Functional divergence of the duplicated AtKIN14a and AtKIN14b genes: critical roles in Arabidopsis meiosis and gametophyte development

Gene duplication is important for gene family evolution, allowing for functional divergence and innovation. In flowering plants, duplicated genes are widely observed, and functional redundancy of closely related duplicates has been reported, but few cases of functional divergence of close duplicates have been described. Here, we show that the Arabidopsis AtKIN14a and AtKIN14b genes encoding highly similar kinesins are two of the most closely related Arabidopsis paralogs, which were formed by a duplication event that occurred after the split of Arabidopsis and poplar. In addition, AtKIN14a and AtKIN14b exhibit varying degrees of coding sequence divergence. Further genetic studies of plants carrying atkin14a and/or atkin14b mutations indicate that, although these two genes have similar functions, there is clear evidence for functional divergence. Although both genes are important for male and female meiosis, AtKIN14a plays a more critical role in male meiosis than AtKIN14b . Moreover, either one of these two genes is necessary and sufficient for gametophyte development, indicating that they are redundant for this function. Therefore, AtKIN14a and AtKIN14b together play important roles in controlling plant reproductive development. Our results suggest that the AtKIN14a and AtKIN14b genes have retained similar functions in gametophyte development and female meiosis, but have evolved partially distinct functions in male meiosis, with AtKIN14a playing a more substantive role.     

Proinflammatory activity of TWEAK on human dermal fibroblasts and synoviocytes: blocking and enhancing effects of anti-TWEAK monoclonal antibodies

Human tumour necrosis factor (TNF)-like weak inducer of apoptosis (hTWEAK) and two anti-hTWEAK mAbs were tested for their ability to elicit or block inflammatory responses in cultured human dermal fibroblasts and synoviocytes. Incubation with hTWEAK increased the production of prostaglandin E₂, matrix metalloproteinase-1 (MMP-1), IL-6, and the chemokines IL-8, RANTES (regulated on activation, normal T expressed and secreted) and interferon-γ-inducible protein-10 (IP-10) in culture supernatant of fibroblasts and synoviocytes. In combination with TNF or IL-1β, hTWEAK further stimulated the secretion of prostaglandin E₂, MMP-1, IL-6 and IL-8 up to fourfold, and IP-10 and RANTES up to 70-fold compared to TNF or IL-1β alone. An anti-hTWEAK mAb, BCB10, blocked the effects of hTWEAK, whereas hTWEAK crosslinked by the anti-hTWEAK mAb, BEB3, further stimulated the inflammatory response of fibroblasts and synoviocytes. The anti-hTWEAK mAbs were ineffective in blocking or increasing the responses of TNF or IL-1β and blocking anti-TNF mAb was ineffective in preventing the responses to TWEAK. These results were also confirmed at the RNA level for MMP-1, macrophage chemoattractant protein-1, RANTES, macrophage inflammatory protein-1α, IP-10 and IL-8. TWEAK in synergism with IL-1 and TNF may be an additional cytokine that plays a role in destructive chronic arthritic diseases.     

Analysis of DNA sequences using a single chemical cleavage procedure

A novel approach to sequence analysis of end-labeled, defined DNA fragments, using a single chemical cleavage procedure and electrophoretic separation in a single lane, has been developed. Prolonged treatment with hot aqueous piperidine results in partial cleavage of the DNA at all positions; the relative propensity for this cleavage is different for the various bases in the DNA. The hydrolysate is resolved on a DNA sequencing gel, and the distribution of radioactivity in the electrophoretic lane is analyzed (a) in terms of differential peak heights of the radioactive bands and (b) in terms of the spacings between successive bands. Simultaneous application of these two base-characteristic criteria allows the deduction of the nucleotide sequence with an accuracy approaching that of the established four-lane methods of DNA sequencing.     

Sequence analysis of end-labeled DNA fragments by solvolysis in hot aqueous piperidine solutions

One-lane DNA sequencing by solvolysis in hot aqueous piperidine solutions, originally described for 5'-32P-labeled DNA (B. Ambrose and R. Pless (1985) Biochemistry 24, 6194-6200), is extended to 3'-labeled fragments. A salt-free sample for electrophoresis can be obtained by using 1 M LiCl in the solvolysis mixture and removing this salt from the dried hydrolysate by washing with ethanol. Rate and distribution of DNA cleavage in hot aqueous piperidine, containing 0.3 M NaCl, are studied in dependence of temperature, solvent, amine concentration, and reaction time. An increase in temperature strongly accelerates overall DNA degradation, but leaves the distribution of cleavage essentially unchanged. When 50% aqueous ethanol is substituted for water as the reaction solvent, the overall cleavage is slower, and scission at G-sites is enhanced relative to cleavage at the other bases. A rise in the piperidine concentration strongly accelerates the reaction, except at very high amine concentration. Cleavage at A-, G-, and C-sites increases steadily with reaction time, while the T-cleavage observed takes place primarily at the very beginning of the solvolysis.     

Further evidence for the protective role of sub-parietal cell membranous secretory product on the cuticle of a pentastomid arthropod parasite developing in its rodent intermediate host

The pentastomid parasite Porocephalus crotali, develops to an infective stage within a granulomatous lesion in the tissues of rodent intermediate hosts. A conspicuous layer of sub-parietal cell (SPC) secretory product, which coats the intermoult cuticle up to a depth of 12 microns, is described. Around the first five nymphal instars this material consists of an amorphous matrix with distinctive electron-lucid lacunae, but that around later instars (six and seven), while retaining much of the original morphology, possesses a significant membranous component. Host effector cells, most notably eosinophils and macrophage/epithelioid cells, are frequently completely enveloped by SPC secretion but invariably appear unreactive to it. Host cells may penetrate to the outermost layer of the epicuticle but again but again cytotoxic activity is absent. During ecdysis, effector cells are recruited to the intercuticular space where widespread degranulation is evident. Some of this is specifically directed against the underside of the cast cuticle, but not against the newly exposed cuticle. Protracted degranulation eventually reduces the cast cuticle to fragments which are endocytosed by giant cells. 1 cm long infective (seventh-stage) nymphs, which retain the sixth stage cuticle as a protective sheath, are largely devoid of membranous secretion and these were dissected from cysts, washed, and surgically transplanted into the body cavities of naive and infected mice. Pronounced differences in the onset and intensity of the subsequent inflammatory response in the two categories of host indicate some form of specific recognition. In both groups of mice though, the cuticle is an eventual target for attack by effector cells, and parasites are killed. The protective function of SPC secretion is discussed.     

Kinase-deficient Pak1 mutants inhibit Ras transformation of Rat-1 fibroblasts.

Among the mechanisms by which the Ras oncogene induces cellular transformation, Ras activates the mitogen-activated protein kinase (MAPK or ERK) cascade and a related cascade leading to activation of Jun kinase (JNK or SAPK). JNK is additionally regulated by the Ras-related G proteins Rac and Cdc42. Ras also regulates the actin cytoskeleton through an incompletely elucidated Rac-dependent mechanism. A candidate for the physiological effector for both JNK and actin regulation by Rac and Cdc42 is the serine/threonine kinase Pak (p65pak). We show here that expression of a catalytically inactive mutant Pak, Pak1(R299), inhibits Ras transformation of Rat-1 fibroblasts but not of NIH 3T3 cells. Typically, 90 to 95% fewer transformed colonies were observed in cotransfection assays with Rat-1 cells. Pak1(R299) did not inhibit transformation by the Raf oncogene, indicating that inhibition was specific for Ras. Furthermore, Rat-1 cell lines expressing Pak1(R299) were highly resistant to Ras transformation, while cells expressing wild-type Pak1 were efficiently transformed by Ras. Pak1(L83,L86,R299), a mutant that fails to bind either Rac or Cdc42, also inhibited Ras transformation. Rac and Ras activation of JNK was inhibited by Pak1(R299) but not by Pak1(L83,L86,R299). Ras activation of ERK was inhibited by both Pak1(R299) and Pak1(L83,L86,R299), while neither mutant inhibited Raf activation of ERK. These results suggest that Pak1 interacts with components essential for Ras transformation and that inhibition can be uncoupled from JNK but not ERK signaling.     

Identification of a ChromosomalShigella flexneriMulti-Antibiotic Resistance Locus Which Shares Sequence and Organizational Similarity with the Resistance Region of the Plasmid NR1

The ampicillin resistance gene fromShigella flexneri2a strain YSH6000 was cloned and shown by Southern hybridization analysis to be closely linked to the previously cloned streptomycin, chloramphenicol, and tetracycline resistance determinants, which are borne on a chromosomally integrated 99-kb element. Analysis of this chromosomal multi-antibiotic resistance locus revealed that it had a high level of sequence and organizational similarity to an equivalent region of theShigellaR-plasmid, NR1. However, the chromosomal locus exhibited several differences, including the presence of two stretches of sequence derived from IS elements, the precise insertion of a β-lactamase encodingoxa1cassette into the Tn21-borne integron In2, a possible 17.5-kb deletion, and the loss or inactivation of the mercury resistance determinant. Based on these data, it is proposed that the chromosomal locus arose following integration of an NR1-like plasmid.     

Terminal Regions Confer Plasticity to the Tetrameric Assembly of Human HspB2 and HspB3

Heterogeneity in small heat shock proteins (sHsps) spans multiple spatiotemporal regimes—from fast fluctuations of part of the protein, to conformational variability of tertiary structure, plasticity of the interfaces, and polydispersity of the inter-converting, and co-assembling oligomers. This heterogeneity and dynamic nature of sHsps has significantly hindered their structural characterization. Atomic coordinates are particularly lacking for vertebrate sHsps, where most available structures are of extensively truncated homomers. sHsps play important roles in maintaining protein levels in the cell and therefore in organismal health and disease. HspB2 and HspB3 are vertebrate sHsps that are found co-assembled in neuromuscular cells, and variants thereof are associated with disease. Here, we present the structure of human HspB2/B3, which crystallized as a hetero-tetramer in a 3:1 ratio. In the HspB2/B3 tetramer, the four α-crystallin domains (ACDs) assemble into a flattened tetrahedron which is pierced by two non-intersecting approximate dyads. Assembly is mediated by flexible “nuts and bolts” involving IXI/V motifs from terminal regions filling ACD pockets. Parts of the N-terminal region bind in an unfolded conformation into the anti-parallel shared ACD dimer grooves. Tracts of the terminal regions are not resolved, most likely due to their disorder in the crystal lattice. This first structure of a full-length human sHsp heteromer reveals the heterogeneous interactions of the terminal regions and suggests a plasticity that is important for the cytoprotective functions of sHsps.