The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains.

The cyclic decapeptide antibiotic tyrocidine is produced by Bacillus brevis ATCC 8185 on an enzyme complex comprising three peptide synthetases, TycA, TycB, and TycC (tyrocidine synthetases 1, 2, and 3), via the nonribosomal pathway. However, previous molecular characterization of the tyrocidine synthetase-encoding operon was restricted to tycA, the gene that encodes the first one-module-bearing peptide synthetase. Here, we report the cloning and sequencing of the entire tyrocidine biosynthesis operon (39.5 kb) containing the tycA, tycB, and tycC genes. As deduced from the sequence data, TycB (404,562 Da) consists of three modules, including an epimerization domain, whereas TycC (723,577 Da) is composed of six modules and harbors a putative thioesterase domain at its C-terminal end. Each module incorporates one amino acid into the peptide product and can be further subdivided into domains responsible for substrate adenylation, thiolation, condensation, and epimerization (optional). We defined, cloned, and expressed in Escherichia coli five internal adenylation domains of TycB and TycC. Soluble His6-tagged proteins, ranging from 536 to 559 amino acids, were affinity purified and found to be active by amino acid-dependent ATP-PPi exchange assay. The detected amino acid specificities of the investigated domains manifested the colinear arrangement of the peptide product with the respective module in the corresponding peptide synthetases and explain the production of the four known naturally occurring tyrocidine variants. The Km values of the investigated adenylation domains for their amino acid substrates were found to be comparable to those published for undissected wild-type enzymes. These findings strongly support the functional integrities of single domains within multifunctional peptide synthetases. Directly downstream of the 3' end of the tycC gene, and probably transcribed in the tyrocidine operon, two tandem ABC transporters, which may be involved in conferring resistance against tyrocidine, and a putative thioesterase were found.     

Isolation and identification of a novel tandemly repeated DNA sequence in the centromeric region of human chromosome 8

EcoRI subclones, designated as 50E1 and 50E4, were independently obtained from a cosmid clone previously mapped to the centromeric region of human chromosome 8. Southern blot hybridization analyses suggested that both subclones contain repetitive DNA sequences different from the chromosome 8 specific alphoid DNA. DNA sequence analysis of the 704 bp insert of 50E1 and the 1, 962 bp insert of 50E4 revealed that both inserts contained tandemly repeated units of 220 bp. Fluorescence in situ hybridization studies confirmed these two subclones to be specifically located on the centromeric region of chromosome 8. A 220 bp consensus sequence, derived from nine monomeric repeats, showed no significant homology to alphoid consensus sequences or to other currently known human centromeric DNA sequence. Furthermore, no significant homology was found with any other DNA sequence deposited in the EMBL or GenBank databases, indicating that this chromosome 8 specific repetitive DNA sequence is novel. From slot blot experiments it was estimated that 0.013% of the human genome comprises 1,750 of these monomeric repeats, residing on the centromeric region of chromosome 8 in tandem array(s).     

Characterization of the Azorhizobium sesbaniae ORS571 genomic locus encoding NADPH-glutamate synthase.

Sixteen independent Azorhizobium sesbaniae ORS571 vector insertion (Vi) mutants defective in ammonium assimilation (Asm-) were selected; genomic DNA sequences flanking the insertion endpoints were cloned directly. Resulting recombinant plasmids were used to identify, by hybridization, corresponding wild-type DNA sequences from an A. sesbaniae lambda EMBL3 genomic library (lambda Asm phages). All 16 Asm- Vi mutants physically mapped to a single genomic locus. Plasmid subclones of recombinant phage lambda Asm152 were able to complement both Escherichia coli gltB and A. sesbaniae Asm- Vi mutants; NADPH-glutamate synthase activity was detected in all such strains complemented to Asm+. Heterologous and homologous complementations required both A. sesbaniae gltA+ and (inferred) gltB+ genes. Eleven A. sesbaniae Asm- Vi mutants mapped to a 4-kilobase-pair (kbp) DNA region that exhibited homology with Bacillus subtilis gltA+. In E. coli maxicell labeling experiments, this 4-kbp DNA region encoded a 165-kilodalton polypeptide that was inferred to be the product of the A. sesbaniae gltA+ gene (glutaminase NADPH-dependent L-glutamate synthase subunit). Site-directed Tn5-lacZ mutagenesis of a glt plasmid subclone identified a region that bisected this locus into (at least) two cistrons. Because the remaining five A. sesbaniae Asm- mutants mapped to a 1.5-kbp region adjacent to gltA+, these mutants probably define a single gltB+ gene (glutamate dehydrogenase NADPH-dependent L-glutamate synthase subunit); this region did not exhibit homology with the B. subtilis gltB+ gene.     

家蝇卵黄蛋白基因的克隆与结构序列分析

家蝇卵黄蛋白基因编码的卵黄蛋白是家蝇胚胎发育的重要营养来源 .根据 3种家蝇卵黄蛋白cDNA保守序列设计引物 ,用PCR技术从家蝇基因组DNA中扩增到大小为 76 8bp的mdYP1基因的部分DNA片段 .经地高辛标记成特异性探针 ,从构建的家蝇基因组文库中筛选出一个阳性克隆 ,并从该克隆中分离到大小为 3991bp的mdYP1基因组基因 .序列分析显示 ,该基因组序列含有约1 6kb的 5′ 上游区和 1 0kb的 3′ 下游区 ,编码区由一个 6 1bp的内含子和大小分别为 2 2 2bp和10 2 8bp的 2个外显子组成 .5′ 上游区含有典型的CAAT TATA盒 .     

Mycoplasma pneumoniae DNA gyrase genes

We have identified a clone from a λEMBL3 library containing a 19kb insert of Mycoplasma pneumoniae DNA which includes the genes that encode both subunits of DNA gyrase. The gyrB gene and the 5’end of the gyrA gene have been subcloned into M13. The gryB gene is 1953bp in length and overlaps the gryA gene by a single base. The nucleotide sequence of these subclones has significant homology to previously reported gyrase genes. In terms of the size of gyrB gene and its proximity to the gyrA gene, M. pneumoniae is more similar to Bacillus subtilis than to Escherichia coli.     

Cloning of clusters of erythromycin biosynthesis genes of Streptomyces erythraeus (Saccharopolyspora erythraea)

The erythromycin resistance gene (ermE) and part of erythromycin biosynthesis genes located in the same cluster with the ermE gene were cloned from S. erythraeus 3 subjected to improvement with respect to erythromycin production. For isolating the erythromycin biosynthesis genes, the plasmid vector pUC18 and the phage vector lambda EMBL3 were used. The ermE gene DNA was used as a labeled probe for analysis of the recombinant plasmids and phages. The recombinant phages lambda ermE1 and ermE4 containing fragments of the chromosomal DNA collinear to the genome DNA of S. erythraeus 3 were analyzed. The size of the cloned fragment of the chromosomal DNA of S. erythraeus 3 was about 20 kb. Subcloning with the vector pUS18 resulted in isolation of plasmids pSU235-pSU244 containing BamHI fragments of chromosomal DNA from S. erythraeus 3. The restriction map of the chromosomal region of S. erythraeus 3 containing the ermE gene was constructed. The cloned genes of erythromycin biosynthesis are useful in the study of their structure and functions, construction of integrative vectors, improvement of cultures producing macrolide antibiotics and isolation of genes responsible for biosynthesis of other polyketide antibiotics.     

Cloning and expression of a Bacillus subtilis division initiation gene for which a homolog has not been identified in another organism.

The Bacillus subtilis 168 division initiation genes defined by the temperature-sensitive mutations ts-1 and ts-12 were cloned into a 10.5-kilobase EcoRI fragment of DNA in the lambda EMBL4 vector. The two genes were separated by approximately 3 kilobases. The gene in which the ts-1 mutation resides was shown to be the same as the B. subtilis homolog of the Escherichia coli ftsZ gene. The other gene was named divIB. It showed no homology to any previously identified gene and coded for a protein of 30.1 kilodaltons which was probably membrane bound.     

Adult chicken alpha-globin gene expression in transfected QT6 quail cells: evidence for a negative regulatory element in the alpha D gene region.

The chicken adult alpha-globin genes, alpha A and alpha D, are closely linked in chromosomal DNA and are coordinately expressed in vivo in an approximate 3:1 ratio, respectively. When subcloned DNAs containing one or the other gene are stably transfected into QT6 quail fibroblasts, the alpha A-globin gene is expressed at measurable RNA levels, but the alpha D gene is not. The alpha A gene expression can be considerably increased by the presence of a linked Rous sarcoma virus long terminal repeat enhancer, but that of the alpha D gene remains undetectable. Transfection with subclones containing both genes, either in cis or in trans, leads to considerably reduced alpha A RNA levels and still no observable alpha D gene expression. Transfection with deleted subclones suggests that maximal expression levels in this system require the alpha A-globin gene promoter, as opposed to that of the alpha D gene, but that such expression is greatly reduced by one or more DNA sequences which lie approximately 2,000 base pairs upstream of the alpha A gene, within the body of the alpha D gene.     

绿色木霉纤维素酶CBHII基因的分子克隆

本文以噬菌体lambda EMBL3 DNA为载体,通过克隆绿色木酶(Trichoderma viride)高分子量基因组DNA的部分酶解片段,并将重组分子进行体外包装后侵染Escherichia.coli K802,由此构建了绿色木霉基因文库。以李氏木霉(Trichoderma reesei)纤维素酶CBHII基因的末端片段为探针,用轮迥噬菌斑原位杂交从文库中筛选出CBHII基因的阳性克隆5个,随机取其中3个克隆用上述探针作斑点杂交,结果进一步证明克隆了全长或近全长的绿色木霉CBHII基因,用李氏木霉CBHI基因的末端片段探针作斑点杂交,结果提示CBHI与CBHII基因的末端序列之间无同源性存在。从斑点杂交的阳性克隆中提取DNA,酶切鉴定插入片段的长度,并克隆于质粒pUC19,Southern杂交结果证明获得了含绿色木霉CBHII基因的重组质粒pCBHII-14。     

Characterization of three genomic loci encoding Rhizobium sp. strain ORS571 N2 fixation genes.

Sixty-five independent, N2 fixation-defective (Nif-) vector insertion (Vi) mutants were selected, cloned, and mapped to the ORS571 genome. The recombinant Nif::Vi plasmids obtained in this way were used as DNA hybridization probes to isolate homologous phages from a genomic library of ORS571 constructed in lambda EMBL3. Genomic maps were drawn for three ORS571 Nif gene loci. Forty-five Nif::Vi mutants in genomic Nif locus 1 defined two gene clusters separated by 8 kilobase pairs (kb) of DNA. In the first cluster, 36 Nif::Vi mutants mapped to a 7-kb DNA segment that showed DNA homology with Klebsiella pneumoniae nifHDKE and encoded at least two Nif operons. In the other cluster, nine Nif::Vi mutants mapped to a 1.5-kb DNA segment that showed homology with K. pneumoniae and Rhizobium meliloti nifA; this DNA segment encoded a separate Nif operon. Fifteen Nif::Vi mutants mapped to a 3.5-kb DNA segment defined as Nif locus 2 and showed DNA homology with the R. meliloti P2 fixABC operon. Nif locus 2 carries a second nifH (nifH2) gene. Four Nif::Vi mutants mapped to a 2-kb DNA segment defined as Nif locus 3 and showed DNA homology with K. pneumoniae nifB. DNA from lambda Nif phages comprising all three genomic Nif loci was subcloned in plasmid vectors able to stably replicate in ORS571. These plasmid subclones were introduced into ORS571 strains carrying physically mapped Nif::Vi insertions, and genetic complementations were conducted. With the exception of certain mutants mapping to the nifDK genes, all mutants could be complemented to Nif+ when they carried plasmid subclones of defined genomic DNA regions. Conversely, most nifDK mutants behaved as pseudodominant alleles.     

Molecular analysis of glycophorin A and B gene structure and expression in homozygous Miltenberger class V (Mi. V) human erythrocytes

In the Miltenberger class V (Mi. V) condition, red cells lack glycophorin A (GPA) and glycophorin B (GPB) but carry instead an unusual glycoprotein thought to be a hybrid molecule produced by the unequal crossing-over between the closely linked genes encoding for GPA and GPB. By Western blot analysis with rabbit anti-GPA antibodies specific for discrete domains of GPA, it was found that the Mi. V glycoprotein (donor F. M.) contains approximately 60 amino acid residues of GPA at its N-terminus. As a preliminary approach to the molecular analysis of this variant the restriction maps of the GPA and GPB genes were established by Southern blot analysis of genomic DNA and from genomic clones isolated from a human leukocyte library constructed in lambda EMBL4. The GPA and GPB genes cover about 30 kb of DNA and are organized into seven exons (A-1-A-7) and five exons (B-1-B-5), respectively. In addition to the normal genes, a third gene (named inv), closely resembling the GPA and GPB genes, was also identified. In the homozygous Mi. V individual the normal GPA and GPB genes were absent, but an unusual form of gene structure was detected by Southern blot analysis. The Mi. V glycoprotein gene was composed of exon B-1 of the GPB gene followed by exons A-2 and A-3 of the GPA gene and the exons B-3, B-4 and B-5 of the GPB gene. Exon B-1 can be distinguished from exon A-1 of GPA since it is located within a different restriction fragment, but both encode the same amino acid sequence (N-terminal region of the signal peptides). Using the polymerase chain reaction, the junction between exon A-3 and exon B-3 was confirmed by amplification of the DNA region where the putative crossing-over has occurred and it was deduced that the Mi. V glycoprotein is a hybrid molecule composed of amino acid residues 1-58 from GPA fused to amino acid residues 27-72 of GPB. In addition, the finding that part of the signal peptide and the 5'-untranslated region are derived from GPB suggests that the genetic background of the Mi. V variant is rather complex and may involve a cascade of recombination or gene conversion events.     

人促红细胞生成素基因组基因的克隆及全序列分析

为了克隆到全长的人促红细胞生成素基因,从而在真核系统中及转基因动物乳腺中进行表达,从一个中国人的血细胞中提取基因组ＤＮＡ,构建了不完全基因组噬菌体文库,文库效价为５×１０４．这种文库的构建方法是用ＢａｍＨⅠ对基因组ＤＮＡ进行完全酶切,回收１０．５ｋｂ左右的酶切片段,插入到λ－噬菌体ＥＭＢＬ３载体中．筛选文库所使用的探针是用ＰＣＲ方法从基因组ＤＮＡ模板中扩增的５５６ｂｐ的ＥＰＯ基因片段．从该文库中筛选到了一个含有完整ＥＰＯ基因的插入片段长度为１２．５ｋｂ的阳性克隆．经全序列分析证实,所克隆的ＥＰＯ基因编码正确,但在５′－侧翼及第一内含子处与国外发表的序列相比较,发现两处核苷酸差异,这两个核苷酸的差异改变了５′－调控区的两个小的开放阅读框——ＯＲＦ１和ＯＲＦ３,其在基因表达调探方面的作用值得进一步探讨．