Map-based cloning of a fertility restorer gene, Rf-1, in rice (Oryza sativa L.)

A rice nuclear gene, Rf-1, restores the pollen fertility disturbed by the BT-type male sterile cytoplasm, and is widely used for commercial seed production of japonica hybrid varieties. Genomic fragments carrying Rf-1 were identified by conducting chromosome walking and a series of complementation tests. Isolation and analysis of cDNA clones corresponding to the fragments demonstrated that Rf-1 encodes a mitochondrially targeted protein containing 16 repeats of the 35-aa pentatricopeptide repeat (PPR) motif. Sequence analysis revealed that the recessive allele, rf-1, lacks one nucleotide in the putative coding region, presumably resulting in encoding a truncated protein because of a frame shift. Rice Rf-1 is the first restorer gene isolated from cereal crops that has the property of reducing the expression of the cytoplasmic male sterility (CMS)-associated mitochondrial gene like many other restorer genes. The present findings may facilitate not only elucidating the mechanisms of male sterility by the BT cytoplasm and its restoration by Rf-1 but also isolating other restorer genes from cereal crops, especially rice.     

Recent trends on the molecular biology of pneumococcal capsules, lytic enzymes, and bacteriophage

Streptococcus pneumoniae has re-emerged as a major cause of morbidity and mortality throughout the world and its continuous increase in antimicrobial resistance is rapidly becoming a leading cause of concern for public health. This review is focussed on the analysis of recent insights on the study of capsular polysaccharide biosynthesis, and cell wall (murein) hydrolases, two fundamental pneumococcal virulence factors. Besides, we have also re-evaluated the molecular biology of the pneumococcal phage, their possible role in pathogenicity and in the shaping of natural populations of S. pneumoniae. Precise knowledge of the topics reviewed here should facilitate the rationale to move towards the design of alternative ways to combat pneumococcal disease.     

Binding site for Robo receptors revealed by dissection of the leucine-rich repeat region of Slit

Recognition of the large secreted protein Slit by receptors of the Robo family provides fundamental signals in axon guidance and other developmental processes. In Drosophila, Slit-Robo signalling regulates midline crossing and the lateral position of longitudinal axon tracts. We report the functional dissection of Drosophila Slit, using structure analysis, site-directed mutagenesis and in vitro assays. The N-terminal region of Slit consists of a tandem array of four independently folded leucine-rich repeat (LRR) domains, connected by disulphide-tethered linkers. All three Drosophila Robos were found to compete for a single highly conserved site on the concave face of the second LRR domain of Slit. We also found that this domain is sufficient for biological activity in a chemotaxis assay. Other Slit activities may require Slit dimerisation mediated by the fourth LRR domain. Our results show that a small portion of Slit is able to induce Robo signalling and indicate that the distinct functions of Drosophila Robos are encoded in their divergent cytosolic domains.     

Transfer of small chromosome fragments of<Emphasis Type="Italic">Agropyron elongatum</Emphasis> to wheat chromosome via asymmetric somatic hybridization

The chromosome constitution of hybrids and chromatin patterns of Agropyron elongatum (Host)Neviski in F₅ somatic hybrid lines -1–3 and I-1-9 between Triticum aestivum L. and A. elongatum were analyzed. Based on the statistic data of pollen mother cells, F₅ I-1-9 and-1-3 had 20–21 bivalents with a frequency of 84.66% and 85.28%, of which, 89.83% and 89.57% were ring bivalents. The result indicated that both hybrid lines were basically stable in the chromosome constitution and behavior. RAPD analysis showed that the two hybrids contained biparental and integrated DNA. GISH (Genome in situ hybridization) revealed that in the form of small chromosome segments, A. elongatum chromatin was scattered on 4–6 wheat chromosomes near by the region of centromere and telomere in the two hybrid lines. SSR analysis indicated that A. elongatum DNA segments were distributed on the 2A, 5B, 6B and 2D wheat chromosomes in the hybrids, which was in accordance with the GISH results that small-segments intercalated poly-site.     

Structural analysis of DNA-PKcs: modelling of the repeat units and insights into the detailed molecular architecture

DNA-dependent protein kinase (DNA-PK) is part of the eukaryotic DNA double strand break repair pathway and as such is crucial for maintenance of genomic stability, as well as for V(D)J (variable-diversity-joining) recombination. The catalytic subunit of DNA-PK (DNA-PKcs) belongs to the phosphatidylinositol-3 (PI-3) kinase-like kinase (PIKK) superfamily and is comprised of approximately 4100 amino acids. We have used a novel repeat detection method to analyse this enormous protein and have identified two different types of helical repeat motifs in the N-terminal region of the sequence, as well as other previously unreported features in this repeat region. A comparison with the ATMs, ATRs, and TORs show that the features identified are likely to be conserved throughout the PIKK superfamily. Homology modelling of parts of the DNA-PKcs sequence has been undertaken and we have been able to fit the models to previously obtained electron microscopy data. This work provides an insight into the overall architecture of the DNA-PKcs protein and identifies regions of interest for further experimental studies.     

Designing repeat proteins: modular leucine-rich repeat protein libraries based on the mammalian ribonuclease inhibitor family

We present a novel approach to design repeat proteins of the leucine-rich repeat (LRR) family for the generation of libraries of intracellular binding molecules. From an analysis of naturally occurring LRR proteins, we derived the concept to assemble repeat proteins with randomized surface positions from libraries of consensus repeat modules. As a guiding principle, we used the mammalian ribonuclease inhibitor (RI) family, which comprises cytosolic LRR proteins known for their extraordinary affinities to many RNases. By aligning the amino acid sequences of the internal repeats of human, pig, rat, and mouse RI, we derived a first consensus sequence for the characteristic alternating 28 and 29 amino acid residue A-type and B-type repeats. Structural considerations were used to replace all conserved cysteine residues, to define less conserved positions, and to decide where to introduce randomized amino acid residues. The so devised consensus RI repeat library was generated at the DNA level and assembled by stepwise ligation to give libraries of 2-12 repeats. Terminal capping repeats, known to shield the continuous hydrophobic core of the LRR domain from the surrounding solvent, were adapted from human RI. In this way, designed LRR protein libraries of 4-14 LRRs (equivalent to 130-415 amino acid residues) were obtained. The biophysical analysis of randomly chosen library members showed high levels of soluble expression in the Escherichia coli cytosol, monomeric behavior as characterized by gel-filtration, and alpha-helical CD spectra, confirming the success of our design approach.