The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly.

Techniques for assembly of designed DNA sequences are important for synthetic biology. So far, a few methods have been developed towards high-throughput seamless DNA assembly in vitro, including both the homologous sequences-based system and the type IIS-mediated system. Here, we describe a novel method designated 'MASTER Ligation', by which multiple DNA sequences can be seamlessly assembled through a simple and sequence-independent hierarchical procedure. The key restriction endonuclease used, MspJI, shares both type IIM and type IIS properties; thus, it only recognizes the methylation-specific 4-bp sites, (m)CNNR (R = A or G), and cuts DNA outside of the recognition sequences. This method was tested via successful assembly of either multiple polymerase chain reaction amplicons or restriction fragments of the actinorhodin biosynthetic cluster of Streptomyces coelicolor (∼29 kb), which was further heterologously expressed in a fast-growing and moderately thermophilic strain, Streptomyces sp. 4F.

[Study on the processing technique of enzyme hydrolysis Eupolyphaga sinensis peptide and innunoregulatory effect].

OBJECTIVE: Using papain to prepare polypeptide from Eupolyphaga sinensis, then study the immune function of polypeptide from Eupolyphaga sinensis in vivo. METHODS: Used hydrolysis degree as index, pH value, enzyme dosage, thermometer reaction time were optimized. Studied the influence of polyeptide on the mice immune functions through mice immune organs index, phagocytic function and the level of IL-2. RESULTS: The optimum enzymolysis condition was as follows: pH 8.0, enzyme 1%, temperature 55 degrees C, reaction time 4. 5 h. In vivo test of mice demonstraed that, Eupolyphaga sinensis could elevate index of thymus and spleen, enhance the phagocytic function of macrophage and promote the level of IL-2 in serum. CONCLUSION: Eupolyphaga sinensis has immunoregulatory effect.

Cloning and characterization of an rRNA methyltransferase from Sorangium cellulosum.

A locus (kmr) responsible for aminoglycosides-resistance of Sorangium cellulosum was cloned and characterized in Myxococcus xanthus. The gene kmr encodes a putative rRNA methyltransferase. Expression of the complete ORF endowed the Myxococcus transformants with the resistance to aminoglycosidic antibiotics of kanamycin, apramycin, gentamycin, neomycin, and tobramycin at an extraordinary high-level (MIC, higher than 500 microg/ml). However, the gene did not function in Escherichia coli cells. In Sorangium genome, the gene kmr was followed by a putative integrase gene, and was highly homologous in different Sorangium strains. The Sorangium rRNA methyltransferase sequence was in low similarity to the reported 16S rRNA methyltransferases, and their resistance spectrums were also different. The results indicate that the rRNA methyltransferase (Kmr) in Sorangium strains is a new member of the rRNA methyltransferases family.

Discovery of the autonomously replicating plasmid pMF1 from Myxococcus fulvus and development of a gene cloning system in Myxococcus xanthus.

Myxobacteria are very important due to their unique characteristics, such as multicellular social behavior and the production of diverse and novel bioactive secondary metabolites. However, the lack of autonomously replicating plasmids has hindered genetic manipulation of myxobacteria for decades. To determine whether indigenous plasmids are present, we screened about 150 myxobacterial strains, and a circular plasmid designated pMF1 was isolated from Myxococcus fulvus 124B02. Sequence analysis showed that this plasmid was 18,634 bp long and had a G+C content of 68.7%. Twenty-three open reading frames were found in the plasmid, and 14 of them were not homologous to any known sequence. Plasmids containing the gene designated pMF1.14, which encodes a large unknown protein, were shown to transform Myxococcus xanthus DZ1 and DK1622 at high frequencies ( approximately 10(5) CFU/microg DNA), suggesting that the locus is responsible for the autonomous replication of pMF1. Shuttle vectors were constructed for both M. xanthus and Escherichia coli. The pilA gene, which is essential for pilus formation and social motility in M. xanthus, was cloned into the shuttle vectors and introduced into the pilA-deficient mutant DK10410. The transformants subsequently exhibited the ability to form pili and social motility. Autonomously replicating plasmid pMF1 provides a new tool for genetic manipulation in Myxococcus.

[Cloning and functional studies of the chromosomal replication origin of Actinomadura yumaensis].

The chromosomal replication origins (oriC) have been investigated in Gram-positive eubacteria actinomycetes, including Streptomyces, Mycobacterium and Amycolatopsis, and reveal various DnaA-boxes and AT-rich sequences between the conserved dnaA and dnaN genes. Actinomadura yumaensis NRRL12515 is a producer of anthelmintic polyether maduramicin. In this paper,cloning, sequencing and functional studies of its oriC have been carried out. A pair of oligonucleotide primers, based on the conserved sequences of dnaA and dnaN, was used to PCR amplification. A-1.3kb DNA band was detected on agarose gel. Subsequently cloning in an E. coli plasmid pBluescript II SK ( + ) and sequencing showed 1265bp,which contained 919bp between dnaA and dnaN genes. 14 DnaA-boxes with conserved 9bp sequence (T/C) (T/C) GTCC (A/C) CA and two 13bp AT-rich regions (GAAAAATCCCAAG, AAGAAAAAACTCA), were found on the sequence,indicating the oriC of A. yumaensis NRRL12515. Phylogenetic trees based on the sequences of oriC and of 16S rRNA genes of the four actinomycetes species show a similar pattern, suggesting that oriC sequences also reflected well the relationship between actinomycetes species. An E. coli plasmid pOR1, containing the oriC, actinomycetes selection markers tsr and melC, was introduced into Streptomyces coelicolor M145 by conjugal transfer. Transformants were obtained,and plasmids DNA were isolated and detected as low copy number, suggesting a functional mini-chromosome in Streptomyces.

Characterization of Nocardia plasmid pXT107.

Nocardia, Rhodococcus and Streptomyces, all members of the actinomycetes family, are Gram-positive eubacteria with high G+C content and able to form mycelium. We report here a newly identified plasmid pXT107 of Nocardia sp. 107, one of the smallest circular plasmids found in Nocardia. The complete nucleotide sequence of pXT107 consisted of 4335 bp with 65% G+C content, and encoded one replication extragenic palindromic (Rep) and six hypothetical proteins. The Rep, double-strand origin and single-strand origin of pXT107 resembled those of typical rolling-circle-replication plasmids, such as pNI100 of Nocardia, pRE8424 of Rhodococcus and pIJ101 of Streptomyces. The Escherichia coli-Nocardia shuttle plasmid pHAQ22, containing the rep gene of pXT107, is able to propagate in Nocardia but not in Streptomyces.

[Sequence and functional analysis of the chromosome replication origin (oriC) of Streptoverticillum caespitosus ATCC27422].

Twenty-two DnaA boxes were identified in the chromosome replication origin (oriC) of Streptoverticillum caespitosus ATCC27422 based upon the characteristics of consensus sequences. The 21st and 22nd DnaA boxes overlapped 8 base pairs each other reversely. Compared with the oriC database of actinomycetes, similar overlapping DnaA boxes were recognized in several species of Streptomyces and Mycobacterium. These overlapping DnaA boxes were composed of the last two DnaA box (21st and 22nd) in the Streptomyces species, but the of 1st and 2nd ones in the Mycobacterium species. The consensus sequence of the overlapping DnaA box is CTGTGCACAA, one base longer than the normal DnaA box sequence presumably due to the overlapping structure. Although the DnaA boxes exist in the 189 792 bp region only, the 1 188 bp and 793 939 bp regions are also important to the DNA replication. Deletion of the 1 188 bp region may cause absolute loss of DNA replication initiation activity measured by the transformation efficiency of plasmids with truncated oriC. When the 793 939 bp region was truncated, the transformation efficiency reduced about 40%. If the oriC was cloned into a vector with partial flanking region sequences (partial dnaA and dnaN gene sequences), the transformation rate was about 4.3-fold lower than that of the construct containing the oriC region only. However, the transformants were much more similar to the host with respect to the morphology of colony and mycelium. The cis-regulatory functions of the flanking sequences, which may influence the initiation efficiency of the chromosome replication and/or the stability of replicon, are thus suggested.

[Isolation and characterization of functional replication origin from Streptomyces avermitilis].

Most eubacteria contain highly conservative gene clusters in the adjacent regions of oriC. According to this principle, a 1.4 kb DNA fragment containing parts of dnaA and dnaN genes of Streptomyces avermitilis was cloned by degenerate PCR. Sequence analysis of this fragment indicated that it encoded two partial genes in the order dnaA (the putative initiator protein) and dnaN (the beta subunit of DNA polymerase III). The intergenic non-coding region between dnaA and dnaN was found to contain 19 putative DnaA boxes, i.e. 9 nt long DnaA protein recognition sequences. It was confirmed that the location, orientation and spacing of DnaA boxes in this intergenic region are conserved among Streptomyces. The consensus sequence of DnaA box identified is (T/C)(T/C)(G/A/C)TCCACA (preferred bases in italic). When this fragment was cloned into Escherichia coli plasmid pQC156, which is otherwise non-replicative in Streptomyces, it exhibited autonomous replication activity in Streptomyces lividans, a closely related Streptomyces strain. Different parts of the oriC contribute unequally to the stability and transformation efficiency. The 3' region of oriC may contain features that support stable autonomous replication. The implications of these results for the understanding of the S. avermitilis oriC replication initiation process and its future application are discussed.

[Cloning of the replication origin (oriC) of Streptoverticillum caespitosus and transformation of Streptomyces lividans ZX7].

Streptoverticillum caespitosus ATCC27422 is a producing strain of mitomycin A for cancer therapy. Taking the advantage of the conserved sequences of genes flanking the oriC of high G + C Gram-positive bacteria, a 1.3 kb DNA fragment containing oriC and its flanking region was cloned by PCR. Nuleotide sequence comparisons revealed that the cloned fragment is more than 80% identical to the same region of S. coelicolor. There are 22 DnaA-boxes in the oriC region, and the conserved sequence of DnaA-box is TTGTCCACA. The plasmid containing the oriC of S. caespitosus was constructed (pMJ9), and it was able to transform the protoplast of Streptomyces lividans ZX7 at the frequency of 3.2 x 10(2) transformants/micrograms plasmid DNA. The colony and mycelia's morphology of the transformants are normal. The constructed plasmid can exist stable in the host as a low copy extra-chromosome replicon. The high rate of the homology and the cross genus replication initiation activity suggests close relationship between Streptomyces and Streptoverticillum in the evolution. While the maximum likelihood phylogenetic tree based upon the oriC of S. caespitosus and several Streptomyces spp. revealed that S. caespitosus differed extensively from the Streptomyces spp. This result supports the separation of Streptoverticillum from Streptomyces.