Fragment of protein L18 from the Escherichia coli ribosome that contains the 5S RNA binding site.

A fragment of ribosomal protein L18 was prepared by limited trypsin digestion of a specific complex of L18 and 5S RNA. It was characterised for sequence and the very basic N-terminal region of the protein was found to be absent. No smaller resistant fragments were produced. 5S RNA binding experiments indicated that the basic N-terminal region, from amino acid residues 1 to 17, was not important for the L18-5S RNA association. Under milder trypsin digestion conditions three resistant fragments were produced from the free protein. The largest corresponded to that isolated from the complex. The smaller ones were trimmed slightly further at both N- and C-terminal ends. These smaller fragments did not reassociate with 5S RNA. It was concluded on the basis of the trypsin protection observations and the 5S RNA binding results that the region extending from residues 18 to 117 approximates to the minimum amount of protein required for a specific and stable protein-RNA interaction. The accessibility of the very basic N-terminal region of L18, in the L18-5S RNA complex, suggests that it may be involved, in some way, in the interaction of 5S RNA with 23S RNA.     

Genomes were forged by massive bombardments with retroelements and retrosequences

Retroposition is an efficient route to move coding regions around the genome ‘in search’ of novel regulatory elements and to shotgun regulatory elements into the genome ‘in search’ of new target genes. The templates for such retrogenes are mRNAs, and for regulatory retronuons (nuon=any definable nucleic acid sequence) usually small non-mRNAs. An example in support of the ‘master gene’ model for SINEs (short interspersed repetive elements) is provided with neuronal BC1 RNA. Furthermore, an alternative explanation of LINE (long interspersed repetive elements) involvement in the generation of SINEs is given. I will also argue that the status of transposable elements with respect to the host resembles more symbiosis than parasitiasis and that host defense is often lenient as if even to ‘tolerate or support’ retronuons. Finally the paradox of evolution's lack of foresight and the future exaptive use of retronuons is being dealt with by referring to W.F. Doolittle's ‘Hierarchical Approaches to Genome Evolution’. This revised version was published online in July 2006 with corrections to the Cover Date.     

Three subunits contribute amino acids to the active site of tetrameric adenylosuccinate lyase: Lys268 and Glu275 are required

Tetrameric adenylosuccinate lyase (ASL) of Bacillus subtilis catalyzes the cleavage of adenylosuccinate to form AMP and fumarate. We previously reported that two distinct subunits contribute residues to each active site, including the His68 and His89 from one and His141 from a second subunit [Brosius, J. L., and Colman, R. F. (2000) Biochemistry 39, 13336-13343]. Glu(275) is 2.8 A from His141 in the ASL crystal structure, and Lys268 is also in the active site region; Glu275 and Lys268 come from a third, distinct subunit. Using site-directed mutagenesis, we have replaced Lys268 by Arg, Gln, Glu, and Ala, with specific activities of the purified mutant enzymes being 0.055, 0.00069, 0.00028, and 0.0, respectively, compared to 1.56 units/mg for wild-type (WT) enzyme. Glu275 was substituted by Gln, Asp, Ala, and Arg; none of these homogeneous mutant enzymes has detectable activity. Circular dichroism and light scattering reveal that neither the secondary structure nor the oligomeric state of the Lys268 mutant enzymes has been perturbed. Native gel electrophoresis and circular dichroism indicate that the Glu275 mutant enzymes are tetramers, but their conformation is altered slightly. For K268R, the K(m)s for all substrates are similar to WT enzyme. Binding studies using [2-3H]-adenylosuccinate reveal that none of the Glu275 mutant enzymes, nor inactive K268A, can bind substrate. We propose that Lys268 participates in binding substrate and that Glu275 is essential for catalysis because of its interaction with His141. Incubation of H89Q with K268Q or E275Q leads to restoration of up to 16% WT activity, while incubation of H141Q with K268Q or E275Q results in 6% WT activity. These complementation studies provide the first functional evidence that a third subunit contributes residues to each intersubunit active site of ASL. Thus, adenylosuccinate lyase has four active sites per enzyme tetramer, each of which is formed from regions of three subunits.     

A key role in catalysis for His89 of adenylosuccinate lyase of Bacillus subtilis

Adenylosuccinate lyase of Bacillus subtilis is a tetrameric enzyme which catalyzes the cleavage of adenylosuccinate to AMP and fumarate. We have mutated His(89), one of three conserved histidines, to Gln, Ala, Glu, and Arg. The enzymes were expressed in Escherichia coli and purified to homogeneity. As compared to a specific activity of 1. 56 micromol of adenylosuccinate converted/min/mg protein for wild-type enzyme, the mutant enzymes exhibit specific activities of 0.0225, 0.0036, 0.0036, and 0.0009 for H89Q, H89A, H89E, and H89R, respectively. Circular dichroism and FPLC gel filtration reveal that mutant enzymes have a similar conformation and oligomeric state to that of wild-type enzyme. In H89Q, the K(M) for adenylosuccinate increases slightly to 2.5-fold that of wild-type, the K(M) for fumarate is elevated 3.3-fold, and the K(M) for AMP is 13 times higher than that observed in wild-type enzyme. The catalytic efficiency of the H89Q enzyme is compromised, with k(cat)/K(M) reduced 174-fold in the direction of AMP formation. These data suggest that His(89) plays a role in both the binding of the AMP portion of the substrate and in correctly orienting the substrate for catalysis. Incubation of H89Q with inactive H141Q enzyme [Lee, T. T., Worby, C., Bao, Z.-Q., Dixon, J. E., and Colman, R. F. (1999) Biochemistry 38, 22-32] leads to a 30-fold increase in activity. This intersubunit complementation indicates that His(89) and His(141) from different subunits participate in the active site and that both are required for catalysis.     

Filamin 2 (FLN2): A muscle-specific sarcoglycan interacting protein

Mutations in genes encoding for the sarcoglycans, a subset of proteins within the dystrophin-glycoprotein complex, produce a limb-girdle muscular dystrophy phenotype; however, the precise role of this group of proteins in the skeletal muscle is not known. To understand the role of the sarcoglycan complex, we looked for sarcoglycan interacting proteins with the hope of finding novel members of the dystrophin-glycoprotein complex. Using the yeast two-hybrid method, we have identified a skeletal muscle-specific form of filamin, which we term filamin 2 (FLN2), as a gamma- and delta-sarcoglycan interacting protein. In addition, we demonstrate that FLN2 protein localization in limb-girdle muscular dystrophy and Duchenne muscular dystrophy patients and mice is altered when compared with unaffected individuals. Previous studies of filamin family members have determined that these proteins are involved in actin reorganization and signal transduction cascades associated with cell migration, adhesion, differentiation, force transduction, and survival. Specifically, filamin proteins have been found essential in maintaining membrane integrity during force application. The finding that FLN2 interacts with the sarcoglycans introduces new implications for the pathogenesis of muscular dystrophy.     

Binding of the S7 Protein with Fragment 926–986/1219–1393 of the 16S rRNA As a Key Step in the Assembly of the Small Subunit of Prokaryotic Ribosomes

Both structural and thermodynamic studies are necessary to understand the ribosome assembly. An initial step was made in studying the interaction between a 16S rRNA fragment and S7, a key protein in assembling the prokaryotic ribosome small subunit. The apparent dissociation constant was obtained for complexes of recombinant Escherichia coliandThermus thermophilusS7 with a fragment of the 3" domain of the E. coli16S rRNA. Both proteins showed high rRNA-binding activity, which was not observed earlier. Since RNA and proteins are conformationally labile, their folding must be considered to correctly describe the RNA–protein interactions.     

The IGF1 small dog haplotype is derived from Middle Eastern grey wolves: a closer look at statistics,sampling, and the alleged Middle Eastern origin of small dogs

This paper is a response to Gray MM, Sutter NB, Ostrander EA, Wayne RK: The IGF1 small dog haplotype is derived from Middle Eastern grey wolves. BMC Biology 2010, 8:16.     

An IGF-I promoter polymorphism modifies the relationships between birth weight and risk factors for cardiovascular disease and diabetes at age 36

BACKGROUND: Biochemical testing for pheochromocytoma by measurement of fractionated plasma metanephrines is limited by false positive rates of up to 18% in people without known genetic predisposition to the disease. The plasma normetanephrine fraction is responsible for most false positives and plasma normetanephrine increases with age. The objective of this study was to determine if we could improve the specificity of fractionated plasma measurements, by statistically adjusting for age. METHODS: An age-adjusted metanephrine score was derived using logistic regression from 343 subjects (including 33 people with pheochromocytoma) who underwent fractionated plasma metanephrine measurements as part of investigations for suspected pheochromocytoma at Mayo Clinic Rochester (derivation set). The performance of the age-adjusted score was validated in a dataset of 158 subjects (including patients 23 with pheochromocytoma) that underwent measurements of fractionated plasma metanephrines at Mayo Clinic the following year (validation dataset). None of the participants in the validation dataset had known genetic predisposition to pheochromocytoma. RESULTS: The sensitivity of the age-adjusted metanephrine score was the same as that of traditional interpretation of fractionated plasma metanephrine measurements, yielding a sensitivity of 100% (23/23, 95% confidence interval [CI] 85.7%, 100%). However, the false positive rate with traditional interpretation of fractionated plasma metanephrine measurements was 16.3% (22/135, 95% CI, 11.0%, 23.4%) and that of the age-adjusted score was significantly lower at 3.0% (4/135, 95% CI, 1.2%, 7.4%) (p < 0.001 using McNemar's test). CONCLUSION: An adjustment for age in the interpretation of results of fractionated plasma metanephrines may significantly decrease false positives when using this test to exclude sporadic pheochromocytoma. Such improvements in false positive rate may result in savings of expenditures related to confirmatory imaging.     

A novel abundant family of retroposed elements (DAS-SINEs) in the nine-banded armadillo (Dasypus novemcinctus)

About half of the mammalian genome is composed of retroposons. Long interspersed elements (LINEs) and short interspersed elements (SINEs) are the most abundant repetitive elements and account for about 21% and 13% of the human genome, respectively. SINEs have been detected in all major mammalian lineages, except for the South American order Xenarthra, also termed Edentata (armadillos, anteaters, and sloths). Investigating this order, we discovered a novel high-copy-number family of tRNA derived SINEs in the nine-banded armadillo Dasypus novemcinctus, a species that successfully crossed the Central American land bridge to North America in the Pliocene. A specific computer algorithm was developed, and we detected and extracted 687 specific SINEs from databases. Termed DAS-SINEs, we further divided them into six distinct subfamilies. We extracted tRNA(Ala)-derived monomers, two types of dimers, and three subfamilies of chimeric fusion products of a tRNA(Ala) domain and an approximately 180-nt sequence of thus far unidentified origin. Comparisons of secondary structures of the DAS-SINEs' tRNA domains suggest selective pressure to maintain a tRNA-like D-arm structure in the respective founder RNAs, as shown by compensatory mutations. By analysis of subfamily-specific genetic variability, comparison of the proportion of direct repeats, and analysis of self-integrations as well as key events of dimerization and deletions or insertions, we were able to delineate the evolutionary history of the DAS-SINE subfamilies.