Cloning, restriction endonuclease mapping and post-transcriptional regulation of rpsA, the structural gene for ribosomal protein S1

Transducing lambda phages have been isolated that carry segments of the Escherichia coli chromosome in the aspC region, 20.5 min on the E. coli map. One of these phages, lambda aspC2, carries rpsA, the structural gene for the ribosomal protein S1. A three kilobase fragment from this phage, cloned into either the plasmid pACYC184 or the plasmid pBR322, was found to express S1. In cells carrying the rpsA gene on the high copy number plasmid pBR322 the rate of rpsA mRNA synthesis was increased 40-fold, whereas the rate of protein S1 synthesis was doubled, in comparison with these rates in an rpsA haploid.     

外源基因pheA、aroG和tyrB在苯丙氨酸合成途径中的共表达

利用基因工程技术提高了短杆菌的苯丙氨酸合成途径中关键酶活性,大幅度地增加了生物合成苯丙氨酸的产时。首先采用聚合酶链反应（ＰＣＲ）从大肠杆菌的氟代苯丙氨酸抗性变异菌株基因组中扩增到与苯丙氨酸合成相关的ａｒｏＧ,ｐｈｅＡ和ｔｙｒＢ３个基因。ａｒｏＧ编码３－脱氧－２－阿拉伯庚酮糖－７－磷酸合成酶（ＤＳ）,ｐｈｅＡ编码双功能酶蛋白－分枝酸变位酶（ＣＭ）和预苯酸脱水酶（ＰＤ）,ｔｙｒＢ编码转氨酶（ＡＴ）。设     

The nucleotide sequence of aroG, the gene for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase (phe) in Escherichia coli K12

We have determined the nucleotide sequence of aroG, the gene coding for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe), one of three isoenzymes that catalyse the first step of the biosynthesis of aromatic amino acids and vitamins in Escherichia coli K12. The DNA sequence agrees with previously published data on the N-terminal sequence, amino acid composition, and subunit molecular weight of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(pne). There is significant identity in the nucleotide sequences of aroG and aroH (the gene for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase (trp), indicating that these two genes have evolved from a common ancestral gene. There is no attenuator structure in the leader region of aroG.     

Regulation and expression of the bacteriophage mu mom gene: mapping of the transactivation (dad) function to the C region

Expression of the bacteriophage Mu mom gene is under tight regulatory control. One of the factors required for mom gene expression is the trans-acting function (designated Dad) provided by another Mu gene. To facilitate studies on the signals mediating mom regulation, we have constructed a mom-lacZ fusion plasmid which synthesizes beta-galactosidase only when the Mu Dad transactivating function is provided. lambda pMu phages carrying different segments of the Mu genome have been assayed for their ability to transactivate beta-galactosidase expression by the fusion plasmid. The results of these analyses indicated that the Dad transactivation function is encoded between the leftmost EcoRI site and the lys gene of Mu; this region includes the C gene, which is required for expression of all Mu late genes. Cloning of an approx. 800-bp fragment containing the C gene produced a plasmid which could complement MuC- phages for growth and could transactivate the mom-lacZ fusion plasmid to produce beta-galactosidase. These results suggest that the C gene product mediates the Dad transactivation function.     

Gene 67, a new,essential bacteriophage T4 head gene codes for a prehead core component,PIP: II. The construction in vitro of unconditionally lethal mutants and their maintenance

We have obtained frameshift mutations of the bacteriophage T4 gene 67 by manipulating restriction cleavage sites within the gene cloned onto small plasmids. When these mutated genes were recombined back into the T4 genome the resulting phages were inviable. They could only be propagated by complementation in strains carrying a cloned, non-mutated copy of the gene on a plasmid. These experiments demonstrate that gene 67 is essential for T4 growth. Electron microscopy of bacteria infected with 67^? phages revealed that phage head morphogenesis was blocked at an early stage and particles resembling abnormal preheads were found in large numbers. The gene 67 product, PIP, is therefore essential for correct prehead assembly.     

Effect of DNA copy number on genetic stability of phage-displayed peptides

A small model peptide, the FLAG epitope, was cloned into two filamentous phage display vectors, f88-4 and fd88-4, creating phages f88-FLAG and fd88-FLAG, respectively. Both vectors have a gene VIII display cassette (in addition to their normal phage gene VIII) and display the cloned peptide on a few percent of the virion's 3000-4000 pVIII (major coat protein) subunits. Vector f88-4 has a replication defect and attains low DNA copy number in infected cells, while vector fd88-4 has no replication defect and attains the normal, high DNA copy number characteristic of wild-type filamentous phage. Almost no loss of displayed peptide was observed during six rounds of propagation of low copy number f88-FLAG phage. In contrast, when high copy number fd88-FLAG phage was similarly propagated, variant clones that did not display the FLAG epitope accumulated gradually. The loss of displayed peptide from the high copy number vector is undoubtedly slow enough to be overcome by even weak affinity selection, and high copy number vectors have important advantages that make their use worth considering, at least when the displayed peptides are small.     

Cloning, mutation, and location of the F origin of conjugal transfer.

pED806 , a pBR322 derivative carrying the origin of transfer ( oriT ) of F, was rapidly lost from cells carrying an F tra+ plasmid. Instability was increased in a RecA- host, and depended in particular upon the Ftra YZ genes that produce the nick at oriT at which transfer is initiated. Instability was also correlated with the orientation of the oriT fragment in the vector plasmid. Mutants of pED806 selected as being stable in the presence of Flac proved to carry cis-dominant oriT mutations. The oriT site was subcloned from pED806 on a HaeII fragment including a HaeII-Bg/II segment of F DNA approximately 385 base pair (bp) long into the 2.25 kilobase (kb) vector plasmid pED825 , giving pED822 . pED822 was fully proficient for oriT function, and recircularised in recipient cells by a recA- and tra-independent oriT -specific ligation/recombination event. ' Phasmids ' constructed by cloning pED806 or an oriT - mutant into a lambda vector were used to confirm that the nick site in lambda oriT phages grown in the presence of Flac tra+ is indeed at oriT . The nick site in a further lambda oriT phage (ED lambda 102) was then located 140 +/- 20 bp from the Bg/II site forming one terminus of the F fragment cloned in pED806 and pED822 .     

Extrachromosomal replication of shuttle vectors in Dictyostelium discoideum.

We cloned a 12.3-kilobase (kb) endogenous plasmid, Ddp1, found in several wild-type and laboratory strains of Dictyostelium discoideum into pBR322. The cloned plasmids have been used to cotransform D. discoideum cells with B10S, a transformation vector carrying a gene fusion conferring resistance to G418. Whereas B10S DNA alone appears to integrate in a tandem array, the cloned Ddp1 plasmids replicate extrachromosomally and are stably maintained in the absence of selection with an average copy number of 50 to 100 copies per cell. The Ddp1-derived plasmids can be directly recovered by transforming Escherichia coli with bulk nuclear DNA from these cells. Preliminary deletion analysis indicates that not all regions of Ddp1 are necessary for stable replication in D. discoideum. Several recombinant vectors which replicate extrachromosomally in D. discoideum were also isolated. One contains the Act6-neor gene fusion from B10S recombined into one of the cloned derivatives of Ddp1 and can be used to directly transform D. discoideum amoebae, selecting for G418 resistance. Another recombinant is only 5.6 kb and resulted from a deletion of a 16.6-kb cloned Ddp1 hybrid plasmid. An analysis of the vector DNAs present in clones derived from single D. discoideum transformants is also described.     

Vectors with restriction-site banks. I. pJRD158, a 3903-bp plasmid containing 28 unique cloning sites

A DNA fragment has been constructed that contains many unique cloning sites not present in currently used Escherichia coli plasmid cloning vehicles. Insertion of this fragment into a modified version of pBR322 results in an AmpRTetR vector (pJRD158) of 3903 bp containing 28 unique cloning sites, four "almost unique" cloning sites, and eight unassigned unique 6-bp palindromes. The plasmid has the additional advantages of very high copy number and altered incompatibility. The latter permits it to be stably maintained in the same host as pBR322.     

Transformation of Chloroplast Ribosomal RNA Genes in Chlamydomonas: Molecular and Genetic Characterization of Integration Events

Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas.     

Mutational analysis of the feedback sites of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase of Escherichia coli.

In Escherichia coli, aroF, aroG, and aroH encode 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isozymes that are feedback inhibited by tyrosine, phenylalanine, and tryptophan, respectively. In vitro chemical mutagenesis of the cloned aroG gene was used to identify residues and regions of the polypeptide essential for phenylalanine feedback inhibition.     

The recN locus of Escherichia coli K12: molecular analysis and identification of the gene product

Summary The recN gene which is necessary for inducible DNA repair and recombination in Escherichia coli has been cloned into the low copy plasmid vector pHSG415. Analysis of the recombinant plasmid, pSP100, revealed a 5.6 Kb HindIII insert of chromosomal DNA. Transposon inactivation of recN function and analysis of a recN::Mu(Ap lac) fusion located the coding region to a 1.4 Kb region within a 2.1 Kb BglII-AvaI DNA fragment transcribed in a clockwise direction with respect to the chromosome map. The gene product was identified in maxicells as a 60,000 dalton protein. Synthesis of this protein was increased in cells lacking LexA activity or in strains carrying recN cloned into the multicopy vector pBR322. Multiple copies of recN increase resistance to ionizing radiation in recN mutants but reduce the survival of a wild-type strain.     

Selection of independent plasmids determining phenol degradation in Pseudomonas putida and the cloning and expression of genes encoding phenol monooxygenase and catechol 1,2-dioxygenase

Long-term cultivation of the Pseudomonas putida multiplasmid strain EST1020 on phenol resulted in the formation of individual PHE plasmids determining phenol degradation. Four types of PHE plasmids, pEST1024, pEST1026, pEST1028, and pEST1029, are characterized. They all contain a transferrable replicon similar to pWWO-8 with a partly duplicated DNA sequence of the 17-kb transposable element of this plasmid and include various amounts of DNA that carry genes encoding phenol degradation (phe genes). We cloned the genes determining phenol monooxygenase and catechol 1,2-dioxygenase from the Pseudomonas sp. parent strain plasmid DNA into the broad host range vector pAYC32 and studied the expression of the cloned DNA. The formation of a new hybrid metabolic plasmid, pEST1354, was demonstrated in P. putida PaW85 as the result of transposition of the 17-kb genetic element from the chromosome of PaW85 into the plasmid carrying cloned phe genes. The target site for the 17-kb transposon was localized in the vector DNA, just near the cloning site. In subcloning experiments we found two regions in the 17-kb DNA stretch that are involved in the expression of the cloned phe genes.     

Restriction map of the Escherichia coli malA region and identification of the malT product.

A series of plaque-forming lambda h80 transducing phages carrying various portions of the malA region were isolated. A 5,800-base pair HindIII-EcoRI DNA fragment from one of these phages was cloned into pBR322 and shown to contain malT, which is the positive regulator gene of the maltose regulon, and most of malP, the structural gene for maltodextrin phosphorylase. A restriction map of the HindIII-EcoRI fragment was established, and it was correlated with the genetic map of the malA region (i) by mapping deletions which had been generated in vitro on the plasmid and (ii) by locating on the restriction map a DNA insertion of known genetic position. A 600-base pair HincII-HaeII segment was shown to contain all or part of the promoters for malT and malP, which are known to be transcribed in opposite directions. Strains carrying gene malT on a plasmid synthesized a 94,000-dalton polypeptide which was not produced by identical strains carrying similar plasmids in which malT was partially deleted. Estimates of the size of the malT gene support the conclusion that the 94,000-dalton polypeptide is the malT product.     

A phagemid vector using the E. coli phage shock promoter facilitates phage display of toxic proteins

Phage display is a powerful tool with which to adapt the specificity of protease inhibitors. To this end, a library of variants of the potato protease inhibitor PI2 was introduced in a canonical phagemid vector. Although PI2 is a natural trypsin inhibitor, we were unable to select trypsin-binding variants from the library. Instead, only mutants carrying deletions or amber stop codons were found. Bacteria carrying these mutations had a much faster growth rate than those carrying the wt PI2-encoding gene, even when the promoter was repressed. To overcome these problems, two new phagemid vectors for g3-mediated phage display were constructed. The first vector has a lower plasmid copy number, as compared to the canonical vector. Bacteria harboring this new vector are much less affected by the presence of the PI2-g3 fusion gene, which appears from a markedly reduced growth retardation. A second vector was equipped with the promoter of the Escherichia coli psp operon, instead of the lac promoter, to control the PI2-g3 gene fusion expression. The psp promoter is induced upon helper phage infection. A phagemid vector with this promoter controlling a PI2-g3 gene fusion did not affect the viability of the host. Furthermore, both new vectors were shown to produce phage particles that display the inhibitor protein and were therefore considered suitable for phage display. The inhibitor library was introduced in both new vectors. Trypsin-binding phages with inhibitory sequences were selected, instead of sequences with stop codons or deletions. This demonstrates the usefulness of these new vectors for phage display of proteins that affect the viability of E. coli.