A comparison of the clavam biosynthetic gene clusters in Streptomyces antibioticus Tü1718 and Streptomyces clavuligerus

The production of clavam metabolites has been studied previously in Streptomyces clavuligerus , a species that produces clavulanic acid as well as 4 other clavam compounds, but the late steps of the pathway leading to the specific end products are unclear. The present study compared the clavam biosynthetic gene cluster in Streptomyces antibioticus , chosen because it produces only 2 clavam metabolites and no clavulanic acid, with that of S.?clavuligerus. A cosmid library of S.?antibioticus genomic DNA was screened with a clavaminate synthase-specific probe based on the corresponding genes from S. clavuligerus, and 1 of the hybridizing cosmids was sequenced in full. A clavam gene cluster was identified that shows similarities to that of S.?clavuligerus but also contains a number of novel genes. Knock-out mutation of the clavaminate synthase gene abolished clavam production in S.?antibioticus, confirming the identity of the gene cluster. Knock-out mutation of a novel gene encoding an apparent oxidoreductase also abolished clavam production. A potential clavam biosynthetic pathway consistent with the genes in the cluster and the metabolites produced by S. antibioticus, and correspondingly different from that of S.?clavuligerus, is proposed.     

Molecular cloning and characterization of the β-catenin gene from fine-wool sheep

β-Catenin is an evolutionarily conserved molecule that functions as a crucial effector in both cell-to-cell adhesion and Wnt signaling. To gain a better understanding of its role in the development of hair follicles, we cloned the cDNA sequence of the β-catenin gene from the skin of Aohan fine-wool sheep and performed a variety of bioinformatics analyses. We obtained the full-length sequence, which was 4573-bp long and contained a 2346-bp open reading frame encoding a protein of 781 amino acids. The protein had a predicted molecular weight of 85.4 kDa and a theoretical isoelectric point of 5.57. Domain architecture analysis of the β-catenin protein revealed an armadillo repeat region, which is a common feature of β-catenin in other species. The ovine β-catenin gene shares 97.91%, 94.25%, 94.59%, 83.89%, and 89.39% sequence identity with its homologs in Bos taurus, Homo sapiens, Sus scrofa, Gallus gallus, and Mus musculus, respectively, while the amino acid sequence is more than 99% identical with each of these species. The expression of β-catenin mRNA was detected in the heart, liver, spleen, lung, kidney, skin, muscle, and adipose tissue. Expression levels were maximal in the lung and minimal in the muscle, and the difference in expression in these tissues was significant (P < 0.01). Western blot analysis revealed the presence of the β-catenin protein in all tissues examined; expression was lowest in the skin and adipose tissues.     

The evolution of the α- and β-globin gene clusters in human populations

Summary DNA analysis of the - and -globin gene clusters has revealed substantial variability between individuals and populations. As well as restriction enzyme site and length polymorphisms, variation in gene copy number and type is observed. Because of this extensive polymorphism DNA analysis offers a highly informative method of studying genetic affinities between human populations. Haplotypes, consisting of a set of restriction enzyme polymorphisms distributed along the cluster, have been developed for both loci. Analysis of the molecular basis of numerous -thalassaemia alleles has revealed, in general, different sets of mutations in different populations, indicating that these postdate the racial divergence. Recent microepidemiological studies on the distribution of -thalassaemia support the hypothesis that this condition, like the {ie16-1}, has been selected because it confers protection against malaria. Population-specific DNA polymorphisms at these and other loci promise to be of considerable value to genetic anthropology.     

Structure of the chicken interferon-γ gene,and comparison to mammalian homologues

The sequence of the chicken interferon-γ (ifn-γ) gene was determined, one of the first non-mammalian cytokine gene structures to be elucidated. Initial genomic clones were amplified from chicken genomic DNA and were used to isolate a cosmid clone covering the entire gene for sequencing. The exon:intron structure of chicken ifn-γ is very similar to those of its mammalian homologues, with the exception of the third intron, which is markedly shorter in the chicken. The first exon contains both 5′ UTR and signal sequence and the first 22 aa of the mature protein. The remainder of the coding region lies in exons 2–4. Exon 4 also encodes the stop codon and the 3′ UTR, including two possible polyadenylation signals. A number of potential regulatory sequences similar to those found in mammals have been identified, in the promoter, in each intron and in the 3′ UTR. In the promoter, these include the TATAATA- and CCAT-boxes, a consensus GATA motif in the reverse orientation and a potential NF-κB binding site. Other regulatory elements identified in the promoters of mammalian ifn-γ genes are absent. Internal to the gene structure, regulatory sequences identified include elements found in the DNase I hypersensitivity region of the first intron of the human ifn-γ gene and several potential NF-κB binding sites. The 3′ UTR contains an AT-rich sequence, including nine repeats of the `instability' motif ATTTA. As in mammals, chicken ifn-γ is a single copy gene. The gene is highly conserved, with no polymorphisms yet identified using either RFLP or SSCP in the coding region. However, promoter sequence polymorphisms between different inbred lines of chickens have been identified, with possible links to disease resistance.     

The offshore jurisdiction of the states of the United States and the provinces of Canada—A comparison

Abstract

The coastal state has jurisdiction over the resources located in the seas and seabed adjacent to its coast. In federal states the question has arisen as to whether the rights in those resources are to be held by the national government or by the subsidiary territorial governments. It has been the focus of political activity and court litigation in the United States and Canada. This paper reviews in detail the status of the offshore claims of the states of the United States and the provinces of Canada. In delimiting these boundaries two systems of law have become relevant—modern public international law and historic common law. Both systems of law have advantages and disadvantages for the courts and interested parties. While the United States has chosen a route that relies very heavily on modern public international law, the Canadian situation is less clear. Litigation now in progress may resolve this issue in Canada by court decree unless a political settlement is reached first. A recent agreement between the Federal Government of Canada and the Gov eminent of Nova Scotia which calls for joint management and revenue sharing may point the way toward such a settlement.     

Phylogenomic analyses of KCNA gene clusters in vertebrates: why do gene clusters stay intact?

Background  

Gene clusters are of interest for the understanding of genome evolution since they provide insight in large-scale duplications events as well as patterns of individual gene losses. Vertebrates tend to have multiple copies of gene clusters that typically are only single clusters or are not present at all in genomes of invertebrates. We investigated the genomic architecture and conserved non-coding sequences of vertebrate KCNA gene clusters. KCNA genes encode shaker-related voltage-gated potassium channels and are arranged in two three-gene clusters in tetrapods. Teleost fish are found to possess four clusters. The two tetrapod KNCA clusters are of approximately the same age as the Hox gene clusters that arose through duplications early in vertebrate evolution. For some genes, their conserved retention and arrangement in clusters are thought to be related to regulatory elements in the intergenic regions, which might prevent rearrangements and gene loss. Interestingly, this hypothesis does not appear to apply to the KCNA clusters, as too few conserved putative regulatory elements are retained.     

Sequence analysis of the upstream regions of Xenopus laevis β-globin genes and arrangement of repetitive elements within the globin gene clusters

The globin gene clusters of Xenopus laevis are interspersed by various different repetitive DNA elements. A specific repeat, the JH12 element, has been mapped by Southern analysis and some of its locations have been subsequently confirmed by nucleotide sequencing. JH12 family members seem to represent mobile genetic elements and display a high degree of divergence. The nucleotide sequences upstream to the adult _I-globin gene and to the two coordinately expressed larval _I- and _II genes have been determined and compared to those of the adult -genes. Besides some repetitive DNA elements and a short sequence of rather weak homology we have found no characteristic sequence motifs to be common to the adult - and -genes. The two larval -genes share one short sequence element being absent from the adult genes. This might reflect completely different sequence requirements for protein interactions and for the regulation of adult and larval globin gene expression.     

Structural organization of the transfer RNA gene clusters of cholera phage φ l49

Vibrio cholerae phage φ l49 codes tRNAs specific for twelve different amino acids. These tRNA genes are contained in two different HindIII fragments 11 and 3.4 kb in size, of the phage genome. The 3.4 kb HindIII fragment was cloned inEscherichia coli using pBR328 as vector. The recombinant plasmid pNR347 produced nine of the twelve tRNA species (arginine, proline, serine, tyrosine, histidine, lysine, leucine, tryptophan and aspartic acid) encoded in the phage genome.     

A comparison of the kinetic properties of free and immobilized Aspergillus oryzae β-galactosidase

The objective of this work was to compare the properties of free and immobilized β-galactosidase (Aspergillus oryzae), entrapped in alginate–gelatin beads and cross-linked with glutaraldehyde. The free and immobilized forms of the enzyme showed no decrease in enzyme activity when incubated in buffer solutions in pH ranges of 4.5–7.0. The kinetics of lactose hydrolysis by the free and immobilized enzymes were studied at maximum substrate concentrations of 90 g/L and 140 g/L, respectively, a temperature of 35 °C and a pH of 4.5. The Michaelis–Menten model with competitive inhibition by galactose fit the experimental results for both forms. The K_m and V_m values of the free enzyme were 52.13 ± 2.8 mM and 2.56 ± 0.3 g_glucose/L min mg_enzyme, respectively, and were 60.30 ± 3.3 mM and 1032.07 ± 51.6 g_lactose/min m³_catalyst, respectively, for the immobilized form. The maximum enzymatic activity of the soluble form of β-galactosidase was obtained at pH 4.5 and 55 °C. Alternatively, the immobilized form was most active at pH 5.0 at 60 °C. The free and immobilized enzymes presented activation energies of 6.90 ± 0.5 kcal/mol and 7.7 ± 0.7 kcal/mol, respectively, which suggested that the immobilized enzyme possessed a lower resistance to substrate transfer.     

Sodium glucose-linked transport in the ruminal epithelium of fallow deer – comparison to sheep

A higher expression of the sodium glucose-linked transporter, SGLT-1, has been previously demonstrated in the intestine of adult fallow deer compared to sheep. The functional presence of SGLT-1 in the rumen of both species was investigated in the present study. Isolated ruminal epithelia were incubated in Ussing chambers. D-Glucose or 3-O-methyl-alpha-D-glucose (3-OMG) were applied at 10 mmol x l-1 to the mucosal side of tissues and the changes in short-circuit current (Delta I(sc)) were recorded. Alternatively, apical uptake of [14C]-D-glucose (200 micro mol x l-1) was determined in the presence or absence of phlorizin (100 micro mol x l-1) or Na+ (115 mmol x l-1). In both species, mucosal D-glucose addition induced a larger ( P<0.05) Delta I(sc) than the mucosal addition of 3-OMG. When comparing the effects of D-glucose or 3-OMG between species, the Delta I(sc) was two-fold larger ( P<0.01) in sheep compared to fallow deer. Accordingly, phlorizin-sensitive D-glucose uptake was approximately 50% smaller ( P<0.05) in fallow deer. It is concluded that D-glucose can be absorbed via SGLT-1 from the forestomachs of both species. However, the intermediate-mixed feeding habit of fallow deer does not seem to coincide with a higher activity of the ruminal SGLT-1.     

Molecular cloning and characterization of the sheep α-TTP gene and its expression in response to different vitamin E status

The α-tocopherol transfer protein (α-TTP) is a ~ 32 kDa protein that exhibits a marked ligand specificity and selectively recognizes of α-tocopherol, which is the most active form of vitamin E. The α-TTP gene has been cloned and its physiological functions have been studied in numbers of species, however, the understanding of sheep α-TTP is still in his infancy. In this study, the full-length cDNA of sheep α-TTP gene was cloned from sheep liver by using of rapid amplification of complementary DNA ends (RACE). As a result, the sheep α-TTP gene was 1098 bp in nucleotide which contained 23 bp 5'-untranslated region (UTR), 226 bp 3'-UTR and 849 bp open reading frame (ORF) that encoded a basic protein of 282 amino acids. Further bioinformatic analysis indicated that the sheep α-TTP gene had a high homologous of both nucleotide and amino acid sequences compared with that of other species and had a Sec14p-like lipid-binding domain which called the CRAL-TRIO domain. Moreover, the expression of sheep α-TTP mRNA and protein in response to different vitamin E supplemented levels were observed according to quantitative real-time PCR (qRT-PCR) and Western blotting analysis. The results showed that dietary vitamin E levels did not affect α-TTP mRNA expression significantly while the low vitamin E supplemented level groups of sheep had significantly higher α-TTP protein compared to high-vitamin E groups.     

Association of HL-A antigens andβ 2-microglobulin at the cellular and molecular level

Surfaces of cultured human lymphoid cells RPMI 1788, RPMI 4098, RPMI 8866, Raji, and WI-L2 were found to contain bothβ ₂-microglobulin (β ₂-μ) and HL-A determinants when tested by direct complement-dependent cytotoxicity andquantitative absorption with different cytotoxic antiβ ₂-μ antisera and specific HL-A alloantisera. The same antigenic specificities were found in 3M KCl extracts of these cultured cells with a sensitiveβ ₂-μ radioimmunoassay and an HL-A antigen blocking assay. Daudi cells provided a contrast, since noβ ₂-μ or HL-A determinants were found on their surfaces or in 3 M KCl extracts prepared from them. Results from specific antibody blocking tests suggest a close association betweenβ ₂-μ and HL-A determinants on plasma membranes of cultured human lymphoid cells. A solid state immunoadsorbent containing antiβ ₂-μ antibodies effectively removed all detectable HL-A antigenic activity from some 3M KCl extracts of cultured human lymphoid cells as well as from some sera. Adsorption of HL-A antigens to these immunoadsorbents was specific since it was blocked only by prior addition ofβ ₂-μ. Once on the antiβ ₂-μ immunoadsorbents, HL-A antigens still reacted specifically with HL-A alloantibodies in quantitative absorption experiments. HL-A antigens andβ ₂-μ could be eluted from antiβ ₂-μ immunoadsorbents with a variety of chaotropic reagents and detergents, but thus far potassium bromide and sodium dodecyl sulfate (SDS) appear to be the most effective. SDS-PAGE of these eluates indicated that HL-A antigens were considerably purified by adsorption to antiβ ₂-μ immunoadsorbents and that two major molecular size fragments were distinguishable, i.e., ∼33,000 for HL-A and ∼ 12,000 forβ ₂-μ.     

A comparison of genetic diversity between the Galápagos Penguin and the Magellanic Penguin

The Galápagos Penguin (Spheniscusmendiculus) is a United States federallylisted endangered species with populations onthe Galápagos Islands of Fernandina andIsabela. Although the waters around theislands are normally productive, lowproductivity during El Niño years resultsin high adult penguin mortality and lowrecruitment in following years. We usedmicrosatellite markers developed for Spheniscus penguins to study the long termgenetic effects of serial bottleneck events inthe Galápagos Penguin, and compared thisvariation to that of its congener, theMagellanic penguin (Spheniscusmagellanicus). The observed heterozygosityfor the Galápagos Penguin was 3%,significantly lower than the 46%heterozygosity of the Magellanic Penguin. Thislow level of heterozygosity is directly relatedto its low effective population size. Whilethis population has survived long term,presumably without high levels of geneticvariation, we feel that the greater frequencyof El Niño events, coupled with increasedhuman impacts such as introduced disease, oildischarge, and competition with fisheries, mayput the species in particular danger ofextinction.     

Mapping of the β-lactoglobulin gene and of an immunoglobulin M heavy chain-like sequence to homoeologous cattle,sheep, and goat chromosomes

With a combination of non-isotopic in situ hybridization and simultaneous fluorescent R-banding, this study presents the first map of the short arm of sheep Chromosome (Chr) 3 and of the homoeologous cattle and goat Chrs 11 with two DNA sequences: the lactoglobulin gene (LGB) and an immunoglobulin M heavy chain-like sequence (IGHML). The results are in agreement with the high degree of banding pattern similarity, previously reported, among cattle, sheep, and goat karyotypes.     

Prion protein gene polymorphisms in sheep in the state of Paraná, Brazil

To determine the polymorphisms of the prion protein gene in sheep from the state of Paraná, Brazil, 323 animals of meat breeds (Suffolk, Hampshire Down, Texel, Ile de France, Dorper, Dorset, Santa Inês and crossbreds) were genotyped by restriction fragment length polymorphism (RFLP) analysis. The most frequent allele was ARQ, with a frequency of 0.61, followed by ARR (0.30). VRQ and AHQ alleles were present at very low frequencies (0.13 and 0.05 respectively), and the ARH allele was not found. Seven genotypes were identified (ARR/ARR, ARR/ARQ, ARQ/ARQ, ARR/VRQ, ARR/AHQ, ARQ/VRQ and ARQ/AHQ), of which ARQ/ARQ was the most frequent (0.41). The Santa Inês breed and crossbred animals showed the highest genotypic variability.