Frequency of deletion formation decreases exponentially with distance between short direct repeats

The effect of distance between 18 bp direct repeats on deletion formation has been examined in Bacillus subtilis. The deletion frequency decreased exponentially by more than 1000-fold as the distance increased from 33 to 2313 bp. This decrease occurred in two distinct phases, which may be determined by DNA-duplex flexibility. A similar relationship between deletion formation and distance was observed in a θ-replicating plasmid and in the chromosome, indicating that this relationship might have a general validity.     

Relations between and inheritance of chlorogenic acid contents in an interspecific cross between Coffea pseudozanguebariae and Coffea liberica var ‘dewevrei’

 Chlorogenic acids (CGA) are phenolic compounds commonly found in green coffee beans. The main CGA classes are caffeoylquinic acids (CQA), dicaffeoylquinic acids (diCQA), and feruloylquinic acids (FQA). Each contains three isomers differing in the number and identity of the acylating residues. An interspecific cross between Coffea pseudozanguebariae (low CGA content) and C. liberica var ‘dewevrei’ (high CGA content) was investigated for CGA contents in F₁ and back-cross hybrids. Relations within and between CGA classes were studied and confirmed the known biosynthesis pathway. A single major gene was noted for the 3-FQA isomer; absence was dominant. Additivity was found for most other isomers either with or without the transformation of variables. Conversely, most ratios were not additive, due to a curvilinear relation between some isomers. The consequences for breeding both in terms of cup taste improvement and disease resistance are discussed. Received: 20 July 1998 / Accepted: 14 August 1998     

Identification of citrullinated alpha-enolase as a candidate autoantigen in rheumatoid arthritis

Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis (RA), but little is understood about their citrullinated target antigens. We have detected a candidate citrullinated protein by immunoblotting lysates of monocytic and granulocytic HL-60 cells treated with peptidylarginine deiminase. In an initial screen of serum samples from four patients with RA and one control, a protein of molecular mass 47 kDa from monocytic HL-60s reacted with sera from the patients, but not with the serum from the control. Only the citrullinated form of the protein was recognised. The antigen was identified by tandem mass spectrometry as alpha-enolase, and the positions of nine citrulline residues in the sequence were determined. Serum samples from 52 patients with RA and 40 healthy controls were tested for presence of antibodies against citrullinated and non-citrullinated alpha-enolase by immunoblotting of the purified antigens. Twenty-four sera from patients with RA (46%) reacted with citrullinated alpha-enolase, of which seven (13%) also recognised the non-citrullinated protein. Six samples from the controls (15%) reacted with both forms. Alpha-enolase was detected in the RA joint, where it co-localised with citrullinated proteins. The presence of antibody together with expression of antigen within the joint implicates citrullinated alpha-enolase as a candidate autoantigen that could drive the chronic inflammatory response in RA.     

Protein interaction networks in bacteria

The complement of expressed cellular proteins - the proteome - is organized into functional, structured networks of protein interactions that mediate assembly of molecular machines and dynamic cellular pathways. Recent studies reveal the biological roles of protein interactions in bacteriophage T7 and Helicobacter pylori, and new methods allow to compare and to predict interaction networks in other species. Smaller scale networks provide biological insights into DNA replication and chromosome dynamics in Bacillus subtilis and Archeoglobus fulgidus, and into the assembly of multiprotein complexes such as the type IV secretion system of Agrobacterium tumefaciens, and the cell division machinery of Escherichia coli. Genome-wide interaction networks in several species are needed to obtain a biologically meaningful view of the higher order organization of the proteome in bacteria.     

Bacillus subtilis ribonucleases J1 and J2 form a complex with altered enzyme behaviour

Ribonucleases J1 and J2 are recently discovered enzymes with dual 5′‐to‐3′ exoribonucleolytic/endoribonucleolytic activity that plays a key role in the maturation and degradation of Bacillus subtilis RNAs. RNase J1 is essential, while its paralogue RNase J2 is not. Up to now, it had generally been assumed that the two enzymes functioned independently. Here we present evidence that RNases J1 and J2 form a complex that is likely to be the predominant form of these enzymes in wild‐type cells. While both RNase J1 and the RNase J1/J2 complex have robust 5′‐to‐3′ exoribonuclease activity in vitro, RNase J2 has at least two orders of magnitude weaker exonuclease activity, providing a possible explanation for why RNase J1 is essential. The association of the two proteins also has an effect on the endoribonucleolytic properties of RNases J1 and J2. While the individual enzymes have similar endonucleolytic cleavage activities and specificities, as a complex they behave synergistically to alter cleavage site preference and to increase cleavage efficiency at specific sites. These observations dramatically change our perception of how these ribonucleases function and provide an interesting example of enzyme subfunctionalization after gene duplication.     

Disruption of LACCASE4 and 17 results in tissue-specific alterations to lignification of Arabidopsis thaliana stems

Peroxidases have been shown to be involved in the polymerization of lignin precursors, but it remains unclear whether laccases (EC 1.10.3.2) participate in constitutive lignification. We addressed this issue by studying laccase T-DNA insertion mutants in Arabidopsis thaliana. We identified two genes, LAC4 and LAC17, which are strongly expressed in stems. LAC17 was mainly expressed in the interfascicular fibers, whereas LAC4 was expressed in vascular bundles and interfascicular fibers. We produced two double mutants by crossing the LAC17 (lac17) mutant with two LAC4 mutants (lac4-1 and lac4-2). The single and double mutants grew normally in greenhouse conditions. The single mutants had moderately low lignin levels, whereas the stems of lac4-1 lac17 and lac4-2 lac17 mutants had lignin contents that were 20 and 40% lower than those of the control, respectively. These lower lignin levels resulted in higher saccharification yields. Thioacidolysis revealed that disrupting LAC17 principally affected the deposition of G lignin units in the interfascicular fibers and that complementation of lac17 with LAC17 restored a normal lignin profile. This study provides evidence that both LAC4 and LAC17 contribute to the constitutive lignification of Arabidopsis stems and that LAC17 is involved in the deposition of G lignin units in fibers.     

Individual differences in impulsivity predict anticipatory eye movements

Impulsivity is the tendency to act without forethought. It is a personality trait commonly used in the diagnosis of many psychiatric diseases. In clinical practice, impulsivity is estimated using written questionnaires. However, answers to questions might be subject to personal biases and misinterpretations. In order to alleviate this problem, eye movements could be used to study differences in decision processes related to impulsivity. Therefore, we investigated correlations between impulsivity scores obtained with a questionnaire in healthy subjects and characteristics of their anticipatory eye movements in a simple smooth pursuit task. Healthy subjects were asked to answer the UPPS questionnaire (Urgency Premeditation Perseverance and Sensation seeking Impulsive Behavior scale), which distinguishes four independent dimensions of impulsivity: Urgency, lack of Premeditation, lack of Perseverance, and Sensation seeking. The same subjects took part in an oculomotor task that consisted of pursuing a target that moved in a predictable direction. This task reliably evoked anticipatory saccades and smooth eye movements. We found that eye movement characteristics such as latency and velocity were significantly correlated with UPPS scores. The specific correlations between distinct UPPS factors and oculomotor anticipation parameters support the validity of the UPPS construct and corroborate neurobiological explanations for impulsivity. We suggest that the oculomotor approach of impulsivity put forth in the present study could help bridge the gap between psychiatry and physiology.     

Rapid and multi-level characterization of trastuzumab using sheathless capillary electrophoresis-tandem mass spectrometry

Monoclonal antibodies (mAbs) are highly complex proteins that display a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product - and time - consuming. This work presents the characterization of trastuzumab sequence using sheathless capillary electrophoresis (referred as CESI) – tandem mass spectrometry (CESI-MS/MS). Using this bottom-up proteomic-like approach, CESI-MS/MS provided 100% sequence coverage for both heavy and light chain via peptide fragment fingerprinting (PFF) identification. The result was accomplished in a single shot, corresponding to the analysis of 100 fmoles of digest. The same analysis also enabled precise characterization of the post-translational hot spots of trastuzumab, used as a representative widely marketed therapeutic mAb, including the structural confirmation of the five major N-glycoforms.