Structure of the genetic code suggested by the hydropathy correlation between anticodons and amino acid residues

The correlation between hydropathies of anticodons and amino acids, detected by other authors utilizing scales of amino acid molecules in solution, was improved with the utilization of scales of amino acid residues in proteins. Three partitions were discerned in the correlation plot with the principal dinucleotides of anticodons (pDiN, excluding the wobble position). (a) The set of outliers of the correlation: Gly-CC, Pro-GG, Ser-GA and Ser-CU. The amino acids are consistently small, hydro-apathetic, stabilizers of protein N-ends, preferred in aperiodic protein conformations and belong to synthetases class II. The pDiN sequences are representative of the homogeneous sector (triplets NRR and NYY), distinguished from the mixed sector (triplets NRY and NYR), that depict a 70% correspondence to the synthetases class II and I, respectively. The triplet pairs proposed to be responsible for the coherence in the set of outliers are of the palindromic kind, where the lateral bases are the same, CCC: GGG and AGA: UCU. This suggests that UCU previously belonged to Ser, adding to other indications that the attribution of Arg to YCU was due to an expansion of the Arg-tRNA synthetase specificity. The other attributions produced two correlation sets. (b) One corresponds to the remaining pDiN of the homogeneous sector, containing both synthetase classes; its regression line overlapped the one formed by the remaining attributions to class II. (c) The other contains the pDiN of the mixed sector and produced steeper slopes, especially with the class I attributions. It is suggested that the correlation was established when the amino acid composition of the protein synthetases became progressively enriched and that the set of outliers were the earliest to have been fixed.     

Spontaneous and 9-aminoacridine-induced frameshift mutagenesis: second-site frameshift mutation within the N-terminal region of the lacI gene of Escherichia coli

Summary A novel forward mutational system, based on the acquisition of an I^q-d dominant phenotype from an initial I^q− recessive state, was used to identify second-site frameshift mutation [±1(±3 _n ) events] within the N-terminal region of thelacI gene ofEscherichia coli. The DNA sequences are described of forty-six spontaneous and twenty 9-aminoacridine(9-AA)-induced second site mutations. Although −1 frameshift events dominate both spectra, the nature and site specificity of these events clearly distinguish two mutational distributions. The spontaneous distribution contains two −(A: T) frameshift hotspots; one within a monotonic A₅ run (9 occurrences), the other at a 5′-CACAACAAC-3′ sequence (12 occurrences). In contrast 17 of the 20 mutations recovered after 9-AA treatment involve the loss of a G: C pair, 14 of which occur at a single site (5′-CGGGC-3′). The striking specificity of the observed mutational hotspots is of interest since this open genetic target contains similar sequences which were infrequently recovered.     

Identification of tobacco proteins associated with the stem-loop 1 RNAs of <Emphasis Type="Italic">Potato virus X</Emphasis>

Potato virus X (PVX) contains five viral proteins as well as cis-acting elements like stem-loop 1 (SL1) RNAs at the 5′ region. SL1 RNAs are involved in PVX RNA replication, encapsidation, translation, and cell-to-cell movement. In this study, we performed two-dimensional electrophoresis Northwestern blot analysis and matrix-assisted laser desorption ionization time of flight mass spectrometry and identified 24 tobacco proteins that interact with SL1 RNAs. Interestingly, one-third of the identified host proteins have been shown to interact with many plant viral proteins. In addition, we demonstrated that PVX capsid protein can bind to both SL1(+/−) RNAs. We further selected three Nicotiana benthamiana proteins including NbMPB2Cb, NbMBF1, and NbCPIP2a, to confirm results of Northwestern blot analysis. Electrophoretic mobility shift assay showed that NbMPB2Cb and NbMBF1 bind to both SL1(+/−) RNAs in vitro. In contrast, NbCPIP2a interacts only SL1(+) RNA. Taken together, we provide a list of host proteins interacting with PVX SL1 RNAs, which would be good candidates for the study of viral RNA-host protein interaction.     

Two modes of sulfite oxidation in the extremely thermophilic and acidophilic archaeon Acidianus ambivalens

Sulfite-oxidizing enzyme activities were analyzed in cell-free extracts of aerobically grown cells of Acidianus ambivalens, an extremely thermophilic and chemolithoautotrophic archaeon. In the membrane and cytoplasmic fractions, two distinct enzyme activities were found. In the membrane fraction, a sulfite:acceptor oxidoreductase activity was found [530 mU (mg protein)^–1; apparent K _m for sulfite, 3.6 mM]. In the cytoplasmic fraction the following enzyme activities were found and are indicative of an oxidative adenylylsulfate pathway: adenylylsulfate reductase [138 mU (mg protein)^–1], adenylylsulfate:phosphate adenyltransferase [“ADP sulfurylase”; 86 mU (mg protein)^–1], adenylate kinase [650 mU (mg protein)^–1], and rhodanese [thiosulfate sulfur transferase, 9.2 mU (mg protein)^–1]. In addition, 5′,5′′′-P¹,P⁴-di(adenosine-5′) tetraphosphate (Ap₄A) synthase and Ap₄A pyrophosphohydrolase activities were detected. Received: 17 August 1998 / Accepted: 29 April 1999     

Mass spectrometry identification of cytochrome P450 2B4 interaction sites for NADPH: Cytochrome P450 reductase

The interaction sites for protein partners, cytochrome P450 2B4 (d-2B4) and NADPH: cytochrome P450 reductase (d-Fp), have been identified. These proteins form complexes during their functioning. Nonspecific covalent cross-linking of the d-2B4 complexes with d-Fp in the Emulgen 913 monomerized system was achieved by 4,4′- dithiobis-phenyl azide. Covalently cross-linked peptides of this complex were identified by ESI-MS/MS. Several binding sites have been identified for these proteins. Based on these sites a model for intermolecular interaction between these proteins has been proposed. This model includes 5 contact sites on d-2B4 for d-Fp (stabilized by the cross-linker); these include the following pairs of corresponding peptides of d-2B4 and d-Fp: 1) d-2B4_324–336 and d-Fp_570–585; 2) d-2B4_423–433 and d-Fp_102–109; 3) d-2B4_327–336 and d Fp_452–464; 4) d-2B4_192–197 and d-Fp_456–464; 5) d-2B4_134–139 and d-Fp_406–425. In the two last cases d-Fp peptides are located in the interdomain cleft and stabilize the protein-protein complex via the cross-linker and so the d 2B4/d-Fp complex formation by these sites may involve amino acid residues of the peptides d-Fp_456–464 and d-Fp_406–425, which surround the interdomain cleft.     

The current state of the Demoiselle crane population in the Transural steppe

The Demoiselle crane population was studied in the southern Chelyabinsk region (52°12′N; 60°21′E) during 1988–2008. Counts of nesting pairs were annually conducted over an area of 100–130 km² in May. The number of breeding pairs varied from 6.5 to 9.5 per 100 km²; on average, there were 8 pairs per 100 km² during 1989–1991. In 2000–2008, the population density increased from 7.5 to 12 pairs per 100 km², on average, 9.3 pairs per 100 km2. Demoiselle crane nesting in steppe pastures and perennial grass fields has been rarely observed for the last decade. Now the main nesting biotopes of the species are stubble fields accounting for up to 70% of all nidification cases documented. The decline in agricultural production in recent decades has resulted in the appearance of long-fallow lands, in which 17 to 35% crane pairs nest.     

A plant protein kinase and plant microsomal H+ transport are stimulated by the ether phospholipid platelet-activating factor

ATP-dependent H⁺ transport in microsomes from zucchini hypocotyls is stimulated by the ether lipid 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor = PAF) known as a hormone-like substance from mammals. The stimulation can only be observed when soluble cytosolic proteins are present. A soluble protein mediating the PAF-dependent H⁺ transport and a PAF-stimulated protein kinase are coeluted by DEAE-Sephacel chromatography. Stimulation of phosphorylation by PAF of a 55 kDa polypeptide without Ca²⁺ and, additionally, of a 35 kDa polypeptide in the presence of Ca²⁺ is observed in zucchini microsomal membranes. This is evidence for a novel phospholipid-stimulated protein kinase in plants. Phosphorylation of regulatory proteins may be involved in the stimulation of in vitro H⁺ transport by PAF.Abbreviations BTP 1,3-bis (tris(hydroxymethyl)-methylamino) propane - DTT dithiotreitol - EGTA ethylene glycol-bis (ß-aminoethyl ether)-N,N,N,N,-tetraacetic acid - Mes 2-(N-morpholino) ethanesulfonic acid - PAF platelet-activating factor = 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine - SDS sodium dodecylsulfate - TCA trichloroacetic acid     

The lipopolysaccharide of recipient cells is a specific receptor for PilV proteins, selected by shufflon DNA rearrangement, in liquid matings with donors bearing the R64 plasmid

Shufflon DNA rearrangement selects one of seven PilV proteins with different C-terminal segments, which then becomes a minor component of the thin pili of Escherichia coli strains bearing the plasmid R64. The PilV proteins determine the recipient specificity in liquid matings. A recipient Escherichia coli K-12 strain was specifically recognized by the PilVA′, -C, and -C′ proteins, while E. coli B was recognized only by the PilVA′ protein. To identify specific PilV receptors in the recipient bacterial cells, R64 liquid matings were performed using various E. coli K-12 waa (rfa) mutants and E. coli B transformants as recipient cells. E. coli K-12 waa mutants lack receptors for specific PilV proteins. E. coli B cells carrying waaJ or waaJKL genes of E. coli K-12 were recognized by donors expressing the PilVC′ protein or the PilVC and -C′ proteins, respectively, in addition to the PilVA′ protein. Addition of E. coli K-12 or B lipopolysaccharide (LPS) specifically inhibited liquid matings. We conclude that the PilV proteins of the thin pili of R64-bearing donors recognize LPS molecules located on the surface of various recipient bacterial cells in liquid matings. Received: 2 September 1999 / Accepted: 18 November 1999     

Genetic probing of the interaction between the translation factor SelB and its mRNA binding element in Escherichia coli

Decoding of the UGA codon in mRNAs for selenoproteins as selenocysteine requires interaction of the translation factor SelB with an mRNA structure, the SECIS element. A genetic analysis of this interaction was performed by selecting for intergenic suppressor mutations in selB which counteracted the detrimental effect of defined mutations in the SECIS element. Both allele-nonspecific and allele-specific mutations, as judged by readthrough of the UGA into the LacZ-encoding segment of fdhF ′-′lacZ fusions and by incorporation of selenium, were isolated. selB genes from ten suppressor mutants were sequenced and the corresponding mutations were localized to five positions within the protein. Four of the suppressors had amino acid exchanges within a 23-amino acid stretch in domain 4b of SelB, which probably represent sites of contact between the protein and the mRNA. A fifth mutation was localized in domain 4a of SelB; it promoted allele-nonspecific readthrough. Since a truncated SelB species lacking domain 4b did not show complex formation with the SECIS element, we speculate that the latter mutation affects the interaction between the tRNA-binding and the mRNA-binding domains. None of the SelB variants was able to promote UGA readthrough when major structural changes that altered the length of the helical part or enlarged the apical loop were introduced into the SECIS element. The results obtained also show that novel pairs of SelB/SECIS derivatives can be generated which may be useful for the targeted insertion of selenocysteine into proteins. Received: 29 June 1999 / Accepted: 10 July 1999     

Isolation and physical characterization of three essential conidiation genes from Aspergillus nidulans

We cloned and characterized three genes from Aspergillus nidulans, designated brlA, abaA and wetA, whose activities are required to complete different stages of conidiophore development. Inactivation of these genes causes major abnormalities in conidiophore morphology and prevents expression of many unrelated, developmentally regulated genes, without affecting expression of nonregulated genes. The three genes code for poly(A)⁺RNAs that begin to accumulate at different times during conidiation. The brlA-and abaA-encoded RNAs accumulate specifically in cells of the conidiophore. The wetA-encoded RNA accumulates in mature conidia. Inactivation of the brlA gene prevents expression of the abaA and wetA genes, whereas inactivation of the abaA gene prevents expression of the wetA gene. Our results confirm genetic predictions as to the temporal and spatial patterns of expression of these genes and demonstrate that these patterns are specified at the level of RNA accumulation.     

Determination of the complete mitochondrial DNA sequence of <Emphasis Type="Italic">Octopus minor</Emphasis>

In this study, we have determined the complete nucleotide sequence of the mitochondrial genome of Octopus minor. It is 15,974 nucleotide pairs and encodes 13 proteins, two ribosomal RNAs and 22 tRNAs of the mitochondrion’s own protein synthesizing system. Seven of thirteen proteins are encoded by the H-strand, while the other six proteins, as well as the two ribosomal RNAs are encoded by the L-strand. The nucleotide composition of the proteins showed a nucleotide bias against G encoded by the H-strand, while they showed a nucleotide bias against A and C encoded by the L-strand. Two of the 13 protein coding genes of O. minor began with the unorthodox translation initiation codon ATA and all others use the standard ATG. In addition, six of thirteen mt proteins of O. minor have unambiguous termination codons. There are four cases where tRNA genes appear to overlap. The long noncoding region (LNCR) of O. minor was 930 nucleotides and no repeated sequences were found in this LNCR. The gene arrangements of O. minor showed remarkable similarity to that of O. ocellatus and O. vulgaris. Phylogenetic analysis demonstrated that O. minor appears as sister taxan to the monophyletic group combined by O. ocellatus and O. vulgaris, suggesting a relative distant genetic relationship between O. minor and the other two octopus species.     

Loss of Introns Along the Evolutionary Diversification Pathway of Snake Venom Disintegrins Evidenced by Sequence Analysis of Genomic DNA from <Emphasis Type="Italic">Macrovipera lebetina transmediterranea</Emphasis> and <Emphasis Type="Italic">Echis ocellatus</Emphasis>

Analysis of cDNAs from Macrovipera lebetina transmediterranea (Mlt) and Echis ocellatus (Eo) venom gland libraries encoding disintegrins argued strongly for a common ancestry of the messengers of short disintegrins and those for precursors of dimeric disintegrin chains. We now report the sequence analysis of disintegrin-coding genes from these two vipers. Genomic DNAs for dimeric disintegrin subunits Ml_G1 and Ml_G2 (Mlt) and Eo_D3 (Eo) contain single 1-kb introns exhibiting the 5′-GTAAG (donor)/3′-AG (acceptor) consensus intron splicing signature. On the other hand, the short RTS-disintegrins Ml_G3 (Mlt) and Eo_RTS (Eo) and the short RGD-disintegrin ocellatusin (Eo) are transcribed from intronless genomic DNA sequences, indicating that the evolutionary pathway leading to the emergence of short disintegrins involved the removal of all intronic sequences. The insertion position of the intron within Ml_G1, Ml_G2, and Eo_D3 is conserved in the genes for vertebrate ADAM (A disintegrin and metalloproteinase) protein disintegrin-like domains and within the gene for the medium-size snake disintegrins halystatins 2 and 3. However, a comparative analysis of currently available disintegrin(-like) genes outlines the view that a minimization of both the gene organization and the protein structure underlies the evolution of the snake venom disintegrin family. [Reviewing Editor: Dr. Bryan Fry] Amine Bazaa and Paula Juárez contributed equally to this work and may both be considered first authors.     

The novel protein KBP regulates mitochondria localization by interaction with a kinesin-like protein

Background  

Members of the Kinesin-3 family of kinesin-like proteins mediate transport of axonal vesicles (KIF1A, KIF1Bβ), distribution of mitochondria (KIF1Bα) and anterograde Golgi to ER vesicle transport (KIF1C). Until now, little is known about the regulation of kinesin-like proteins. Several proteins interact with members of this protein family. Here we report on a novel, KIF1 binding protein (KBP) that was identified in yeast two-hybrid screens.     

Genetic variability in relocated Père David’s deer (<Emphasis Type="Italic">Elaphurus davidianus</Emphasis>) populations—Implications to reintroduction program

Since 1985, China has established three breeding herds of Père David’s deer: the Beijing Père David’s Deer Park (39°07′N, 116°03′E), the Dafeng Père David’s Deer Nature Reserve (33°05′N, 120°49′E) and Shishou (Tianezhou) Père David’s Deer Nature Reserve (29°49′N, 112°33′E), through reintroductions of about 30–40 founders. Since establishment, all three populations have grown steadily. However, genetic backgrounds in those populations are still unknown. We studied the genetic diversity in Père David’s deer and genetic consequences of population relocations in China. We revealed that genetic diversity was extremely low in Père David’s deer populations in China. Only a single mtDNA D-loop haplotype was found in the deer, furthermore, only five polymorphic microsatellite loci were screened out from 84 pairs of species-transferred primers. Genetic makeup in the three Père David’s deer populations were significantly different (P < 0.01). H _E and allelic richness in the Tianezhou population were the highest (0.54, 2.60, n = 31), Beijing population (0.52, 2.4, n = 125) showed the second highest measures, while the Dafeng population (0.46, 2.39, n = 39) measured lowest. Our results suggest that effective management of a species of low genetic diversity like the Père David’s deer should consider the genetic background of each founder to make sure genetic variations are preserved in both source population and relocated population. Now, the Tianezhou population is the most appropriate source population in China when establishing new Père David deer populations in the wild.

An analysis of genetic variation across the MBL2 locus in Dutch Caucasians indicates that 3′ haplotypes could modify circulating levels of mannose-binding lectin

Mannose-binding protein (MBL) is a critical component of innate immunity and provides first-line protection against pathogens. Both circulating MBL serum levels and functional activity have been correlated with common genetic variants in the MBL2 gene. Associations between MBL deficiency and severe infections have been reported in immuno-incompetent patients and for autoimmune disorders; however, measured MBL serum levels do not fully correlate with the ‘secretor haplotypes’. Previously, the MBL2 locus was resequenced and determined that a recombination hotspot divides MBL2 into two haplotype blocks. It was sought to investigate whether additional variants, in either block structure could associate with MBL serum levels. Therefore, 31 common variants were analysed across the locus in 212 DNA samples of healthy Caucasian individuals with known MBL serum concentrations. The additional 5′ variants were in strong linkage to the elements of the ‘secretor haplotypes’; functional alleles B, C and D also lie on restricted haplotypes. Four variants in the 3′ block (Ex4-1483T>C, Ex4-1067G>A, Ex4-901G>A and Ex4-710G>A) are components of a distinct haplotype block. The results of this study suggest that additional 5′ variants as well as markers of distinct 3′ haplotype blocks in MBL2 may contribute to circulating protein levels, but further studies are required to confirm these observations. Last, there could be a selective advantage for diversification of the 3′ region of the gene.Electronic Supplementary Material Supplementary material is available for this article at