Genotypic effects of calpain 1 and calpastatin on the tenderness of cooked M. longissimus dorsi steaks from Jersey x Limousin, Angus and Hereford-cross cattle

Single nucleotide polymorphisms (SNPs) in the calpain 1 (CAPN1) and calpastatin (CAST) genes were studied to determine their effects on meat tenderness in Bos taurus cattle. Strip loins (M. longissimus dorsi) were removed from cattle in four resource populations after slaughter (n = 1042), aged under controlled conditions until fixed times after rigor mortis, cooked and measured using a tenderometer. Animals were genotyped for the CAPN1 SNP c.947C>G (p.Ala316Gly; AF252504) and for the CAST SNP c.2959A>G (AF159246). Frequencies of CAPN1 C alleles ranged from 23% to 68%, and CAST A alleles from 84% to 99.5%. From all data combined, the CAPN1 CC genotype (compared with the GG genotype) was associated with a 20.1 +/- 1.7% reduced average shear force at intermediate stages of ageing (P < 0.001) and with a 9.5 +/- 1.3% reduction near ultimate tenderness (P < 0.001). The heterozygote was intermediate. For CAST, corresponding values for AA compared with AG genotypes were reductions of 8.6 +/- 2.0% and 5.1 +/- 1.6% respectively (both P < 0.001), but there were too few GG genotypes for comparison. There were small interactions between the CAPN1 and CAST genotypes. For the CAPN1 and CAST genotypes combined, the maximal genotype effect in average shear force was 25.7 +/- 5.5% (P < 0.001) at intermediate stages and 15.2 +/- 4.8% near ultimate tenderness (P < 0.01).     

Calcification rates of the massive coral Siderastrea siderea and crustose coralline algae along the Florida Keys (USA) outer-reef tract

Coral reefs are degrading on a global scale, and rates of reef-organism calcification are predicted to decline due to ocean warming and acidification. Systematic measurements of calcification over space and time are necessary to detect change resulting from environmental stressors. We established a network of calcification monitoring stations at four managed reefs along the outer Florida Keys Reef Tract (FKRT) from Miami to the Dry Tortugas. Eighty colonies (in two sequential sets of 40) of the reef-building coral, Siderastrea siderea, were transplanted to fixed apparatus that allowed repetitive detachment for buoyant weighing every 6 months. Algal-recruitment tiles were also deployed during each weighing interval to measure net calcification of the crustose coralline algal (CCA) community. Coral-calcification rates were an order of magnitude greater than those of CCA. Rates of coral calcification were seasonal (summer calcification was 53 % greater than winter), and corals in the Dry Tortugas calcified 48 % faster than those at the other three sites. Linear extension rates were also highest in the Dry Tortugas, whereas percent area of the coral skeletons excavated by bioeroding fauna was lowest. The spatial patterns in net coral calcification revealed here correlate well with Holocene reef thickness along the FKRT and, in part, support the “inimical waters hypothesis” proposed by Ginsburg, Hudson, and Shinn almost 50 yrs ago to explain reef development in this region. Due to the homogeneity in coral-calcification rates among the three main Keys sites, we recommend refinement of this hypothesis and suggest that water-quality variables (e.g., carbonate mineral saturation state, dissolved and particulate organic matter, light attenuation) be monitored alongside calcification in future studies. Our results demonstrate that our calcification monitoring network presents a feasible and worthwhile approach to quantifying potential impacts of ocean acidification, warming, and/or deteriorating water quality on the process of calcification.     

Low-pH-induced effects on patterns of protein synthesis and on internal pH in Escherichia coli and Salmonella typhimurium

Escherichia coli and Salmonella typhimurium were grown in a supplemented minimal medium (SMM) at a pH of 7.0 or 5.0 or were shifted from pH 7.0 to 5.0. Two-dimensional gel electrophoretic analysis of proteins labeled with H2(35)SO4 for 20 min during the shift showed that in E. coli, 13 polypeptides were elevated 1.5- to 4-fold, whereas in S. typhimurium, 19 polypeptides were increased 2- to 14-fold over the pH 7.0 control. Upon long-term growth at pH 5.0, almost double the number of polypeptides were elevated twofold or more in S. typhimurium compared with E. coli. In E. coli, there was no apparent induction of heat shock proteins upon growth at pH 5.0 in SMM. However, growth of E. coli in a complex broth to pH 5.0, or subsequent growth of fresh E. coli cells in the filtrate from this culture, showed that a subset of five polypeptides is uniquely induced by low pH. Two of these polypeptides, D60.5, the inducible lysyl-tRNA synthetase, and C62.5, are known heat shock proteins. Measurements of the internal pH (pHi) and growth rates of both organisms were made during growth in SMM at pH 7.0, pH 5.0, and upon the pH shift. The data show that the pHi of E. coli decreases more severely than that of S. typhimurium at an external pH of 5.0; the growth rate of E. coli is about one-half that of S. typhimurium at this pH, whereas the two organisms have the same growth rate at pH 7.0. The two-dimensional gel, growth, and pHi experiments collectively suggest that, at least in SMM, S. typhimurium is more adaptive to low-pH stress than is E. coli.     

A bacterial genome in flux: the twelve linear and nine circular extrachromosomal DNAs in an infectious isolate of the Lyme disease spirochete Borrelia burgdorferi

We have determined that Borrelia burgdorferi strain B31 MI carries 21 extrachromosomal DNA elements, the largest number known for any bacterium. Among these are 12 linear and nine circular plasmids, whose sequences total 610 694 bp. We report here the nucleotide sequence of three linear and seven circular plasmids (comprising 290 546 bp) in this infectious isolate. This completes the genome sequencing project for this organism; its genome size is 1 521 419 bp (plus about 2000 bp of undetermined telomeric sequences). Analysis of the sequence implies that there has been extensive and sometimes rather recent DNA rearrangement among a number of the linear plasmids. Many of these events appear to have been mediated by recombinational processes that formed duplications. These many regions of similarity are reflected in the fact that most plasmid genes are members of one of the genome's 161 paralogous gene families; 107 of these gene families, which vary in size from two to 41 members, contain at least one plasmid gene. These rearrangements appear to have contributed to a surprisingly large number of apparently non-functional pseudogenes, a very unusual feature for a prokaryotic genome. The presence of these damaged genes suggests that some of the plasmids may be in a period of rapid evolution. The sequence predicts 535 plasmid genes >/=300 bp in length that may be intact and 167 apparently mutationally damaged and/or unexpressed genes (pseudogenes). The large majority, over 90%, of genes on these plasmids have no convincing similarity to genes outside Borrelia, suggesting that they perform specialized functions.     

Exploitation of Plasmid pMRC01 To Direct Transfer of Mobilizable Plasmids into Commercial Lactococcal Starter Strains

Genetic analysis of the 60.2-kb lactococcal plasmid pMRC01 revealed a 19.6-kb region which includes putative genes for conjugal transfer of the plasmid and a sequence resembling an origin of transfer (oriT). This oriT-like sequence was amplified and cloned on a 312-bp segment into pCI372, allowing the resultant plasmid, pRH001, to be mobilized at a frequency of 3.4 × 10⁻⁴ transconjugants/donor cell from an MG1363 (recA mutant) host containing pMRC01. All of the resultant chloramphenicol-resistant transconjugants contained both pRH001 and genetic determinants responsible for bacteriocin production and immunity of pMRC01. This result is expected, given that transconjugants lacking the lacticin 3147 immunity determinants (on pMRC01) would be killed by bacteriocin produced by the donor cells. Indeed, incorporation of proteinase K in the mating mixture resulted in the isolation of transformants, of which 47% were bacteriocin deficient. Using such an approach, the oriT-containing fragment was exploited to mobilize pRH001 alone to a number of lactococcal hosts. These results demonstrate that oriT of pMRC01 has the potential to be used in the development of mobilizable food-grade vectors for the genetic enhancement of lactococcal starter strains, some of which may be difficult to transform.     

Amylase gene expression in intraspecific and interspecific somatic transformants of Drosophila.

The Amylase locus in Drosophila melanogaster normally contains two copies of the structural gene for alpha-amylase, a centromere-proximal copy, Amy-p, and a distal copy, Amy-d. Products of the two genes may display discrete electrophoretic mobilities, but many strains known to carry the Amy duplication are characterized by a single amylase electromorph, e.g., Oregon-R, which produces the mobility variant AMY-1. A transient expression assay was used in somatic transformation experiments to test the functional status of the Amy genes from an Oregon-R strain. Plasmid constructs containing either the proximal or distal copy were tested in amylase-null hosts. Both genes produced a functional AMY-1 isozyme. Constructs were tested against an AMY-3 reference activity produced by a coinjected plasmid that contains the Amy-d3 allele from a Canton-S strain. With reference to the internal control, the Amy-p and Amy-d genes from Oregon-R expressed different relative activity levels for AMY-1 in transient assays. The transient expression assay was successfully used to test the functional status of Amy-homologous sequences from strains of other species of Drosophila characterized by a single amylase elctromorph, namely, Drosophila pseudoobscura ST and Drosophila miranda S 204. The amylase-null strain of D. melanogaster provided the hosts for these interspecific somatic transformation experiments.     

Degradation of mono-, di-, and trihalogenated benzoic acids by Pseudomonas aeruginosa JB2.

Pseudomonas aeruginosa JB2 was isolated from a polychlorinated biphenyl-contaminated soil by enrichment culture containing 2-chlorobenzoate as the sole carbon source. Strain JB2 was subsequently found also to grow on 3-chlorobenzoate, 2,3- and 2,5-dichlorobenzoates, 2,3,5-trichlorobenzoate, and a wide range of other mono- and dihalogenated benzoic acids. Cometabolism of 2,4-dichlorobenzoate was also observed. Chlorocatechols were the central intermediates of all chlorobenzoate catabolic pathways. Degradation of 2-chlorobenzoate was routed through 3-chlorocatechol, whereas 4-chlorocatechol was identified from the metabolism of both 2,3- and 2,5-dichlorobenzoate. The initial attack on chlorobenzoates was oxygen dependent and most likely mediated by dioxygenases. Although plasmids were not detected in strain JB2, spontaneous mutants were detected in 70% of glycerol-grown colonies. The mutants were all of the following phenotype: benzoate+, 3-chlorobenzoate+, 2-chlorobenzoate-, 2,3-dichlorobenzoate-, 2,5-dichlorobenzoate-. While chlorocatechols were oxidized by the mutants at wild-type levels, oxidation of 2-chloro- and 2,3- and 2,5-dichlorobenzoates was substantially diminished. These findings suggested that strain JB2 possessed, in addition to the benzoate dioxygenase, a halobenzoate dioxygenase that was necessary for the degradation of chlorobenzoates substituted in the ortho position.     

Persistent gene expression in mouse nasal epithelia following feline immunodeficiency virus-based vector gene transfer

Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 10(6) transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 10(7) to 10(9) TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (approximately 10(9) TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for approximately 1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity.     

Thermophilic prokaryotes have characteristic patterns of codon usage, amino acid composition and nucleotide content

A number of recent studies have shown that thermophilic prokaryotes have distinguishable patterns of both synonymous codon usage and amino acid composition, indicating the action of natural selection related to thermophily. On the other hand, several other studies of whole genomes have illustrated that nucleotide bias can have dramatic effects on synonymous codon usage and also on the amino acid composition of the encoded proteins. This raises the possibility that the thermophile-specific patterns observed at both the codon and protein levels are merely reflections of a single underlying effect at the level of nucleotide composition. Moreover, such an effect at the nucleotide level might be due entirely to mutational bias. In this study, we have compared the genomes of thermophiles and mesophiles at three levels: nucleotide content, codon usage and amino acid composition. Our results indicate that the genomes of thermophiles are distinguishable from mesophiles at all three levels and that the codon and amino acid frequency differences cannot be explained simply by the patterns of nucleotide composition. At the nucleotide level, we see a consistent tendency for the frequency of adenine to increase at all codon positions within the thermophiles. Thermophiles are also distinguished by their pattern of synonymous codon usage for several amino acids, particularly arginine and isoleucine. At the protein level, the most dramatic effect is a two-fold decrease in the frequency of glutamine residues among thermophiles. These results indicate that adaptation to growth at high temperature requires a coordinated set of evolutionary changes affecting (i) mRNA thermostability, (ii) stability of codon-anticodon interactions and (iii) increased thermostability of the protein products. We conclude that elevated growth temperature imposes selective constraints at all three molecular levels: nucleotide content, codon usage and amino acid composition. In addition to these multiple selective effects, however, the genomes of both thermophiles and mesophiles are often subject to superimposed large changes in composition due to mutational bias.     

Impact of heparan sulfate chains and sulfur-mediated bonds on the mechanical properties of bovine lens capsule

We assessed the importance of glycosaminoglycans and sulfur-mediated bonds for the mechanical properties of lens capsules by comparing the stress-strain responses from control and treated pairs of bovine source. No significant change in mechanical properties was observed upon reduction of disulfide bonds. However, removal of glycosaminoglycan chains resulted in a significantly stiffer lens capsule, whereas high concentrations of reducing agent, which is expected to reduce the recently reported sulfilimine bond of collagen IV, resulted in a significantly less stiff lens capsule. A comparison of the diffraction patterns of the control and strongly reduced lens capsules indicated structural rearrangements on a nanometer scale.