Cloning of chicken lysozyme structural gene sequences synthesized in vitro.

Double-stranded chicken lysozyme cDNA was synthesized from an oviduct mRNA fraction enriched for lysozyme mRNA. The ds-cDNA was inserted into the BamHI site of plasmid pBR322 using chemically synthesized DNA linker molecules containing the BamHI restriction endonuclease cleavage site. After bacterial transformation, colonies carrying lysozyme DNA were identified by hybridization with highly purified lysozyme cDNA. The 555 base pairs long cloned DNA fragment of one recombinant plasmid was isolated and characterized by restriction endonuclease digestion. The DNA sequence of selected parts of the inserted DNA is as predicted from the amino acid sequence of prelysozyme. The sequence data allows the unambiguous location of the coding region within lysozyme mRNA.     

Leghemoglobin-like sequences in the DNA of four actinorhizal plants

Summary A cloned cDNA partial copy of a soybean leghemoglobin mRNA was used to probe genomic DNA of four species of actinorhizal plants. Southern blot hybridization revealed the presence of sequences with homology to the leghemoglobin probe in DNA from Alnus glutinosa, Casuarina glauca, Ceanothus americanus and Elaeagnus pungens. The hybridization patterns of the restriction fragments revealed some fragment size conservation between the DNA of soybean and the DNA of four actinorhizal plants which are taxonomically unrelated to soybean or to each other. The results presented here indicate that globin gene sequences are much more widely distributed in the plant kingdom than has previously been thought. Furthermore, if sequence conservation is actually as high as the restriction fragment patterns suggest, the evolution of the DNA surrounding the globin sequences has been highly constrained.     

大豆血红蛋白基因lba转化根瘤菌工程菌株的构建

以土著大豆根瘤菌接种大豆幼苗45 d后获得的根瘤为材料,提取其总RNA并反转录成cDNA,采用同源序列克隆法扩增大豆血红蛋白基因lba编码区序列。利用DNA重组技术,将lba基因连到lac启动子的下游,利用带有发光酶标记基因luxAB的质粒载体pTR102构建表达载体pTR-Plac-lba。采用三亲本杂交的方式,将表达载体pTR-Plac-lba及作为对照的空载体pTR102分别转化土著大豆根瘤菌,获得根瘤菌工程菌株SFH(pTR-Plac-lba)和SFH(pTR102)。盆栽试验发现,接种SFH(pTR-Plac-lba)的大豆植株各生理指标明显高于接种SFH(pTR102)、土著根瘤菌以及未接菌的大豆植株各生理指标。试验证明,导入大豆血红蛋白基因lba的根瘤菌工程菌株SFH(pTR-Plac-lba)对于提高大豆根瘤的固氮酶活性,增加大豆产量起到显著效果。     

Primary structure of the soybean nodulin-23 gene and potential regulatory elements in the 5''-flanking regions of nodulin and leghemoglobin genes.

The nodulin-23 gene of soybean is one of the most abundantly transcribed genes induced during symbiosis with Rhizobium. Using a plasmid (pNod25) from a nodule cDNA library, we have isolated the nodulin-23 gene from a soybean genomic library. Nucleotide sequence analysis of the cDNA and of the genomic clone indicated that the coding region of this gene is 669 bp long and is interrupted by a single intron of about 530 bp. The deduced protein sequence suggests that nodulin-23 may have a signal sequence. The 5'-flanking sequence of two other nodulin genes, nodulin-24 encoding for a membrane polypeptide and one of the leghemoglobin genes (LbC3), were obtained. Comparison of these sequences revealed three conserved regions, one of which, an octanucleotide (GTTTCCCT), has 100% homology. The conserved sequences are arranged in a unique fashion and have a spatial organization with respect to order and position, which may suggest a potential regulatory role in controlling the expression of nodulin and leghemoglobin genes during symbiosis.     

The nucleotide sequences of two leghemoglobin genes from soybean.

We present the complete nucleotide sequences of two leghemoglobin genes isolated from soybean DNA. Both genes contain three intervening sequences in identical positions. Comparison of the coding sequences with known amino-acid sequences of soybean leghemoglobins suggest that the two genes correspond to leghemoglobin C2 and leghemoglobin C3, respectively.     

The primary structures of two leghemoglobin genes from soybean

We present the complete nucleotide sequences of two leghemoglobin genes isolated from soybean DNA. Both genes contain three intervening sequences which interrupt the two coding sequences in identical positions. The 5' and 3' flanking sequences in both genes contain conserved sequences similar to those found in corresponding positions in other eukaryotic genes. Thus, the general DNA sequence organization of these plant genes is similar to that of other eukaryotic genes.     

胰蛋白酶抑制剂基因siRNA表达体系的构建

以大豆基因组DNA为模板,利用聚合酶链式反应(PCR)技术克隆了大豆胰蛋白酶抑制剂基因KSTI3的全长DNA片段,并将其构建到pMD18-T vector上。核苷酸序列测定结果表明:该基因片段全长654bp,与已发表的KSTI3基因序列同源性达99%。将反义 正义基因片段插入到pBI121 35S启动子下,构建重组质粒pBIKSTI3。通过冻融法将该重组质粒转入农杆菌EHA105中,获得了siRNA表达体系。利用农杆菌介导法将带有pBIKSTI3的菌株转化大豆,从2棵再生植株中得到2 100bp的特异性扩增条带,而未转化的植株中无该片段的产生。     

Molecular cloning of DNA complementary to a mouse α-fetoprotein mRNA sequence

DNA complementary to mouse yolk sac messenger RNA has been inserted at the PstI site of the plasmid pBR322 by annealing of the oligo(dG)-tailed plasmid DNA with the oligo(dC)-tailed mouse DNA. Transformation of Escherichia coli strain RRI with this annealed DNA yielded clones bearing recombinant plasmids. The clones were screened for DNA complementary to mouse a-fetoprotein (AFP) messenger RNA sequences by hybridization with a cDNA probe transcribed from an AFP mRNA of over 90% purity. Out of nine plasmids that were isolated and analyzed by restriction mapping, all had homologous insert DNA of various lengths. The plasmid with the longest insert, pAF6, contained 1.65 kb of added DNA, which is about 70% of the AFP mRNA. This clone was positively identified by a hybridization-translation procedure to contain a cDNA sequence for AFP. A restriction map of this clone and the orientation of the message are presented.     

Chromosomal arrangement of leghemoglobin genes in soybean.

A cluster of four different leghemoglobin (Lb) genes was isolated from AluI-HaeIII and EcoRI genomic libraries of soybean in a set of overlapping clones which together include 45 kilobases (kb) of contiguous DNA. These four genes, including a pseudogene, are present in the same orientation and are arranged in the order: 5'-Lba-Lbc1-Lb psi-Lbc3-3'. The intergenic regions average 2.5 kb. In addition to this main Lb locus, there are other Lb genes which do not appear to be contiguous to this locus. A sequence probably common to the 3' region of Lb loci was found flanking the Lbc3 gene. The 3' flanking region of the main Lb locus also contains a sequence that appears to be expressed more abundantly in root tissue. Another sequence which is primarily expressed in root and leaf is found 5' to two Lb loci. Overall, the main leghemoglobin locus is similar in structure to the mammalian globin gene loci.     

Cloning and sequence analysis of a cDNA encoding ferric leghemoglobin reductase from soybean nodules.

A cDNA encoding soybean (Glycine max [L.] Merr) ferric leghemoglobin reductase (FLbR), an enzyme that is postulated to play an important role in maintaining leghemoglobin in its functional ferrous state, has been cloned and characterized. A group of highly degenerate oligonucleotides deduced from the N-terminal amino acid sequence of FLbR was used to prime the polymerase chain reaction (PCR) on soybean nodule mRNA and cDNA. A full-length clone of FLbR cDNA was isolated by screening a lambda gt11 soybean nodule cDNA library using the specific PCR-amplified FLbR cDNA fragment as a probe. The cDNA contained about 1.8 kb and had a coding sequence for 523 amino acids with a predicted molecular mass of 55,729 D, which included a putative 30-residue signal peptide and a 493-residue mature protein. Computer-aided analysis of the deduced FLbR amino acid sequence showed considerable homology (varied from 20-50% with enzymes and species) to dihydrolipoamide dehydrogenase (EC 1.8.1.4), glutathione reductase (EC 1.6.4.2), mercuric reductase (EC 1.16.1.1), and trypanothione reductase (EC 1.6.4.8) in a superfamily of pyridine nucleotide-disulfide oxidoreductases from various organisms. Northern blot analysis using FLbR cDNA as a probe showed that the FLbR gene was expressed in soybean nodules, leaves, roots, and stems, with a greater level of expression in nodules and leaves than in roots and stems. Southern blot analysis of the genomic DNA showed the presence of two homologous FLbR genes in the soybean genome.     

小鼠Nanog基因的克隆及其在大肠杆菌中的表达

按照nanog基因编码序列设计合成引物,利用RT-PCB从小鼠的囊胚期胚胎中扩增得到该 基因,并将该基因克隆到pET-28b(+)载体上,获得pET-28b(+)-nanog原核表达重组质粒,限制 性酶分析和DNA序列测定均证实该克隆插入片段为nanog基因编码序列。重组质粒转化大肠杆 菌BL21(DE3),经IPTG诱导表达,在大肠杆菌表达系统中获得了高效表达,western杂交证实该 蛋白具有6-His抗原活性,从而证实目的蛋白为Nanog蛋白。     

单纯疱疹病毒2型特异性DNA片段的克隆和鉴定

用核酸限制性内切酶BamHI对单纯疱疹病毒2型(HSV—2)的DNA进行酶解,回收位于基因组中的反向重复序列区的Bam HIG片段,然后将其克隆在载体质粒PUC 8的Bam HI切点上,进一步用核酸限制性内切酶Eco RI和KPNI对这一重组质粒联合酶解,移去EcoRI—KPNI小片段,经末端修饰后,将其连接得到新的重组质粒pRC102,它含有一小段HSV—2的DNA序列。以此质粒为探针,分别与HSV—1、HSV—2及细胞DNA进行斑点杂交;与HSV—1和HSV—2酶解后的DNA片段进行Southern转印系交。两组实验结果显示,pRC102质粒DNA只与HSV—2 DNA特异性杂交,其HSV—2的型特异性良好。     

C型产气荚膜梭菌α毒素基因的克隆与表达

利用PCR技术,从C型产气荚膜梭菌染色体基因组中扩增了1．2kb的α毒素基因,将纯化的PCR产物与载体pGEM-T连接,转化至受体菌JM109中,经NcoI／EcoRI和BamHI／EcoRI酶切鉴定及核苷酸序列测定证实,重组质粒pXCPAl中含有α毒素全基因。随后用NcoI／EcoRI酶切质粒pXCPAl,回收α毒素基因片段,插入到事先经同样酶切处理的载体pET-28c中相应酶切位点,构建了表达质粒pETXAl,经NcoI／EcoRI和BamHI／EcoRI酶切鉴定及核苷酸序列测定证实,表达质粒含有α毒素基因且基因序列和阅读框架正确。重组菌株BL21(DE3)(pETXAl)表达产物经ELISA检测和SDS-PAGE分析,重组菌株表达的α毒素蛋白能够被α毒素单抗识别,其表达量占菌体总蛋白相对含量的16．28％。     

Molecular cloning of lupin leghemoglobin cDNA

Poly(A)⁺RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences specific for nodules were selected by differential colony hybridization using³²P-labeled cDNA synthesized either from nodule poly(A)⁺RNA or from poly(A)⁺RNA of uninfected root as probes. Among the recombinant plasmids, the cDNA gene for leghemoglobin was identified. The protein structure derived from its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)⁺RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules.     

Restriction endonucleases and their applications

Restriction endonucleases are deoxyribonucleases which cleave double-stranded DNA into fragments. With only one exception, all restriction endonucleases recognize short, non-methylated DNA sequences. Restriction endonucleases can be divided into two groups based on the position of the cleavage site relative to the recognition sequence. Class I restriction endonucleases cleave double-stranded DNA at positions outside the recognition sequence and generate fragments of random size. The cleavage sites of Class II restriction endonucleases are located, in most cases, within the recognition sequence. Most of the Class II restriction endonucleases recognize 4, 5, or 6 base pair palindromes and generate fragments with either flush ends or staggered ends. DNA fragments with staggered ends contain 3, 4, or 5 nucleotide single-stranded tails called ‘sticky ends’. DNA fragments produced by Class II restriction endonuclease cleavage can be separated on gels according to their molecular weight. The fragments can be isolated from the gel and used for sequence analysis to elucidate genetic information stored in DNA. Further, an isolated fragment can be inserted into a small extrachromosomal DNA, e.g. plasmid, phage or viral DNA, and its replication and expression can be studied in clones of prokaryotic or eukaryotic cells. Restriction endonucleases and cloning technology are powerful modern tools for attacking genetic problems in medicine, agriculture and industrial microbiology.     

Characterization of the genes coding for the Eco RV restriction and modification system of Escherichia coli

A plasmid encoding the recently described Eco RV restriction and modification system has been isolated and characterized. This plasmid, pLB1 , is 6.2 kb long and carries only the Eco RV genes. A subclone of 3 kb has been inserted in pBR322. The relative positions of the endonuclease and the methylase genes were determined by the construction of a set of overlapping deletions generated by Bal31 resection. The DNA sequence of a 2.2 kb fragment containing the two genes was determined. The two genes are transcribed divergently from a 310 bp region and the assignment of the coding region has been confirmed by direct aminoacid sequence analysis. Possible mechanisms of regulation of the endonuclease gene expression at the translational level are proposed and discussed.     

pYEMF,a pUC18-Derived <Emphasis Type="Italic">Xcm</Emphasis>I T-Vector for Efficient Cloning of PCR Products

A 1330-bp DNA sequence with two XcmI cassettes was inserted into pUC18 to construct an efficient XcmI T-vector parent plasmid, pYEMF. The large size of the inserted DNA fragment improved T-vector cleavage efficiency, and guaranteed good separation of the molecular components after restriction digestion. The pYEMF-T-vector generated from parent plasmid pYEMF permits blue/white colony screening; cloning efficiency analysis showed that most white colonies (>75%) were putative transformants which carried the cloning product. The sequence analysis and design approach presented here will facilitate applications in the fields of molecular biology and genetic engineering.     

Isolation and characterization of a gene coding for glyceraldehyde-3-phosphate dehydrogenase from Saccharomyces cerevisiae.

A yeast glyceraldehyde-3-phosphate dehydrogenase gene has been isolated from a collection of Escherichia coli transformants containing randomly sheared segments of yeast genomic DNA. Complementary DNA, synthesized from partially purified glyceraldehyde-3-phosphate dehydrogenase messenger RNA, was used as a hybridization probe for cloning this gene. The isolated hybrid plasmid DNA has been mapped with restriction endonucleases and the location of the glyceraldehyde-3-phosphate dehydrogenase gene within the cloned segment of yeast DNA has been established. There are approximately 4.5 kilobase pairs of DNA sequence flanking either side of the glyceraldehyde-3-phosphate dehydrogenase gene in the cloned segment of yeast DNA. The isolated hybrid plasmid DNA has been used to selectively hybridize glyceraldehyde-3-phosphate dehydrogenase messenger RNA from unfractionated yeast poly(adenylic acid)-containing messenger RNA. The nucleotide sequence of a portion of the isolated hybrid plasmid DNA has been determined. This nucleotide sequence encodes 29 amino acids which are at the COOH terminus of the known amino acid sequence of yeast glyceraldehyde-3-phosphate dehydrogenase.