Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences

Large DNA constructs of arbitrary sequences can currently be assembled with relative ease by joining short synthetic oligodeoxynucleotides (oligonucleotides). The ability to mass produce these synthetic genes readily will have a significant impact on research in biology and medicine. Presently, high-throughput gene synthesis is unlikely, due to the limits of oligonucleotide synthesis. We describe a microfluidic PicoArray method for the simultaneous synthesis and purification of oligonucleotides that are designed for multiplex gene synthesis. Given the demand for highly pure oligonucleotides in gene synthesis processes, we used a model to improve key reaction steps in DNA synthesis. The oligonucleotides obtained were successfully used in ligation under thermal cycling conditions to generate DNA constructs of several hundreds of base pairs. Protein expression using the gene thus synthesized was demonstrated. We used a DNA assembly strategy, i.e. ligation followed by fusion PCR, and achieved effective assembling of up to 10 kb DNA constructs. These results illustrate the potential of microfluidics-based ultra-fast oligonucleotide parallel synthesis as an enabling tool for modern synthetic biology applications, such as the construction of genome-scale molecular clones and cell-free large scale protein expression.     

Single-strand conformation polymorphisms (SSCPs) detected in five bovine genes

Summary
Polymerase chain reaction (PCR) primers designed to amplify bovine specific sequences of the arginine-vasopressin ( ARVP ), glycoprotein hormone alpha ( CGA ), cytochrome oxidase c subunit IV pseudogene ( COXP ), prochymosin ( CYM ), coagulation factor X ( F10 ), inhibin βA ( INHBA ), low density lipoprotein receptor ( LDLR ) and oxytocin ( OXT ) genes in hybrid cells were used in a search for single strand conformation polymorphisms. DNA from 75 animals comprising crossbred and 7 purebred breeds were analysed. ARVP, COXP, CYM, LDLR and OXT were found to be polymorphic while CGA, F10 and INHBA were not. Polymorphic regions were identified within 206 bp of exon 1 of ARVP , 582 bp of the pseudogene COXP , 253 bp of exon 9 of CYM , 519 bp of LDLR cDNA and 160 bp of the upstream regulatory region of OXT. This is the first report of bovine polymorphisms for these genes and an important step in our goal to incorporate type I comparative anchor loci into the bovine linkage map. Polymorhic loci were subsequently analysed in pedigreed full-sib families and shown to be inherited in a Men-delian fashion.     

牛凝乳酶原基因在乳酸乳球菌中的表达

【目的】利用乳酸乳球菌nisin诱导基因表达系统(the NIsin Controlled gene Expression system,NICE)表达牛凝乳酶原。【方法】从克隆载体pS19-PPC中获得牛凝乳酶原基因,将该基因与表达载体pNZ8148连接并电转化乳酸乳球菌NZ9000,转化子经酶切、PCR和测序鉴定后,用nisin进行诱导表达,表达产物利用SDS-PAGE和Western blot鉴定,表达产物纯化后检测凝乳活性。【结果】重组牛凝乳酶原与天然牛凝乳酶原比较,其分子量大小、免疫性质、生物活性和抑制剂敏感性没有发现显著差异,其凝乳活性可达2×103IMCU/mL。【结论】在乳酸乳球菌中表达了具有凝乳活性的牛凝乳酶原,同时乳酸乳球菌作为发酵剂和凝乳酶产生菌双重角色的实现,为奶酪加工提供了新思路和新方法。     

Patch oligodeoxynucleotide synthesis (POS): a novel method for synthesis of long DNA sequences and full-length genes

Synthesis of long DNA fragments is often associated with mutations and requires multiple DNA manipulation steps. A novel DNA synthesis method, referred to as patch oligodeoxynucleotide synthesis (POS) to assembly long DNA fragments is presented here. This method involves connection of two types of oligodeoxynucleotides: long constructional oligonucleotides (COs) and short patch oligonucleotides (POs). Long COs were connected by a ligase with the aid of POs, which were complementary to both adjacent COs to help remove secondary structures during assembly. The partial double-stranded DNA template that was formed was then amplified by PCR. Accordingly, we synthesized SV40 polyadenylation signal sequences (187 bp), a codon-optimized yellow fluorescent protein gene (678 bp), and Rattus norvegicus catenin β1 (2,352 bp). This presented method can be broadly applied to synthesize DNA fragments of varying lengths with great convenience.     

Structure of the human genomic region homologous to the bovine prochymosin-encoding gene

Two human genomic libraries were probed with bovine prochymosin (bPC) cDNA. Recombinant clones covering a genomic region homologous to the entire coding region and flanking sequences of the bPC gene were isolated. Human sequences homologous to exons of the bPC gene are distributed in a DNA fragment of 10 kb. Alignment of the human sequences and the exons of bPC reveals that the human 'exons' 1-3, 5 and 7-9 have sizes identical to the corresponding bovine exons, but a nucleotide (nt) has been deleted in the human exon 4 and two nt in the human exon 6. The aligned human sequence and the coding part of bPC gene share 82% nt homology, the value ranging, in separate exons, from 76 (exon 1) to 84% (exons 5 and 6). 150 bp of 5'-flanking sequence of the human gene has 75% homology to the corresponding region of bPC gene and contains a TATA-box in a similar position. A 1-nt deletion in the human exon 4 would shift the translational reading frame of a putative human PC mRNA relative to bPC mRNA, and result in an in-phase terminator spanning codons 163 and 164 in bPC mRNA. Another terminator in-phase with the amino-acid sequence encoded by the bPC gene occurs in the human exon 5 and the second frameshift mutation in exon 6. Thus, the nt sequence analysis of the human genomic region has revealed the presence of mutations that have rendered it unable to produce a full-length protein homologous to bPC and, therefore, we refer to this gene as a human prochymosin pseudogene (hPC psi). Blot-hybridization analysis of human genomic DNA indicates that hPC psi is a single gene in the human genome.     

Complete secretion of activable bovine prochymosin by genetically engineered L forms of Proteus mirabilis.

To circumvent problems encountered in the synthesis of active chymosin in a number of bacteria and fungi, a recombinant DNA L-form expression system that directed the complete secretion of fully activable prochymosin into the extracellular culture medium was developed. The expression plasmid constructions involved the in-frame fusion of prochymosin cDNA minus codons 1 to 4 to streptococcal pyrogenic exotoxin type A gene (speA') sequences, including the speA promoter, ribosomal binding site, and signal sequence and five codons of mature SpeA. Secretion of fusion prochymosin enzymatically and immunologically indistinguishable from bovine prochymosin was achieved after transformation of two stable protoplast type L-form strains derived from Proteus mirabilis. The secreted proenzyme was converted by autocatalytic processing to chymosin showing milk-clotting activity. In controlled laboratory fermentation processes, a maximum specific rate of activable prochymosin synthesis of 0.57 x 10(-3)/h was determined from the time courses of biomass dry weight and product formation. Yields as high as 40 +/- 10 micrograms/ml were obtained in the cell-free culture fluid of strain L99 carrying a naturally altered expression plasmid of increased segregational stability. The expression-secretion system described may be generally useful for production of recombinant mammalian proteins synthesized intracellularly as aberrantly folded insoluble aggregates.     

Complete secretion of activable bovine prochymosin by genetically engineered L forms of Proteus mirabilis

To circumvent problems encountered in the synthesis of active chymosin in a number of bacteria and fungi, a recombinant DNA L-form expression system that directed the complete secretion of fully activable prochymosin into the extracellular culture medium was developed. The expression plasmid constructions involved the in-frame fusion of prochymosin cDNA minus codons 1 to 4 to streptococcal pyrogenic exotoxin type A gene (speA') sequences, including the speA promoter, ribosomal binding site, and signal sequence and five codons of mature SpeA. Secretion of fusion prochymosin enzymatically and immunologically indistinguishable from bovine prochymosin was achieved after transformation of two stable protoplast type L-form strains derived from Proteus mirabilis. The secreted proenzyme was converted by autocatalytic processing to chymosin showing milk-clotting activity. In controlled laboratory fermentation processes, a maximum specific rate of activable prochymosin synthesis of 0.57 x 10(-3)/h was determined from the time courses of biomass dry weight and product formation. Yields as high as 40 +/- 10 micrograms/ml were obtained in the cell-free culture fluid of strain L99 carrying a naturally altered expression plasmid of increased segregational stability. The expression-secretion system described may be generally useful for production of recombinant mammalian proteins synthesized intracellularly as aberrantly folded insoluble aggregates.     

牛凝乳酶原基因克隆及序列的进化分析

利用RT-PCR克隆获得牛凝乳酶原基因的cDNA序列, 测序后与GenBank中凝乳酶原基因进行序列比对和生物信息学分析。序列比对统计分析显示, 该基因为牛凝乳酶原B基因, 与已知牛和其他哺乳动物的凝乳酶原基因具有很高的同源性,18种哺乳动物凝乳酶原基因密码子一、二、三位点的碱基偏倚度分别为：6.227、1.042和1.456。这表明该基因具有整体保守性和突变位点偏倚性两个特征, 可以作为哺乳动物系统进化的研究对象。采用多种方法构建的该基因系统进化树一致表明, 偶蹄动物与灵长动物的亲缘关系比偶蹄动物与啮齿动物的亲缘关系更近, 比偶蹄动物与食肉动物的亲缘关系更远, 并且18种哺乳动物的亲缘关系与动物种系进化关系一致, 为哺乳动物系统进化关系研究提供了分子水平的佐证和依据。     

A simple and accurate two-step long DNA sequences synthesis strategy to improve heterologous gene expression in pichia

In vitro gene chemical synthesis is a powerful tool to improve the expression of gene in heterologous system. In this study, a two-step gene synthesis strategy that combines an assembly PCR and an overlap extension PCR (AOE) was developed. In this strategy, the chemically synthesized oligonucleotides were assembled into several 200-500 bp fragments with 20-25 bp overlap at each end by assembly PCR, and then an overlap extension PCR was conducted to assemble all these fragments into a full length DNA sequence. Using this method, we de novo designed and optimized the codon of Rhizopus oryzae lipase gene ROL (810 bp) and Aspergillus niger phytase gene phyA (1404 bp). Compared with the original ROL gene and phyA gene, the codon-optimized genes expressed at a significantly higher level in yeasts after methanol induction. We believe this AOE method to be of special interest as it is simple, accurate and has no limitation with respect to the size of the gene to be synthesized. Combined with de novo design, this method allows the rapid synthesis of a gene optimized for expression in the system of choice and production of sufficient biological material for molecular characterization and biotechnological application.     

牛凝乳酶原基因在大肠杆菌中表达调控的研究

Shine-Dalgarno序列与起始密码子之问的距离与组成对凝乳酶原基因表达有明显的影响,可导致其表达水平有15倍之差。SD序列至ATG之间为15bp不利于表达,表达质粒中sD-ATG在7-11bp之间都有可能获得高效表达;但决定因素不是简单的长度,而是RBS附近可能的二级结构即△G的大小、SD序列及ATG中参与配对的碱基数目。将pTLC23中凝乳酶原cDNA3＇端端非翻译区插入终止密码子TGA与转录终止子rrnBT₁T₂之间适当位置可提高凝乳酶原基因的表达,这可能是因为这段序列能形成由53个碱基对和8个碱基组成的稳定的mRNA二级结构,起到转录终止子的作用,而一般认为串联终止子对终止转录更为有效。     

Transposons inactivate biosynthesis of the nonribosomal peptide microcystin in naturally occurring Planktothrix spp

The filamentous cyanobacteria Planktothrix spp. occur in the temperate region of the Northern hemisphere. The red-pigmented Planktothrix rubescens bacteria occur in deep, physically stratified, and less eutrophic lakes. Planktothrix is a known producer of the toxic heptapeptide microcystin (MC), which is produced nonribosomally by a large enzyme complex consisting of peptide synthetases and polyketide synthases encoded by a total of nine genes (mcy genes). Planktothrix spp. differ in their cellular MC contents as well as the production of MC variants; however, the mechanisms favoring this diversity are not understood. Recently, the occurrence of Planktothrix strains containing all mcy genes but lacking MC has been reported. In this study, 29 such strains were analyzed to find out if mutations of the mcy genes lead to the inability to synthesize MC. Two deletions, spanning 400 bp (in mcyB; one strain) and 1,869 bp (in mcyHA; three strains), and three insertions (IS), spanning 1,429 bp (in mcyD; eight strains), 1,433 bp (in mcyEG; one strain), and 1,433 bp (in mcyA; one strain), were identified. Though found in different genes and different isolates and transcribed in opposite directions, IS were found to be identical and contained conserved domains assigned to transposable elements. Using mutation-specific primers, two insertions (in mcyD and mcyA) and one deletion (in mcyHA) were found regularly in populations of P. rubescens in different lakes. The results demonstrate for the first time that different mutations resulting in inactivation of MC synthesis do occur frequently and make up a stable proportion of the mcy gene pool in Planktothrix populations over several years.     

Novel cAMP regulatory elements in the promoter region of bovine P-450(11 beta) gene

In order to elucidate the cAMP regulatory elements in the promoter region of bovine P-450(11 beta) genes, we analyzed the promoter region using chloramphenicol acetyltransferase (CAT) gene as the reporter. Various deletion plasmids were constructed using the promoter region of CB11 beta-7, which is one of the two normal genes. Examination of the effects of Bt2cAMP on the CAT activities of mouse adrenal tumor cells (Y-1 cells) transfected with these deletion plasmids suggested that two elements named Ad3 and Ad4 play major roles in the induction by cAMP. Ad3(AAGATAAGGCACCCATCCATCTT) is located at -306 bp to -284 bp and Ad4 (CCAAGGTC) is located at -331 bp to -324 bp in the promoter region of the P-450(11 beta) gene. Deletions of both Ad3 and Ad4 resulted in a large decrease of the induction ratio from 9- to 3-fold. Coexistence of Ad3 and Ad4 is essential for their function, because any mutations introduced into either one of them resulted in a decrease of the cAMP induction ratio. These two elements are highly conserved among bovine, mouse, and human P-450(11 beta) genes and have no similarity with known cAMP regulatory elements. DNase I footprint analysis indicated that factors which specifically bind to the two elements exist in the nuclear extract of bovine adrenal cortex cells. Ad3 and Ad4 showed different patterns in gel shift analysis using probes which contained Ad3 or Ad4 sequence, suggesting their interaction with different nuclear factors. We also found two other protected regions by DNase I footprint analysis of the promoter regions of P-450(11 beta) gene, and named them Ad5 and Ad6.(ABSTRACT TRUNCATED AT 250 WORDS)     

The triple-helical domain of alpha 2(VI) collagen is encoded by 19 short exons that are multiples of 9 base pairs

We have analyzed the structure of the gene coding for the alpha 2(VI) subunit of chicken type VI collagen. The triple-helical domain of this polypeptide is encoded by 19 short exons distributed over 10 kilobase pairs of genomic DNA. These exons begin with the codon for glycine and end with the codon for the Y amino acid of the collagenous triplet Gly-X-Y. The sizes of the exons are integral multiples of 9 base pairs (bp) (27, 36, 45, 54, 63, and 90 bp), the predominant one being 63 bp. The organization of this type VI collagen gene is therefore quite different from that of the fibrillar collagen genes which have evolved by duplication of a primordial 54-bp unit. It also differs from that of the basement membrane collagen genes whose exon/intron boundaries often split the codons for amino acids.     

Fermentation conditions for high-level expression of the tac-promoter-controlled calf prochymosin cDNA in Escherichia coli HB101

Escherichia coli HB101 harboring an expression plasmid that bears the calf prochymosin gene controlled by the tac promoter was cultivated under different conditions in order to find an optimal fermentation arrangement that would lead to maximal prochymosin yield. Our results indicate that it is advantageous to use lactose in the double role of inducer and carbon/energy source when foreign gene expression is controlled by the tac promoter and the gene product is only moderately toxic owing to its accumulation in the form of an intracellular body. Glucose, on the other hand, may be used when expression should be repressed. Growth temperature substantially influenced the specific rate of prochymosin and beta-lactamase gene expression and the plasmid copy number. Three phases were distinguished in the time course of the fermentation on lactose: exponential growth practically without prochymosin synthesis, linear growth with prochymosin synthesis, and prochymosin synthesis without growth of biomass. The synthesis of prochymosin in the form of intracellular inclusion body was accompanied by the loss of respiratory activity of the cell and the loss of its ability to multiply. Sixteen hours cultivation at 37 degrees C in a complex medium with lactose as inducer and carbon/energy source resulted in up to 30% of the volume and 48% of the total protein of biomass being accumulated for as prochymosin inclusion bodies. The concentration of extractable enzymatically active chymosin in the culture reached 12 mg/L.