Partial nucleotide sequence of a bovine major histocompatibility class II DRβ-like gene

Summary. A genomic clone containing a bovine DRβ-like gene, BoDRβ II , was isolated from a bovine genomic library and characterized by restriction enzyme mapping and nucleotide sequencing of exon regions. Alignment of this sequence with the human DRβ cDNA sequence allowed identification of exon/intron boundaries. The clone contains a 13.3-kilobase (kb) insert, and includes 1.3kb 5' of the β₁ exon and 6.7kb 3' of the transmembrane (TM) exon. Open reading frames were present in the BoDRβ exons sequenced. Nucleotide identities of the bovine β₁, β₂ and TM exons with the corresponding human DRβ exons were 73, 91 and 83%, respectively. Nucleotide identities of these exons with those of a previously described bovine DRβ-like pseudogene, BoDRβ I , were 69, 95 and 81%, respectively. Although a limited amount of sequence data was obtained for the intron regions, a 71% identity was found within a 514-nucleotide region immediately 3' to the β₂ exons in BoDRβ I and BoDRβ II . A series of GT residues followed by a longer series of GA residues began about 35 nucleotides 3' of the β₁ exon in both BoDRβ I and BoDRβ II .     

Cloning and genetic organization of the gene cluster encoding F71 fimbriae of a uropathogenic Escherichia coli and comparison with the F72 gene cluster

Abstract The gene cluster coding for expression of F7₁ fimbriae of the uropathogenic Escherichia coli strain AD110 has been cloned by a cosmid-cloning procedure. A positive clone was further subcloned to a plasmid of 17.5 kilobases (kb), pPIL110-75. Analysis of pPIL110-75 showed that at least six genes are present encoding proteins with apparent M _rs of 75 000, 36 000, 23 000, 20 000, 17 000 and 14 000. The 20-kDa protein, encoding the F7₁ fimbrial subunit is dispensable for expression of the MRHA phenotype. Complementation experiments of mutants in the F7₂ gene cluster by gene products of the F7₁ gene cluster show that the two gene clusters are related.     

Identification of trehalose and glycine betaine as compatible solutes in the moderately halophilic sulfate reducing bacterium, Desulfovibrio halophilus

Abstract A gene ( ERG11 ) encoding cytochrome P450 sterol 14α-demethylase (P450_14DM) was isolated from the maize pathogen, Ustilago maydis , by amplifying part of the coding region of the gene using PCR and by employing the amplified DNA fragment as a hybridization probe to recover the complete gene from an U. maydis λEMBL3 genomic library. The deduced amino acid sequence of the U. maydis gene showed homology to P450_14DMs from other organisms and contained specific motifs which were hallmarks of P450s. Expression of the gene in an U. maydis mutant (A20) deficient in P450_14DM led to only a partial restoration of P450_14DM activity. Accumulation of ergosta-7,22-dienol and ergost-7-enol in A20 transformants containing the ERG11 gene implied that an additional mutation affecting sterol Δ ^5,6-desaturase activity accompanied the P450_14DM lesion.     

Turkey microsatellite DNA loci amplified by chicken-specific primers

Forty-eight primer-pairs complementary to unique DNA sequences flanking chicken (genus Gallus ) genomic (TG)_n microsatellite repeats were previously designed. These primer-pairs were used in the polymerase chain reaction to amplify turkey (genus Meleagris ) genomic DNA loci. Results indicated that the majority (92%) of these primer-pairs generated amplification products in turkey genomic DNA. Hybridization using end-labelled (TG)₈ as a probe showed that, out of 41 primer-pairs tested, only 14 generated an amplification product that also contained a detectable (TG)_n microsatellite repeat when turkey DNA was the template. Among 18 primerpairs tested for polymorphism, using three commercial turkey lines, five were found to exhibit length polymorphism, three of which did not contain a detectable TG repeat. Therefore, a significant portion of chicken microsatellite markers can be useful for genomic mapping and linkage analysis in the turkey, reducing the costs involved in producing turkey-specific microsatellite markers.     

Analysis of genetic structure of a Suillus grevillei population in a Larix kaempferi stand by polymorphism of inter-simple sequence repeat (ISSR)

Clones of ectomycorrhizal fungi can colonize new areas through production of vegetative mycelium or spore dispersal, but the relative importance of these processes in nature is not known. In this study, sporocarps of an ectomycorrhizal fungus, Suillus grevillei , were mapped and sampled from a Larix kaempferi stand at the foot of Mt Fuji. DNA was extracted directly from each sporocarp, and DNA polymorphism was analysed by polymerase chain reaction (PCR) amplification of inter-simple sequence repeat (ISSR) regions primed by (GTG)₅, (GCC)₅ and (GACA)₄. Different sensitivities to detect polymorphism were found among the three primers, with (GACA)₄ showing the highest sensitivity. Forty seven sporocarps were analysed by the three ISSR primers and divided into 34 genets based on combination of PCR fingerprints. In the population 28 genets were represented by individual sporocarps. In most cases, sporocarps grown in aggregation (within a circle of 50 cm diameter) showed some different ISSR band patterns. These results suggest that genets of S. grevillei at the test site are relatively small. The genetic similarities between the 34 genets were also calculated and similarity groups were determined by the criterion that all similarity F values of genets within a group were not <80%. In general, the genets within a similarity group located close to each other. The results of multiple different but highly related genets in a small area suggest that the population of S. grevillei in this stand is not spread and maintained by clonal mycelium extension but is reproduced by spore dispersal.     

Genomic DNA fingerprinting of clinical isolates of Helicobacter pylori using short oligonucleotide probes containing repetitive sequences

The ability of oligonucleotide probes containing short repetitive sequence motifs to differentiate between isolates of Helicobacter pylori was investigated. Genomic DNA preparations from H. pylori were digested with the restriction enzyme Hind III, electrophoresed in agarose gels and transferred to nylon filters. Five separate oligonucleotide probes were tested for hybridization sequentially to fingerprint the digested DNA from a panel of 29 clinical isolates and one type strain of H. pylori , and their relative discriminatory abilities were assessed. Four probes, (GACA)₄, (GT)₈, (GTG)₅ and (GGAT)₄, were each shown to yield highly informative hybridization band profiles allowing differentiation of H. pylori isolates. The DNA fingerprints of individual isolates obtained with each probe were distinct and reproducible. Direct comparison with ribotyping revealed that oligonucleotide fingerprinting had far superior discriminatory power. Computer-assisted similarity analysis of (GGAT)₄-generated hybridization profiles of pairwise combinations of H. pylori isolates revealed that there was no correlation between ribotype and oligonucleotide fingerprint patterns. The results of this study demonstrate that oligonucleotide probes containing microsatellite sequences provide a new and powerful tool for isolate discrimination of H. pylori .     

Isolation and genetic characterization of the porcine apolipoprotein E gene

The present report describes the isolation and genetic characterization of the porcine apolipoprotein E (ape-E) gene. A single positive recombinant phage clone containing a 10·7-kb insert was isolated from a porcine genomic library, and a 4·2-kb fragment was subcloned and sequenced. The 4·2-kb fragment contained the entire apo-E gene in addition to upstream and downstream sequences (GenBank accession no. 470240). The porcine apo-E gene is made up of four exons and three introns, and encodes a preapo-E protein comprised of a signal peptide of 18 amino acids and a mature protein of 299 amino acids. The porcine apo-E gene contains a (CG)₁₃ microsatellite marker within intron three. This microsatellite is moderately polymorphic, and at least four alleles were evident at this locus among 10 animals from each of the Yorkshire, Hampshire, Landrace and Duroc breeds. Finally, localization of the porcine apo-E gene to chromosome 6 band q2·1 was determined by fluorescent in situ hybridization and confirmed by genetic linkage analysis.     

Cloning and Characterization of a Mouse σ1 Receptor

Abstract: A cDNA clone (S2-1a) isolated from a mouse brain cDNA library, using a guinea pig σ₁ cDNA as probe, has high homology to the predicted protein sequence of the guinea pig (88%) and human (90%) σ₁ receptors. Northern analysis revealed a major mRNA of ∼1.8 kb in a wide range of mouse tissues, with highest levels in brain, liver, kidney, and thymus. Southern analysis and chromosomal mapping in the mouse suggested a single-copy gene in region A5-B2 of chromosome 4. Expression of the clone in MCF-7 and CHO cells led to a pronounced increase in (+)-[³H]pentazocine binding with a selectivity profile consistent with σ₁ receptors. In vitro translation yielded a protein of ∼28 kDa, as did transfection of a probe containing the hemagglutinin (HA) epitope (S2-1a.HA) into CHO cells, as determined by western analysis using an antibody directed against HA. (+)-[³H]-Pentazocine binding to immunopurified HA-tagged receptor demonstrated conclusively that S2-1a.HA encodes a high-affinity (+)-[³H]pentazocine binding site with characteristics of a murine σ₁ receptor. An antisense oligodeoxynucleotide designed from S2-1a potentiated opioid analgesia in vivo.     

A Chemical Indicator for the Rapid Measurement of F0 Values

A new chemical indicator for monitoring steam sterilization processes has been calibrated in F ₀ units. The effective temperature range for F ₀ measurements using this device has been shown to lay between 115 and 123°C. The effective F ₀ range of the device has been shown to be 4–23 F ₀ units. Using the device, measurements can be made within 0.5 units of conventionally calculated F ₀ values.     

Copper acquisition by the SenC protein regulates aerobic respiration in Pseudomonas aeruginosa PAO1

Actinophage TG1 forms stable lysogens by integrating at a unique site on chromosomes of Streptomyces strains. The phage ( attP _TG1 ) and bacterial ( attB _TG1 ) attachment sites for TG1 were deduced from comparative genomic studies on the TG1-lysogen and nonlysogen of Streptomyces avermitilis . The attB _TG1 was located within the 46-bp region in the dapC gene (SAV4517) encoding the putative N -succinyldiaminopimelate aminotransferase. TG1-lysogens of S. avermitilis , however, did not demand either lysine or diaminopimelate for growth, indicating that the dapC annotation of S. avermitilis requires reconsideration. A bioinformatic survey of DNA databases using the fasta program for the attB _TG1 sequence extracted possible integration sites from varied streptomycete genomes, including Streptomyces coelicolor A3(2) and Streptomyces griseus . The gene encoding the putative TG1 integrase ( int _TG1 ) was located adjacent to the attP _TG1 site. TG1 integrase deduced from the int _TG1 gene was a protein of 619 amino acids having a high sequence similarity to φC31 integrase, especially at the N-terminal catalytic region. By contrast, sequence similarities at the C-terminal regions crucial for the recognition of attachment sites were moderate or low. The site-specific recombination systems based on TG1 integrase were shown to work efficiently not only in Streptomyces strains but also in heterologous Escherichia coli .     

Plant response to destruxins visualized by imaging of chlorophyll fluorescence

Kinetic fluorescence imaging was used to set a new detection limit for plant exposure to low levels of destruxins – phytotoxins of Alternaria brassicae . A general experimental algorithm is presented that can be used to identify the combination of fluorescence parameters providing the highest contrast between the affected and unaffected plants or plant segments. Leaves of canola ( Brassica napus ) and white mustard ( Sinapis alba ) were exposed to various concentrations of destruxins and images of key fluorescence signals ( F ₀, F _M, F _P, and of F _S) were captured in a single kinetic experiment. Contrast was quantified within these images between the leaf areas exposed to destruxins and the untreated areas. The highest contrast was found in the image constructed by pixel-to-pixel division of images F ₀ by F _P and F ₀ by F _M. Using the F ₀/ F _M ratio image, we were able to detect exposure to destruxin concentration as low as approximately 0.05 mg l⁻¹ applied to canola leaf and approximately 10 mg l⁻¹ when applied to mustard. The detection limits were significantly lower than those obtained by optical microscopy indicating that kinetic chlorophyll fluorescence imaging can be used as a diagnostic tool in screening for varieties with an enhanced resistance to destruxins of Alternaria brassicae .     

The use of (GACA)4 PCR to sex Old World vultures (Aves: Accipitridae)

A PCR method was developed employing a single primer (GACA)₄ to sex Black vultures ( Aegypius monachus ), Lappet-faced vultures ( Torgos tracheliotus ), and Griffon vultures ( Gyps fulvus ). Using the (GACA)₄ primer several PCR products were generated. One or more PCR products displayed a sex-specific pattern, i.e. they were only present in females (probably corresponding to repetitive DNA on the W chromosome) but absent in males. The sex ratio of 85 Griffon vultures from Spain was almost 1.     

Salinity effects on water relations in Lycopersicon esculentum and its wild salt-tolerant relative species L. pennellii

Cultivated tomato Lycopersicon esculentum (L.) Mill. cv. P-73 and its wild salt tolerant relative L. pennellii (Correll) D'Arcy accession PE-47, were grown during spring-summer 1989 under unheated plastic greenhouse conditions. Plants were submitted to two different salt treatments using 0 and 140 mM NaCI irrigation water. In both tomato species, salinity caused a proportionally larger reduction in leaf area than in leaf weight and, in L. esculentum , a proportionally larger decrease in stem weight than in leaf weight. Daily variations in leaf water potential (Ψ₁) were fundamentally due to changes in the evaporative demand of the atmosphere. Reductions in Ψ₁ due to salinity were consistent only in L. esculentum . In all the conditions studied, leaf turgor was maintained. Leaf conductance (g₁)was higher in L. esculentum than in L. pennellii .Salinity induced a clear reduction in g₁ levels in L. esculentum whereas, in L. pennellii , this reduction was noted only in May. In both species the Ψ^o_s (leaf osmotic potential at full turgor) levels were reduced by salinity. The bulk modulus of elasticity (E) and relative water content at turgor loss point (RWC_tlp) were not affected by salinity. The RWC_tlp values in L. pennellii seem to be controlled by E values.     

D21S215 is a (GT)n polymorphic marker close to centromeric alphoid sequences on chromosome 21.

A plasmid, AWZ1, that contained a dinucleotide (GT)n repeat was identified from a chromosome 21-specific genomic library. When amplified by PCR from human genomic DNA, the repeat length was highly polymorphic between individuals; its location, D21S215, was mapped in the CEPH pedigrees by linkage analysis to the pericentromeric region of chromosome 21. It is the closest polymorphic marker to alphoid sequences on this chromosome.     

Acute toxicity of copper, zinc and manganese in single and mixed salt solutions to juvenile longfin dace, Agosia chrysogaster

The acute toxicity of copper, zinc and manganese and copper-zinc and copper-manganese mixtures were determined for juvenile longfin dace, Agosia chrysogaster in hard water bioassays (mean=218 mg 1⁻¹ CaCO₃). Copper-zinc was the most lethal toxicant (96-h L.c.₅₀= 0.21 mg 1⁻¹ copper and 0.28 mg 1⁻¹ zinc) and exhibited a more than additive toxicity which was in contrast to the additive toxicity of copper-manganese mixtures (96-h L.c.₅₀= 0.45 mg 1⁻¹ copper and 64.0 mg 1⁻¹ manganese). The toxicity of copper (96-h L.c.₅₀= 0.86 mg 1⁻¹) and zinc (96-h L.c.₅₀= 0.79 mg 1⁻¹) to the fish was similar but both were considerably more lethal than manganese (96-h L.c.₅₀= 130 mg 1⁻¹).     

Age, growth and reproduction of the barrelfish Hyperoglyphe perciformis (Mitchill) in the western North Atlantic

Otoliths ( n = 847) and gonads ( n = 817) were collected from barrelfish Hyperoglyphe perciformis that were captured by commercial fishermen in the waters off South Carolina and Georgia in 1995, 1997 and 2001–2006. Of the otoliths collected, 97% were aged successfully, and specimens sampled ranged from 5 to 85 years, with a median age of 12 years. The von Bertalanffy growth parameters yielded the equation: L_t = 857·8{1 − e^{−0·0985[ t −(−8·95)]}}, where L_t is fork length ( L _F) at time t . Through histological examination, 94% of the gonads assessed were assigned to a sex and reproductive class. Females spawned from September to May with a peak from November to January. Males spawned year round, but had a peak from September to April. The sex ratio (M:F) for this population was 1:1·34. The smallest mature female was 605 mm L _F and the youngest immature female was 697 mm L _F. Estimates of L _F and age at 50% maturity ( L ₅₀ and A ₅₀) for females were 660 mm L _F (95% CI = 633–667 mm L _F) and 6·08 years (95% CI = 3·50–7·27 years), respectively. The youngest mature male was 575 mm L _F and the oldest immature male was 762 mm L _F, and no estimates of L ₅₀ or A ₅₀ were made for males. It was determined that barrelfish exhibit the typical characteristics of long life span, slow growth and high age at maturity seen in other deepwater fishes, and that care should be taken to manage this species accordingly.