The natural non-protein amino acid N-β-methylamino-l-alanine (BMAA) is incorporated into protein during synthesis

N-β-methylamino-l-alanine (BMAA) is an amino acid produced by cyanobacteria and accumulated through trophic levels in the environment and natural food webs. Human exposure to BMAA has been linked to progressive neurodegenerative diseases, potentially due to incorporation of BMAA into protein. The insertion of BMAA and other non-protein amino acids into proteins may trigger protein misfunction, misfolding and/or aggregation. However, the specific mechanism by which BMAA is associated with proteins remained unidentified. Such studies are challenging because of the complexity of biological systems and samples. A cell-free in vitro protein synthesis system offers an excellent approach for investigation of changing amino acid composition in protein. In this study, we report that BMAA incorporates into protein as an error in synthesis when a template DNA sequence is used. Bicinchoninic acid assay of total protein synthesis determined that BMAA effectively substituted for alanine and serine in protein product. LC–MS/MS confirmed that BMAA was selectively inserted into proteins in place of other amino acids, but isomers N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobutyric acid (DAB) did not share this characteristic. Incorporation of BMAA into proteins was significantly higher when genomic DNA from post-mortem brain was the template. About half of BMAA in the synthetic proteins was released with denaturation with sodium dodecylsulfonate and dithiothreitol, but the remaining BMAA could only be released by acid hydrolysis. Together these data demonstrate that BMAA is incorporated into the amino acid backbone of proteins during synthesis and also associated with proteins through non-covalent bonding.     

Transfer RNA genes ofZea mays chloroplast DNA

A minimum of 37 genes corresponding to tRNAs for 17 different amino acids have been localized on the restriction endonuclease cleavage site map of theZea mays chloroplast DNA molecule. Of these, 14 genes corresponding to tRNAs for 11 amino acids are located in the larger of the two single-copy regions which separate the two inverted copies of the repeat region. One tRNA gene is in the smaller single-copy region. Each copy of the large repeated sequence contains, in addition to the ribosomal RNA genes, 11 tRNA genes corresponding to tRNAs for 8 amino acids. The genes for tRNA₂ ^Ile and tRNA^Ala map in the ribosomal spacer sequence separating the 16S and 23S ribosomal RNA genes. The three isoaccepting species for the tRNAs^Leu and the three for tRNAs^Ser, as well as the two isoaccepting species for tRNA^Asn, tRNA^Gly, tRNAs^Ile, tRNAs^Met, tRNAs^Thr, are shown to be encoded at different loci. Two independent methods have been used for the localization of tRNA genes on the physical map of the maize chloroplast DNA molecule: (a) cloned chloroplast DNA fragments were hybridized with radioactively-labelled total 4S RNAs, the hybridized RNAs were then eluted, and identified by two-dimensional polyacrylamide gel electrophoresis, and (b) individual tRNAs were³²P-labelledin vitro and hybridized to DNA fragments generated by digestion of maize chloroplast DNA with various restriction endonucleases.     

Cytometric analysis of growth-regulator-dependent transcription and cell-cycle progression in Petunia protoplast cultures

Acridine orange simultaneously stains DNA and RNA. Using flow cytometry, synthesis of these nucleic acids can be related throughout a culture time-course. This technique has been used with nuclei isolated from Petunia hybrida protoplasts during 48 h of culture. Nuclear RNA content has been evaluated with respect to DNA levels, namely the cell-cycle phase. Nuclear RNA synthesis was not dependent upon exogeneous hormones during the first 18 h of culture, but either auxin (2,4-dichlorophenoxyacetic acid, 2,4-D) or cytokinin (N⁶-benzyladenine) were necessary for entry into the S phase. Cytokinin alone could stimulate maximal RNA synthesis within each cell-cycle phase up to 24 h. In complete medium, DNA synthesis only began from a phase “G₁B” having substantial RNA, although a subnormal amount of RNA (in protoplasts cultivated only with 2,4-D) did not prevent protoplast entry into the S phase. However, both hormones were necessary for highest RNA levels and G₂ frequencies after 48 h. As in mammalian cells, the mean RNA level in plant 4C nuclei is double that of 2C nuclei. G₂ nuclei are larger than G₁ nuclei, but upon activation G₁ nuclei in fact diminsh in size. This study aimed to identify restriction points in the cell cycle as affected by growth regulators and the specific synthesis of nucleic acids. For example, the RNA levels induced by N⁶-benzyladenine, although similar to those in complete medium, were not sufficient to induce mitosis. Conversely, 2,4-D action was probably limited by low nucleotide synthesis in the absence of cytokinin.     

Effects of NDA,a new plant growth retardant,on cell culture growth ofZea mays L.

A new plant growth retardant, the norbornenodiazetine derivative 5-(4-chlorophenyl) - 3,4,5,9,10 - pentaaza - tetracyclo - 5,4,1,0^2.6,0^8.11- dodeca - 3,9 - diene (NDA) was tested for its effects on growth ofZea mays suspension cultures. It was shown that NDA could inhibit cell division almost completely at a concentration of 5× 10^?5 M, while 80% of cells could be considered viable. Tracer experiments revealed that NDA inhibited thymidine, uridine, and leucine uptake into cells after 30 min of application. In contrast, amino acid incorporation into proteins was reduced only after one day of treatment and incorporation of precursors into DNA and RNA still later. Since NDA stimulated DNase, RNase, and protease activity in the cells simultaneously, an enhancement of DNA and RNA in cells possibly was prevented. That NDA affected protein synthesis indirectly seemed to be proved by the late point in time of its action on leucine incorporation and by only slight effects on cell free translation. An explanation of these findings could be an alteration in or inhibition of sterol biosynthesis caused by NDA, because it is known that sterols play an important role in controlling permeability of plant membranes as well as in maintaining normal protein synthesis. Thus we tested NDA for its effects on sterol production in maize cells and demonstrated that the composition of the sterol fraction, mainly stigmasterol and β-sitosterol, was clearly changed qualitatively as well as quantitatively.     

Deoxyribonucleic acid base composition of some micrococci and sarcinae

The strains designated in this paper asMicrococcus lysodeikticus, M. sodonensis, M. flavus, Sarcina flava, S. pelagia, S. variabilis, S. marginata, S. subflava, S. citrea, S. lutea andStaphylococcus afermentans have similar DNA base compositions. The mole % GC (guanine plus cytosine) contents in DNA of these strains ranged from 71.8 to 73.3 as calculated from the denaturation temperature (Tm). They may be, therefore, closely related. However, at variance with Kocur and Martinec (1962) they do not seem to be identical withMicrococcus luteus (Schroeter 1872) Cohn 1872, because the neotype culture of the latter species has a different content of guanine and cytosine in its DNA (GC=66.3%). Sarcina aurantiaca, Micrococcus dentrificans andM. luteus have a similar DNA base composition. However, they are not identical as they differ from each other in several physiological characters. In the strains designated asStaphylococcus roseus andSarcina erythromyxa the content of GC varies within the range 72–72.8%. These species do not differ from each other physiologically. They form a pink pigment, reduce nitrates, do not hydrolyze casein and gelatin, and do not produce urease. They seem, therefore, to be identical, which confirms the conclusion of Kocur and Martinec (1962) who designated them asMicrococcus roseus Flügge 1886. Micrococcus conglomeratus differs significantly in DNA base composition from almost all strains of the groupM. lysodeikticus—Staphylococcus afermentans, also fromMicrococcus luteus, M. roseus andM. denitrificans. It differs fromSarcina aurantiaca only physiologically.     

Analysis of amino acids by HPLC/electrospray negative ion tandem mass spectrometry using 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) derivatization

A new method for the determination of amino acids is presented. It combines established methods for the derivatization of primary and secondary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) with the subsequent amino acid specific detection of the derivatives by LC–ESI–MS/MS using multiple reaction monitoring (MRM). The derivatization proceeds within 5 min, and the resulting amino acid derivatives can be rapidly purified from matrix by solid-phase extraction (SPE) on HR-X resin and separated by reversed-phase HPLC. The Fmoc derivatives yield several amino acid specific fragment ions which opened the possibility to select amino acid specific MRM transitions. The method was applied to all 20 proteinogenic amino acids, and the quantification was performed using l-norvaline as standard. A limit of detection as low as 1 fmol/µl with a linear range of up to 125 pmol/µl could be obtained. Intraday and interday precisions were lower than 10 % relative standard deviations for most of the amino acids. Quantification using l-norvaline as internal standard gave very similar results compared to the quantification using deuterated amino acid as internal standards. Using this protocol, it was possible to record the amino acid profiles of only a single root from Arabidopsis thaliana seedlings and to compare it with the amino acid profiles of 20 dissected root meristems (200 μm).     

On the possible origin and evolution of the genetic code

The genetic code is examined for indications of possible preceding codes that existed during early evolution. Eight of the 20 amino acids are coded by ‘quartets’ of codons with four-fold degeneracy, and 16 such quartets can exist, so that an earlier code could have provided for 15 or 16 amino acids, rather than 20. If two-fold degeneracy is postulated for the first position of the codon, there could have been 10 amino acids in the code. It is speculated that these may have been phenylalanine, valine, proline, alanine, histidine, glutamine, glutamic acid, aspartic acid, cysteine and glycine. There is a notable deficiency of arginine in proteins, despite the fact that it has six codons. Simultaneously, there is more lysine in proteins than would be expected from its two codons, if the four bases in mRNA are equiprobable and are arranged randomly. It is speculated that arginine is an ‘intruder’ into the genetic code, and that it may have displaced another amino acid such as ornithine, or may even have displaced lysine from some of its previous codon assignments. As a result, natural selection has favored lysine against the fact that it has only two codons. The introduction of tRNA into protein synthesis may have been a cataclysmic and comparatively sudden event, since duplication of tRNA takes place readily, and point mutations could rapidly differentiate members of the family of duplicates from each. Two tRNAs for different amino acids may have a common ancestor that existed more recently than the separation of the prokaryotes and eukaryotes. This is shown by homology of twoE. coli tRNAs for glycine and valine, and two yeast tRNAs for arginine and lysine.     

Nitratireductor shengliensis sp. nov., Isolated from an Oil-Polluted Saline Soil

Two Gram-negative, non-motile, short-rod-shaped bacterial isolates, designated 110399^T and 110248, were isolated from an oil-polluted saline soil in Shengli Oilfield, Eastern China. The two strains shared 99.9 % 16S rRNA gene sequence similarity with the DNA–DNA relatedness value being 80.0 %. They were both capable to grow at 20–40 °C, pH 7–9, and 1–9 % (w/v) NaCl with the optimum growth happened at 30 °C, pH 8, and 2–6 % (w/v) NaCl. The phylogenetic analysis based on 16S rRNA gene sequences revealed that the two strains were members of Nitratireductor and most closely related to Nitratireductor pacificus pht-3B^T and N. basaltis J3^T with the 16S rRNA gene sequence similarities being 97.1 and 97.0 %. The DNA–DNA relatedness between the novel strains and two type strains were below 27 ± 7 %. The strains 110399^T and 110248 also differed from N. pacificus and N. basaltis in nitrate reduction, salt tolerance, enzyme activities, and utilization of carbon sources. The major cellular fatty acids of strain 110399^T were C_19:0ω8c cyclo (10.5 %) and Summed Feature 8 (C_18:1ω7c and/or C_18:1ω6c, 41.5 %) which are typical in the genus Nitratireductor. The predominant ubiquinone was Q-10. The genome DNA G+C content of strain 110399^T and 110248 was 61.1 and 61.7 mol%. On the basis of genetic, phenotypic, and chemotaxonomic analyses, strains 110399^T and 110248 represent a novel species within the genus Nitratireductor, for which the name Nitratireductor shengliensis sp. nov. is proposed. The type strain is 110399^T (=CGMCC 1.12519^T = LMG 27405^T).     

Identification of proteolytic bacteria from the Arctic Chukchi Sea expedition cruise and characterization of cold-active proteases

Following collection of seawater samples during an Arctic Chukchi Sea expedition cruise of the Korean icebreaker Araon in 2012, a total of 15,696 bacteria were randomly isolated from Marine Broth 2216 agar plates. Of these, 2,526 (16%) showed proteolytic activity and were identified as mainly Alteromonas (31%), Staphylococcus (27%), and Pseudoalteromonas (14%). Among the proteolytic strains, seven were selected based on their significant ability to grow and produce a halo on skim milk plates at low temperatures (<5°C) owing to cold-active proteases. These strains were affiliated with the genus Pseudoalteromonas and were divided into three groups based on phylogenetic analysis of the 16S rRNA genes. Profiling cell membrane fatty acids confirmed the 16S rRNA-based differentiation and revealed the accordance between the two analyses. Seven genes for serine protease precursors were amplified from the corresponding strains, and based on sequence similarities, these genes were divided into three groups that were identical to those identified by the 16S rRNA phylogenetic analysis. Three protease genes from the representative strains of each group were composed of 2,127–2,130 bp, encoding 708–709 amino acids, and these genes yielded products with calculated molecular weights of approximately 72.3–72.8 kDa. Amino acid sequence analysis suggested that the precursors are members of the subtilase serine endo- and exo-peptidase clan and contain four domains (signal peptide, N-terminal prosequence, catalytic domain, and two pre-peptidase C-terminal domains). Upon expression in E. coli, each recombinant protease exhibited proteolytic activity on zymogram gels.     

New antibiotics produced by Bacillus subtilis strains

Two Bacillus subtilis strains isolated from the fruiting body of a basidiomycete fungus Pholiota squarrosa exhibited a broad range of antibacterial activity, including those against methicillin-resistant Staphylococcus aureus INA 00761 (MRSA) and Leuconostoc mesenteroides VKPM B-4177 resistant to glycopeptide antibiotics, as well as antifungal activity. The strains were identified as belonging to the “B. subtilis” complex based on their morphological and physiological characteristics, as well as by sequencing the 16S rRNA gene fragments. Both strains (INA 01085 and INA 01086) produced insignificant amounts of polyene antibiotics (hexaene and pentaene, respectively). Strain INA 01086 also produced a cyclic polypeptide antibiotic containing Asp, Gly, Leu, Pro, Tyr, Thr, Trp, and Phe, while the antibiotic of strain INA 01085 contained, apart from these, two unidentified nonproteinaceous amino acids. Both polypeptide antibiotics were new compounds efficient against gram-positive bacteria and able to override the natural bacterial antibiotic resistance.     

DNA Base composition of soil arthrobacters and other coryneforms from cheese and sea fish

The DNA base composition of 34 coryneforms isolated from different sources, and those of 20 named cultures of the generaArthrobacter, Brevibacterium,Mycobacterium, Corynebacterium andNocardia has been determined. A preliminary study of the morphological and physiological characteristics of the new isolates, and some named cultures, led to a division into three groups:

1. Soil coryneforms identified as arthrobacters, completed with theArthrobacter globiformis strains ATCC 8602 and 8010.
2. Orange coryneforms and one white isolate from cheese, and orange coryneforms including one yellow isolate from sea fish, completed with twoBrevibacterium linens strains ATCC 9174 and 9175.
3. Non-orange cheese coryneforms.

DNA base composition of group (1) ranges from 65.3 to 67.0 molar % GC, suggesting that this group is genetically homogeneous. % GC values of group (2) range from 62.6 to 64.0 except for one isolate (65.6), suggesting that this group is also homogeneous. DNA base composition of group (3) ranges from 65.5 to 66.9 % GC, except for three isolates (56.5, 60.1, 60.6). It is concluded that as far as their % GC is concerned, the strains of group (3), except the threem entioned ones, may be closely related to the arthrobacters of group (1). The strains of group (2) are probably less closely related to those of the groups (1) and (3).