Cloning and characterization of the histidine biosynthetic gene cluster of Streptomyces coelicolor A3(2)

Biochemical and genetic data indicate that in Streptomyces coelicolor A3(2) the majority of the genes involved in the biosynthesis of histidine are clustered in a small region of the chromosome [Carere et al., Mol. Gen. Genet. 123 (1973) 219-224; Russi et al., Mol. Gen. Genet. 123 (1973) 225-232]. To investigate the structural organization and the regulation of these genes, we have constructed genomic libraries from S. coelicolor A3(2) in pUC vectors. Recombinant clones were isolated by complementation of an Escherichia coli hisBd auxotroph. A recombinant plasmid containing a 3.4-kb fragment of genomic DNA was further characterized. When cloned in the plasmid vector, pIJ699, this fragment was able to complement S. coelicolor A3(2) hisB mutants. Overlapping clones spanning a 15-kb genomic region were isolated by screening other libraries with labeled DNA fragments obtained from the first clone. Derivative clones were able to complement mutations in four different cistrons of the his cluster of S. coelicolor A3(2). Nucleotide sequence analysis of a 4-kb region allowed the identification of five ORFs which showed significant homology with the his gene products of E. coli. The order of the genes in S. coelicolor A3(2) (5'--hisD-hisC-hisBd-hisH-hisA-3') is the same as in the his operon of E. coli.     

Genetic analysis of the rnc operon of Escherichia coli.

RNase III, an Escherichia coli double-stranded endoribonuclease, is known to be involved in maturation of rRNA and regulation of several bacteriophage and Escherichia coli genes. Clones of the region of the E. coli chromosome containing the gene for RNase III (rnc) were obtained by screening genomic libraries in lambda with DNA known to map near rnc. A phage clone with the rnc region was randomly mutagenized with a delta Tn10 element, and the insertions were recombined onto the chromosome, generating a series of strains with delta Tn10 insertions in the rnc region. Two insertions that had Rnc- phenotypes were located. One of them lay in the rnc gene, and one was in the rnc leader sequence. Polarity studies showed that rnc is in an operon with two other genes, era and recO. The sequence of the recO gene beyond era indicated it could encode a protein of approximately 26 kilodaltons and, like rnc and era, had codon usage consistent with a low level of expression. Experiments using antibiotic cassettes to disrupt the genes rnc, era, and recO showed that era is essential for E. coli growth but that rnc and recO are dispensable.     

In vivo selection of better self-splicing introns in Escherichia coli: the role of the P1 extension helix of the Tetrahymena intron

In vivo selection was used to improve the activity of the Tetrahymena pre-rRNA self-splicing intron in the context of heterologous exons. The intron was engineered into a kanamycin nucleotidyltransferase gene, with the pairing between intron bases and the 5' and 3' splice sites maintained. The initial construct failed to confer kanamycin resistance on Escherichia coli, although the pre-mRNA was active in splicing in vitro. Random mutation libraries were constructed to identify active intron variants in E. coli. All the active mutants sequenced contained mutations disrupting a base-paired region above the paired region P1 (referred to as the P1 extension region or P1ex) that involves the very 5' end of the intron. Subsequent site-directed mutagenesis confirmed that these P1ex mutations are responsible and sufficient to activate the intron splicing in E. coli. Thus, it appears that too strong of a secondary structure in the P1ex element can be inhibitory to splicing in vivo. In vitro splicing assays demonstrated that two P1ex mutant constructs splice six to eight times faster than the designed construct at 40 microM GTP concentration. The relative reaction rates of the mutant constructs compared to the original design are further increased at a lower GTP concentration. Possible mechanisms by which the disrupted P1ex structure could influence splicing rates are discussed. This study emphasizes the value of using libraries of random mutations to improve the activity of ribozymes in heterologous contexts in vivo.     

Import-defective colicin B derivatives mutated in the TonB box

The pore-forming colicin B is taken up into Escherichia coli by a receptor and TonB-dependent process. The receptor and colicin B both contain a similar amino acid sequence, close to the N-terminal end, termed the TonB box. Point mutations were introduced into the TonB-box region of the colicin B structural gene cba resulting in colicin B derivatives which were partially or totally inactive against E. coli cells. All derivatives still bound to the receptor. An inactive derivative killed cells when translocated across the outer membrane by osmotic shock treatment, and formed pores in planar lipid bilayer membranes identical to the wild-type colicin. Some of the mutations were partially suppressed by mutations in the tonB structural gene. It was concluded that the TonB-box mutations define a region that is involved in the uptake of colicin B across the outer membrane.     

A vector for construction of gene libraries and the expression of heterologous genes in Saccharomyces cerevisiae

We have constructed a convenient new vector, YEp-DE, for the construction of gene libraries and the expression of heterologous genes in Saccharomyces cerevisiae. The vector contains the yeast LEU2 gene, the 2 mu origin of replication, and a region from pUC18 that includes the ampr gene, the Escherichia coli origin of replication (ori), and the LacZ gene with multiple cloning sites. Five sites (Sac1, Sma1, BamH1, Sal1, Sph1) in this region are unique. This vector has advantages over similar yeast-E. coli shuttle vectors: small size (7291 bp, entirely sequenced), convenient cloning sites, and lacZ selection for detecting recombinant plasmids.     

Isolation of the Candida tropicalis gene for P450 lanosterol demethylase and its expression in Saccharomyces cerevisiae

We have isolated the gene for cytochrome P450 lanosterol 14 alpha-demethylase (14DM) from the yeast Candida tropicalis. This was accomplished by screening genomic libraries of strain ATCC750 in E. coli using a DNA fragment containing the yeast Saccharomyces cerevisiae 14DM gene. Identity of this gene was confirmed by a) observing a heme binding region common to all P450s after sequencing the 3' portion of the gene, and b) based upon tests of its expression in strains of Saccharomyces cerevisiae.     

Cloning, sequencing and overexpression of the mannitol-specific enzyme-III-encoding gene of Staphylococcus carnosus

The gene which encodes the mannitol-specific enzyme III (EIIImtl) of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus carnosus, has been cloned. Genomic libraries of S. carnosus DNA were constructed using the expression vector pUC19 and EIIImtl-producing clones were identified using rabbit polyclonal antiserum. A 700-bp Dde I fragment, containing the complete gene encoding EIIImtl, was sequenced by the dideoxy chain-termination technique. Upstream from the ORF for EIIImtl one can find a sequence analogous to that of the Escherichia coli promoter. This region acts as a strong promoter when subcloned into the promoter test vector M13HDL17. EIIImtl was overproduced using the inducible T7 polymerase system and purified to homogeneity. Amino acid sequence comparison confirmed a 38% similarity to the hydrophilic enzyme-III-like portion of enzyme IImtl of E. coli. There is also a 36% similarity to the N terminus of the fructose-specific phospho-carrier protein from E. coli.     

Screening of Environmental DNA Libraries for the Presence of Genes Conferring Na+(Li+)/H+ Antiporter Activity on Escherichia coli: Characterization of the Recovered Genes and the Corresponding Gene Products

Environmental DNA libraries prepared from three different soils were screened for genes conferring Na(+)(Li(+))/H(+) antiporter activity on the antiporter-deficient Escherichia coli strain KNabc. The presence of those genes was verified on selective LK agar containing 7.5 mM LiCl. Two positive E. coli clones were obtained during the initial screening of 1,480,000 recombinant E. coli strains. Both clones harbored a plasmid (pAM1 and pAM3) that conferred a stable Li(+)-resistant phenotype. The insert of pAM2 (1,886 bp) derived from pAM1 contained a gene (1,185 bp) which encodes a novel Na(+)/H(+) antiporter belonging to the NhaA family. The insert of pAM3 harbored the DNA region of E. coli K-12 containing nhaA, nhaR, and gef. This region is flanked by highly conserved insertion elements. The sequence identity with E. coli decreased significantly outside of the insertion sequence elements, indicating that the unknown organism from which the insert of pAM3 was cloned is different from E. coli. The products of the antiporter genes located on pAM2 and pAM3 revealed functional homology to NhaA of E. coli and enabled the antiporter-deficient E. coli mutant to grow on solid media in the presence of up to 450 mM NaCl or 250 mM LiCl at pH 8.0. The Na(+)/H(+) antiporter activity in everted membrane vesicles that were derived from the E. coli strains KNabc/pAM2 and KNabc/pAM3 showed a substantial increase between pHs 7 and 8.5. The maximal activity was observed at pHs 8.3 and 8.6, respectively. The K(m) values of both antiporters for Na(+) were approximately 10-fold higher than the values for Li(+).     

Rapid generation of incremental truncation libraries for protein engineering using alpha-phosphothioate nucleotides

Incremental truncation for the creation of hybrid enzymes (ITCHY) is a novel tool for the generation of combinatorial libraries of hybrid proteins independent of DNA sequence homology. We herein report a fundamentally different methodology for creating incremental truncation libraries using nucleotide triphosphate analogs. Central to the method is the polymerase catalyzed, low frequency, random incorporation of alpha-phosphothioate dNTPs into the region of DNA targeted for truncation. The resulting phosphothioate internucleotide linkages are resistant to 3'-->5' exonuclease hydrolysis, rendering the target DNA resistant to degradation in a subsequent exonuclease III treatment. From an experimental perspective the protocol reported here to create incremental truncation libraries is simpler and less time consuming than previous approaches by combining the two gene fragments in a single vector and eliminating additional purification steps. As proof of principle, an incremental truncation library of fusions between the N-terminal fragment of Escherichia coli glycinamide ribonucleotide formyltransferase (PurN) and the C-terminal fragment of human glycinamide ribonucleotide formyltransferase (hGART) was prepared and successfully tested for functional hybrids in an auxotrophic E.coli host strain. Multiple active hybrid enzymes were identified, including ones fused in regions of low sequence homology.     

Chloroplast ribosomal protein L13 is encoded in the nucleus and is considerably larger than its bacterial homologue. Construction, immunoisolation, and nucleotide sequence (including transit peptide) its cDNA clone from an angiosperm

Chloroplast ribosomes of higher plants are of the prokaryotic ribosome motif but, unlike in bacteria, their ribosomal protein (r-protein) genes are distributed between the organelle and the nucleus. In order to isolate some of the nuclear-encoded r-protein genes, we have raised antibodies to several spinach chloroplast r-proteins and constructed spinach cDNA expression libraries in lambdagt11. Screening the libraries with one of the antisera yielded three cDNA clones for r-protein L13, an early 50 S subunit assembly protein essential for RI50 formation. The cDNA clone encodes, beginning with a Met codon in the consensus plant initiator context, a polypeptide of 250 amino acid residues. The NH2-terminal 60 residues bear the characteristic features of a chloroplast transit peptide. The putative mature L13 protein, which has common immunoepitopes with Escherichia coli L13, is 34% longer than the E. coli homologue. It has 56% sequence identity with E. coli L13 in the homologous region, but no identity to any known protein in the extra stretch. There are two neighboring ATG codons in the 5' region and two putative plant polyadenylation signals in the 3'-untranslated region of the cDNA. Their possible effect to increase translational efficiency is discussed, and the importance of encoding a RI50 protein in the nuclear genome for possible nuclear control of chloroplast protein synthesis is noted.     

Characterization and mapping of microdissected genomic clones from the adenomatous polyposis coli (APC) region.

The gene associated with adenomatous polyposis coli (APC) has been mapped to the long arm of chromosome 5. To saturate the APC region with DNA markers, two independent microdissection libraries with an emphasis on 5q21.2-21.3 and 5q22 have been constructed from GTG-banded human metaphase chromosomes. PCR-amplified insert DNA of the primary amplificate used as a probe in chromosomal in situ suppression (CISS) hybridization of human metaphase spreads revealed region-specific signals at the chromosomal site that was excised for cloning. One hundred forty-two inserts, derived from both libraries, have been characterized in more detail. Deletion mapping analysis was performed with 17 single-copy clones on a hamster-human hybrid cell panel. Seven of these clones were located within two interstitial deletions of 6-8 Mb from APC-affected individuals around chromosome bands 5q21-22. The identification of new microclones mapping into these deletions and their use in isolating YAC clones should contribute to the construction of a contiguous physical map of the APC region.     

Toward structural and functional genomics of Agrobacterium tumefaciens: linkage map of the left region of linear chromosome.

Genome of A. tumefaciens contains a linear and a circular chromosome. As an initial step of elucidating the structural and functional genomics of this bacterium, linkage map of the left region of its linear chromosome was constructed. Total genomic libraries of A. tumefaciens MAFF301001 were constructed in BAC vectors namely, pFOS1 and pBeloBAC11. Upon construction of sub-libraries, minimum overlapping clones needed to cover the left region was determined. So far, four contigs have been assembled with a total of 19 overlapping clones. Detailed EcoRI physical map of contig III was constructed and it covers a 110 kb region of the Pme5 fragment of the linear chromosome. Seven end regions of the linking clones were partially sequenced but no gene existence was determined due to low homology.     

Cloning and expression in Escherichia coli of the gene encoding Aspergillus flavus urate oxidase.

Amino acid sequencing of peptides obtained after proteolytic hydrolysis of Aspergillus flavus urate oxidase (uricase) permitted the design of oligodeoxynucleotide probes that were used to obtain 1.2- and 5-kilobase pair DNA fragments from A. flavus cDNA and genomic libraries, respectively. The cDNA fragment contained the entire coding region for uricase, and comparison with the genomic fragment revealed the presence of two short introns in the coding region of the gene. A. flavus uricase has around 40% overall identity with uricases from higher organisms but with many conserved amino acids. Hitherto highly conserved consensus patterns found in other uricases were found to be modified in the A. flavus enzyme, notably the sequence Val-Leu-Lys-Thr-Thr-Gln-Ser near position 150, which in the filamentous fungus is uniquely modified to Val-Leu-Lys-Ser-Thr-Asn-Ser. Silent mutations were introduced by cassette mutagenesis near the 5'-extremity of the coding sequence in order to conform with Escherichia coli codon usage, and the uricase was expressed in the E. coli cytoplasm in a completely soluble, biologically active form.     

Molecular cloning and DNA sequencing of the radC gene of Escherichia coli K-12.

The radC102 mutation that sensitizes E. coli K-12 cells to ultraviolet light, ionizing radiations and alkylating agents was localized between the fpg and pyrE genes at 81.7 min on the bacterial chromosome. E. coli strain BH20 (radC+, fpg-1::KnR) has a 10.5-kb EcoRI/KpnI DNA fragment spanning the region from pyrE to the insertion mutation fpg-1::KnR. The proximity of the radC gene to this insertion mutation provided a strategy to isolate the radC+ gene based on the cloning of radC+ and fpg-1::KnR on the same DNA fragment using the resistance to kanamycin as a selector. A library of EcoRI/KpnI DNA fragments of E. coli strain BH20 was inserted into pUC19. One recombinant plasmid conferring resistance to kanamycin was selected and named pRCV10. The pRCV10 plasmid partially restores the resistance to UV-radiation when transformed into SR1187 (radC102), but sensitizes the wild-type strain to the same treatment. The radC102 complementing region was localized on a 1.2-kb BglII/BglII DNA fragment which was sequenced. The DNA sequence complementing the radC102 mutation contained an ATG translation start codon with an open reading frame of 297 base pairs which encodes a polypeptide of Mr 11,500. The order of the genes in this region of the E. coli chromosome is: fpg--rpmBG--radC--pyrE.     

Chromosomal genes essential for stable maintenance of the mini-F plasmid in Escherichia coli

We have isolated mutants of Escherichia coli which do not support stable maintenance of mini-F plasmids (delta ccd rep+ sop+). These host mutations, named hop, were classified into five linkage groups on the E. coli chromosome. Genetic analyses of these hop mutations by Hfr mating and P1 transduction showed their loci on the E. coli genetic map to be as follows: hopA in the gyrB-tnaA region, hopB in the bglB-oriC region, hopD between 8 and 15 min, and hopE in the argA-thyA region. Kinetics of stability of the sop+ and delta sop mini-F plasmids in these hop mutants suggest that the hopA mutants are defective in partitioning of mini-F rather than in plasmid replication. The hopB, hopC, and hopD mutants were partially defective in replication of mini-F. The physical structure of the plasmid DNA was normal in hopA, B, C, and D mutants. Large amounts of linear multimers of plasmid DNA accumulated in mutants of the fifth linkage group (hopE). None of the hop mutations in any linkage group affected the normal growth of cells.     

乙型肝炎病毒表面抗原前S区和谷胱甘肽巯基转移酶融合基因在大肠杆菌中的表达

我们构建了谷胱甘肽巯基转移酶（ＧＳＴ）和完整的或部分缺失的乙型肝炎病毒表面抗原前Ｓ区的融合基因,并在大肠杆菌中进行了表达。融合蛋白的产量随着前Ｓ区长度的增加而迅速降低,而且融合蛋白的前Ｓ区有严重的降解,主要降解位点在ｐｒｅＳ１区的ａ．ａ．７５和ｐｒｅＳ２区的ａ．ａ．１３０和ａ．ａ．１６５左右。利用蛋白降解酶系缺陷型菌株进行的研究表明,这种降解酶存在于多个大肠杆菌株中而且和大肠杆菌中的两个主要的蛋白降解酶系Ｌｏｎ和ｈｔｐＲ无关。具有重要生物学功能的前Ｓ区肽段（ｐｒｅＳ１ａ．ａ．１－６５）因含有阻止分泌的滞留顺序而无法在哺乳动物细胞和酵母中大量表达,但滞留顺序的存在并没有影响含有这一肽段的融合蛋白在大肠杆菌中的表达和产物的纯化。ＧＳＴ融合表达系统产量高,纯化快速简便。用这一方法大量表达并得到的这一肽段不仅是研究乙型肝炎病毒的分子生物学的重要材料,而且可以作为新一代乙型肝炎疫苗的主要组成成分。     

Characterization of the genes and proteins of a two-component system from the hyperthermophilic bacterium Thermotoga maritima.

As a step towards studying representative members of the two-component family of signal transduction proteins, we have cloned genes encoding a histidine protein kinase and a response regulator from the hyperthermophilic bacterium Thermotoga maritima. The genes have been designated HpkA and drrA, respectively. The deduced HpkA sequence contains all five characteristic histidine protein kinase motifs with the same relative order and spacing found in the mesophilic bacterial proteins. A hydropathy profile indicates that HpkA possesses only one membrane-spanning segment located at the extreme N terminus. The N-terminal region of DrrA exhibits all of the characteristics of the conserved domains of mesophilic bacterial response regulators, and the C-terminal region shows high similarity to the OmpR-PhoB subfamily of DNA-binding proteins. Recombinant T. maritima proteins, truncated HpkA lacking the putative membrane-spanning N- terminal amino acids and DrrA, were expressed in Escherichia coli. Partial purification of T. maritima proteins was achieved by heat denaturation of E. coli host proteins. In an in vitro assay, truncated HpkA protein was autophosphorylated in the presence of ATP. Thus, the N-terminal hydrophobic region is not required for kinase activity. Phosphotransfer between truncated HpkA and DrrA was demonstrated in vitro with the partially purified proteins. The phosphorylation reactions were strongly temperature dependent. The results indicate that the recombinant T. maritima two-component proteins overexpressed in E. coli are stable as well as enzymatically active at elevated temperatures.     

Active site amino acid sequence of the bovine O6-methylguanine-DNA methyltransferase.

An O6-methylguanine-DNA methyltransferase has been partially purified from calf thymus by conventional biochemical techniques. The enzyme was specifically radioactively labelled at the cysteine residue of the active site and further purified by attachment to a solid support. Following digestion with trypsin, a radioactive peptide containing the active site region of the protein was purified by size fractionation, ion exchange chromatography and reverse phase HPLC. The technique yielded an essentially homogeneous oligopeptide which was subjected to amino acid sequencing. The sequence adjacent to the acceptor cysteine residue of the bovine protein exhibits striking homology to the C-terminal methyl acceptor site of the E. coli Ada protein and the proposed acceptor sites of the E. coli Ogt and the B. subtilis Dat1 proteins.