop. This mutation has been found in patients with autosomal dominant retinitis pigmentosa (RP) and results in misfolding of rhodopsin expressed in vitro. Thus, ENU mutagenesis is capable of replicating mutations that occur in human patients and is useful for generating de novo models of human inherited eye disease. Furthermore, the availability of the mouse genomic sequence and extensive DNA polymorphisms made the rapid identification of this gene possible, demonstrating that the use of ENU-induced mutations for functional gene identification is now practical for individual laboratories.

In HeLa cells, ribonuclease P (RNase P), the tRNA processing enzyme consists of an RNA subunit (H1 RNA) associated with at least nine protein subunits, Rpp14, Rpp20, Rpp21, Rpp29 (hPop4), Rpp30, Rpp38, Rpp40, hPop1, and hPop5 (18.8 kDa). We report here the cloning and immuno-biochemical analysis of Rpp25, another protein subunit of RNase P. Polyclonal rabbit antibodies raised against recombinant Rpp25 recognize their corresponding antigens in RNase P-containing fractions purified from HeLa cells, and they also precipitate active holoenzyme. Furthermore, this protein has general RNA binding properties.