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Designing and building a minimal genome A goal in biology is to understand the molecular and biological function of every gene in a cell. One way to approach this is to build a minimal genome that includes only the genes essential for life. In 2010, a 1079-kb genome based on the genome of Mycoplasma mycoides (JCV-syn1.0) was chemically synthesized and supported cell growth when transplanted into cytoplasm. Hutchison III et al. used a design, build, and test cycle to reduce this genome to 531 kb (473 genes). The resulting JCV-syn3.0 retains genes involved in key processes such as transcription and translation, but also contains 149 genes of unknown function. Science , this issue p. 10.1126/science.aad6253

Let There Be Life The DNA sequence information from thousands of genomes is stored digitally as ones and zeros in computer memory. Now, Gibson et al. (p. 52 , published online 20 May; see the cover; see the Policy Forum by Cho and Relman ) have brought together technologies from the past 15 years to start from digital information on the genome of Mycoplasma mycoides to chemically synthesize the genomic DNA as segments that could then be assembled in yeast and transplanted into the cytoplasm of another organism. A number of methods were also incorporated to facilitate testing and error correction of the synthetic genome segments. The transplanted genome became established in the recipient cell, replacing the recipient genome, which was lost from the cell. The reconstituted cells were able to replicate and form colonies, providing a proof-of-principle for future developments in synthetic biology.

Rolling circle amplification (RCA) of genomic or plasmid DNA has found wide used in technique academic and biotechnology reseach. In this study, we have successfully found what caused the nonspecific product in the reaction, thus designing an improved RCA which overcomes false priming at sites where primer dimers are formed. The results demonstrated that PEG or PEI could interact with the components of the improved RCA system, and help increase the reaction rate and yield of the improved RCA system, and dramatically reduces the nonspecific RCA products. This low-cost and efficient method dramatically overcomes nonspecific product amplification phenomenon in the RCA reaction which is problematic and has existed for a long time.

The synergistic effects on rheological properties for a series of aqueous solution of κ -carrageenan isolated from Hypnea charoides L amouroux and galactomannan (locust-bean gum) were investigated. At a concentration of 0.4% of total gums, gelation did not occur at room temperature, but it did at a low temperature (0°C). The maximum dynamic modulus was obtained with a series of the samples composed of K-salt of κ -carrageenan and locust-bean gum in the mixing ratio of 1:1 at low temperature (0°C). The less synergistic effect on the dynamic modulus was obtained in mixture solutions with Na-salt of κ -carrageenan and locust-bean gum. At about 25°C, gel-sol transition was observed in the mixing ratio of κ -carrageenan (K-salt) to locust-bean gum of 3:1 and 4:1. A possible association site between K-salt of κ -carrageenan and locust-bean gum was proposed.

G protein-coupled receptors (GPCRs) mediate effects of extracellular signaling molecules in all the body's cells. These receptors are encoded by scarce mRNAs; therefore, detecting their transcripts with conventional microarrays is difficult. We present a method based on multiplex PCR and array detection of amplicons to assay GPCR gene expression with as little as 1 mug of total RNA, and using it, we profiled three human bone marrow stromal cell (BMSC) lines.

The timing of vaccine availability is essential for an effective response to pandemic influenza. In 2009, vaccine became available after the disease peak, and this has motivated the development of next generation vaccine technologies for more rapid responses. The SAM(®) vaccine platform, now in pre-clinical development, is based on a synthetic, self-amplifying mRNA, delivered by a synthetic lipid nanoparticle (LNP). When used to express seasonal influenza hemagglutinin (HA), a SAM vaccine elicited potent immune responses, comparable to those elicited by a licensed influenza subunit vaccine preparation. When the sequences coding for the HA and neuraminidase (NA) genes from the H7N9 influenza outbreak in China were posted on a web-based data sharing system, the combination of rapid and accurate cell-free gene synthesis and SAM vaccine technology allowed the generation of a vaccine candidate in 8 days. Two weeks after the first immunization, mice had measurable hemagglutinin inhibition (HI) and neutralizing antibody titers against the new virus. Two weeks after the second immunization, all mice had HI titers considered protective. If the SAM vaccine platform proves safe, potent, well tolerated and effective in humans, fully synthetic vaccine technologies could provide unparalleled speed of response to stem the initial wave of influenza outbreaks, allowing first availability of a vaccine candidate days after the discovery of a new virus.

The complete amino acid sequence of a hemorrhagic factor, HR2b, from the venom of Okinawa habu was determined. The hemorrhagic factor was fragmented by CNBr cleavage, trypsin, staphylococcal protease V8 and lysyl endopeptidase digestions. The resulting peptides were purified on high performance chromatography, and sequenced by Edman degradation. HR2b was composed of 204 amino acids with pyroglutamyl residue at the amino terminus, and the calculated mol. wt based on the amino acid composition was 23,335. There are three disulfide linkages in the primary structure. The consensus sequence (His-Glu-Xaa-Xaa-His) for zinc-binding site of zinc-requiring metalloproteinases was found in the structure. The primary structure of HR2b shows a significant similarity with that of HR2a of Amami habu venom; 98.5% identity.

Thirteen peptides, homologous to one of the five domains of human alpha 1B-glycoprotein (alpha 1BG), were isolated from a mongoose antihemorrhagic factor (AHF1); four of them were generated by BrCN cleavage, and three and six peptides by the digestions with lysyl endopeptidase and staphylococcal protease V8, respectively. The purified peptides covered 75.9% of the whole sequence of human alpha 1BG (359/474 residues) and showed 46.4% identity (167/360 amino acids) with the sequence of human alpha 1BG including cysteine residues forming disulfide linkages. One of the sugar binding sites of human alpha 1BG was also conserved in AHF1. These results suggest that AHF1 is a protein homologous to human alpha 1BG and a supergene family of immunoglobulins.

Z.-Q. Qi , K. Yonaha , Y. Tomihara and S. Toyama . Characterization of the antihemorrhagic factors of mongoose ( Herpestes edwardsii ). Toxicon 32, 1459–1469, 1994.—Three antihemorrhagic factors (AHF1, AHF2 and AHF3) isolated from the serum of mongoose ( Herpestes edwardsii ) are glycoproteins of monomer structure with the same mol. wt (about 65,000), which contain 4.2%, 13.6% and 6.0% carbohydrates as glucose, respectively. All are composed of about 600 amino acids of similar composition. The 32 amino terminal amino acid sequences of three antihemorrhagic factors were determined, and sequence homologies were examined. AHF1 and AHF2 were of the same amino acid sequence, and showed high homologies to AHF3, oprin (opossum proteinase inhibitor) and human α1B-glycoprotein; 68.7%, 42.3% and 50.0% identity, respectively. AHF1 completely inhibited the hemorrhagic activity of HR2b, the hemorrhagic factor of habu snake, at the concentration of five-fold molar excess, although incomplete inhibition (50%) of proteinase activity of the hemorrhagic factor was observed even at the concentration of 20-fold molar excess of antihemorrhagic factor. Incubation of HR2b with AHF1, and analysis of the reaction products by chromatography on TSK gel G-3000SW and on the ultracentrifuge did not show formation of an inactive enzyme inhibitor complex. However, the complex formation between AHF1 and HR2b was observed by a BIAcore analysis and TSK gel SP-5PW column chromatography. No alteration in the primary or the secondary structure of both factors was demonstrated by SDS-PAGE and circular dichroism spectrum at the far-UV wavelength before and after incubation of both factors, respectively

Protein phosphatase-1, originally studied as phosphorylase phosphatase, is one of the major ser/thr protein phosphatases. It has a long history and a complex enzymology. It consists of a catalytic subunit of 37 kDa, which is bound to a number of different regulatory or targeting subunits. These are believed to restrict its activity to its immediate microenvironment and thus define its specificity, as well as acting to regulate phosphatase activity. The existence of multiple protein phosphatase-1 binding proteins provides the mechanism whereby phosphatase-1 activity can be involved in a diverse range of cellular functions, and reflects a novel strategy for its evolutionary development.