Specific association of iduronic acid-rich dermatan sulphate with the extracellular matrix of human skin fibroblasts cultured on collagen gels.

Human skin fibroblasts cultured on collagen gels produced two dermatan sulphate species, one, enriched in iduronic acid residues, that bound specifically to the collagenous fibres of the gel, the other, enriched in glucuronic acid, that accumulated in the culture medium. Collagen-binding and collagen-non-binding dermatan sulphates were also produced by cells grown on plastic surfaces, but in these cultures each constituent was released into the growth medium. Net synthesis of dermatan sulphate was 3-fold higher in cells maintained on collagen gels. In contrast, heparan sulphate synthesis was not influenced by the nature of the culture surface. The concentration of heparan sulphate in surface-membrane extracts was similar for cells grown on plastic and on collagen gels, but cells cultured on collagen showed a notable increase in the content of surface-membrane dermatan sulphate. The patterns of synthesis and distribution of sulphated glycosaminoglycans observed in skin fibroblasts maintained on collagen gels may reflect differentiated cellular functions.     

In vitro reconstitution of Cascade‐mediated CRISPR immunity in Streptococcus thermophilus

Clustered regularly interspaced short palindromic repeats (CRISPR)‐encoded immunity in Type I systems relies on the Cascade (CRISPR‐associated complex for antiviral defence) ribonucleoprotein complex, which triggers foreign DNA degradation by an accessory Cas3 protein. To establish the mechanism for adaptive immunity provided by the Streptococcus thermophilus CRISPR4‐Cas (CRISPR‐associated) system (St‐CRISPR4‐Cas), we isolated an effector complex (St‐Cascade) containing 61‐nucleotide CRISPR RNA (crRNA). We show that St‐Cascade, guided by crRNA, binds in vitro to a matching proto‐spacer if a proto‐spacer adjacent motif (PAM) is present. Surprisingly, the PAM sequence determined from binding analysis is promiscuous and limited to a single nucleotide (A or T) immediately upstream (?1 position) of the proto‐spacer. In the presence of a correct PAM, St‐Cascade binding to the target DNA generates an R‐loop that serves as a landing site for the Cas3 ATPase/nuclease. We show that Cas3 binding to the displaced strand in the R‐loop triggers DNA cleavage, and if ATP is present, Cas3 further degrades DNA in a unidirectional manner. These findings establish a molecular basis for CRISPR immunity in St‐CRISPR4‐Cas and other Type I systems.     

Transposable Element ISHp608 of Helicobacter pylori: Nonrandom Geographic Distribution, Functional Organization, and Insertion Specificity

A new member of the IS605 transposable element family, designated ISHp608, was found by subtractive hybridization in Helicobacter pylori. Like the three other insertion sequences (ISs) known in this gastric pathogen, it contains two open reading frames (orfA and orfB), each related to putative transposase genes of simpler (one-gene) elements in other prokaryotes; orfB is also related to the Salmonella virulence gene gipA. PCR and hybridization tests showed that ISHp608 is nonrandomly distributed geographically: it was found in 21% of 194 European and African strains, 14% of 175 Bengali strains, 43% of 131 strains from native Peruvians and Alaska natives, but just 1% of 223 East Asian strains. ISHp608 also seemed more abundant in Peruvian gastric cancer strains than gastritis strains (9 of 14 versus 15 of 45, respectively; P = 0.04). Two ISHp608 types differing by approximately 11% in DNA sequence were identified: one was widely distributed geographically, and the other was found only in Peruvian and Alaskan strains. Isolates of a given type differed by < or = 2% in DNA sequence, but several recombinant elements were also found. ISHp608 marked with a resistance gene was found to (i) transpose in Escherichia coli; (ii) generate simple insertions during transposition, not cointegrates; (iii) insert downstream of the motif 5"-TTAC without duplicating target sequences; and (iv) require orfA but not orfB for its transposition. ISHp608 represents a widespread family of novel chimeric mobile DNA elements whose further analysis should provide new insights into transposition mechanisms and into microbial population genetic structure and genome evolution.     

Domain-specific modification of heparan sulfate by Qsulf1 modulates the binding of the bone morphogenetic protein antagonist Noggin

We have reported previously that Noggin is a heparin-binding protein and associates with the cell surface through heparan sulfate proteoglycans, where it remains functional for the binding of bone morphogenetic proteins (BMPs). Here we report that the binding of Noggin to the cell surface is highly selective for heparan sulfate and that specific structural features are required for the interaction. Noggin binds most efficiently to heparin sequences composed of 10 or more monosaccharides; N-, 6-O-, and 2-O-sulfates contribute to this interaction. In addition, we have shown that the developmentally regulated endosulfatase Qsulf1 selectively removes sulfate groups from the 6-O position of sugars within the most highly sulfated S domains of heparan sulfate, whereas 6-O-sulfates in the NA/NS domains are not substrates for the enzyme. The activity of Qsulf1 in cells in culture results in the release of Noggin from the cell surface and a restoration of BMP responsiveness to the cells. This shows that Noggin binds to the S domains of heparan sulfate and provides evidence that, in addition to modulating Wnt signaling in vivo by the release of heparan sulfate bound Wnt, Qsulf1 also modulates BMP signaling by the release of surface-bound Noggin.     

Evolutionary dynamics of insertion sequences in Helicobacter pylori

Prokaryotic insertion sequence (IS) elements behave like parasites in terms of their ability to invade and proliferate in microbial gene pools and like symbionts when they coevolve with their bacterial hosts. Here we investigated the evolutionary history of IS605 and IS607 of Helicobacter pylori, a genetically diverse gastric pathogen. These elements contain unrelated transposase genes (orfA) and also a homolog of the Salmonella virulence gene gipA (orfB). A total of 488 East Asian, Indian, Peruvian, and Spanish isolates were screened, and 18 and 14% of them harbored IS605 and IS607, respectively. IS605 nucleotide sequence analysis (n = 42) revealed geographic subdivisions similar to those of H. pylori; the geographic subdivision was blurred, however, due in part to homologous recombination, as indicated by split decomposition and homoplasy tests (homoplasy ratio, 0.56). In contrast, the IS607 populations (n = 44) showed strong geographic subdivisions with less homologous recombination (homoplasy ratio, 0.2). Diversifying selection (ratio of nonsynonymous change to synonymous change, >1) was evident in approximately 15% of the IS605 orfA codons analyzed but not in the IS607 orfA codons. Diversifying selection was also evident in approximately 2% of the IS605 orfB and approximately 10% of the IS607 orfB codons analyzed. We suggest that the evolution of these elements reflects selection for optimal transposition activity in the case of IS605 orfA and for interactions between the OrfB proteins and other cellular constituents that potentially contribute to bacterial fitness. Taken together, similarities in IS elements and H. pylori population genetic structures and evidence of adaptive evolution in IS elements suggest that there is coevolution between these elements and their bacterial hosts.     

Quantitative and qualitative assessment of serum- and inflammatory mediator-induced migration of carp (Cyprinus carpio) head kidney neutrophils in vitro

A quantitative transmigration system, permitting the harvest of transmigrated cells for further analysis, was used to study carp head kidney (HK) granulocyte migration in vitro. Pooled carp serum and leukotriene B4 (LTB-4), but not recombinant human C-X-C chemokine ligand 8 (rhCXCL8), recombinant human complement component 5a (rhC5a) or N-formyl-methionyl-leucyl-phenylalanine (FMLP) induced strong migration (up to 70%) of carp HK granulocytes. The transmigrated cells were viable (>or=96%) and uniform (purity >or=97%). After serum- as well as LTB-4-induced transmigration granulocytes produced the same amounts of reactive oxygen species (ROS) as non-migrated cells in HK cell suspension. Their morphology, staining characteristics and flow cytometric scatter characteristics, plus their ability to produce ROS characterised the transmigrated granulocytes as neutrophils. The quantitative transmigration system described here could also serve as an excellent tool for the selective attraction and isolation of highly purified carp neutrophils from HK cell suspensions.     

Tetrameric restriction enzymes: expansion to the GIY-YIG nuclease family

The GIY-YIG nuclease domain was originally identified in homing endonucleases and enzymes involved in DNA repair and recombination. Many of the GIY-YIG family enzymes are functional as monomers. We show here that the Cfr42I restriction endonuclease which belongs to the GIY-YIG family and recognizes the symmetric sequence 5′-CCGC/GG-3′ (‘/’ indicates the cleavage site) is a tetramer in solution. Moreover, biochemical and kinetic studies provided here demonstrate that the Cfr42I tetramer is catalytically active only upon simultaneous binding of two copies of its recognition sequence. In that respect Cfr42I resembles the homotetrameric Type IIF restriction enzymes that belong to the distinct PD-(E/D)XK nuclease superfamily. Unlike the PD-(E/D)XK enzymes, the GIY-YIG nuclease Cfr42I accommodates an extremely wide selection of metal-ion cofactors, including Mg²⁺, Mn²⁺, Co²⁺, Zn²⁺, Ni²⁺, Cu²⁺ and Ca²⁺. To our knowledge, Cfr42I is the first tetrameric GIY-YIG family enzyme. Similar structural arrangement and phenotypes displayed by restriction enzymes of the PD-(E/D)XK and GIY-YIG nuclease families point to the functional significance of tetramerization.     

Design of a CRISPR-Cas system to increase resistance of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> to bacteriophage SPP1

Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated (cas) genes form an adaptive prokaryotic immune system which provides acquired resistance against viruses and plasmids. Bacillus subtilis presently is the best-characterized laboratory model for Gram-positive bacteria and also widely used for industrial production of enzymes, vitamins and antibiotics. In this study, we show that type II-A CRISPR-Cas system from Streptococcus thermophilus can be transferred into B. subtilis and provides heterologous protection against phage infection. We engineered a heterologous host by cloning S. thermophilus Cas9 and a spacer targeting bacteriophage SPP1 into the chromosome of B. subtilis, which does not harbor its own CRISPR-Cas systems. We found that the heterologous CRISPR-Cas system is functionally active in B. subtilis and provides resistance against bacteriophage SPP1 infection. The high efficiency of the acquired immunity against phage could be useful in generation of biotechnologically important B. subtilis strains with engineered chromosomes.