Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance.

The nucleotide sequence of pC194, a small plasmid from Staphylococcus aureus which is capable of replication in Bacillus subtilis, has been determined. The genetic determinant of chloramphenicol (CAM) resistance, which includes the chloramphenicol acetyl transferase (CAT) structural gene, the putative promoter and controlling element of this determinant, have been mapped functionally by subcloning a 1,035-nucleotide fragment which specifies the resistance phenotype using plasmid pBR322 as vector. Expression of CAM resistance is autogenously regulated since the 1,035-nucleotide fragment containing the CAT gene sequence and its promoter cloned into pBR322 expresses resistance inducibly in the Escherichia coli host. A presumed controlling element of CAT expression consists of a 37-nucleotide inverted complementary repeat sequence that is located between the -10 and ribosome-loading sequences of the CAT structural gene. Whereas the composite plasmid containing the minimal CAT determinant cloned in pBR322 could not replicate in B. subtilis, ability to replicate in B. subtilis was seen if the fragment cloned included an extension consisting of an additional 300 nucleotides beyond the 5' end of the single pC194 MspI site associated with replication. This 5' extension contained a 120-nucleotide inverted complementary repeat sequence similar to that found in pE194 TaqI fragment B which contains replication sequences of that plasmid. pC194 was found to contain four opening reading frames theoretically capable of coding for proteins with maximum molecular masses, as follows: A, 27,800 daltons; B, 26,200 daltons; C, 15,000 daltons; and D, 9,600 daltons. Interruption or deletion of either frame A or D does not entail loss of ability to replicate or to express CAM resistance, whereas frame B contains the CAT structural gene and frame C contains sequences associated with plasmid replication.     

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.     

Cloning of the type VII trimethoprim-resistant dihydrofolate reductase gene and identification of a specific DNA probe

Abstract A 1.3 kb Hin III fragment encoding the type VII trimethoprim-resistant dihydrofolate reductase gene was cloned into pBR322. Unidirectional deletion of this cloned fragment with exonuclease III identified the start of the dihydrofolate reductase gene. An internal 300bp Eco RV fragment was identified which could be used as a specific non-radioactive DNA probe to distinguish bacteria carrying the type VII gene from those carrying genes encoding other known dihydrofolate reductase types.     

Plasmid-determined enzymatic degradation of nylon oligomers.

The nylon oligomer (6-aminohexanoic acid cyclic dimer) degradation genes on plasmid pOAD2 of Flavobacterium sp. KI72 were cloned into Escherichia coli vector pBR322. The locus of one of the genes, the structural gene of 6-aminohexanoic acid linear oligomer hydrolase, was determined by constructing various deletion plasmids and inserting the lacUV5 promoter fragment of E. coli into the deletion plasmid. Two kinds of repeated sequences (RS-I and RS-II) were detected on pOAD2 by DNA-DNA hybridization experiments. These repeated sequences appeared five times (RS-I) or twice (RS-II) on pOAD2. One of the RS-II regions and the structural gene of the hydrolase overlapped.     

Tissue-specific and developmentally regulated expression of a chimeric actin-globin gene in transgenic mice.

A chimeric plasmid containing about 2/3 of the rat skeletal muscle actin gene plus 730 base pairs of its 5' flanking sequences fused to the 3' end of a human embryonic globin gene (D. Melloul, B. Aloni, J. Calvo, D. Yaffe, and U. Nudel, EMBO J. 3:983-990, 1984) was inserted into mice by microinjection into fertilized eggs. Eleven transgenic mice carrying the chimeric gene with or without plasmid pBR322 DNA sequences were identified. The majority of these mice transmitted the injected DNA to about 50% of their progeny. However, in transgenic mouse CV1, transmission to progeny was associated with amplification or deletion of the injected DNA sequences, while in transgenic mouse CV4 transmission was distorted, probably as a result of insertional mutagenesis. Tissue-specific expression was dependent on the removal of the vector DNA sequences from the chimeric gene sequences prior to microinjection. None of the transgenic mice carrying the chimeric gene together with plasmid pBR322 sequences expressed the introduced gene in striated muscles. In contrast, the six transgenic mice carrying the chimeric gene sequences alone expressed the inserted gene specifically in skeletal and cardiac muscles. Moreover, expression of the chimeric gene was not only tissue specific, but also developmentally regulated. Similar to the endogenous skeletal muscle actin gene, the chimeric gene was expressed at a relatively high level in cardiac muscle of neonatal mice and at a significantly lower level in adult cardiac muscle. These results indicate that the injected DNA included sufficient cis-acting control elements for its tissue-specific and developmentally regulated expression in transgenic mice.     

Heterologous transformation ofClaviceps purpurea

Summary A transformation system based on dominant selection markers was established for an industrialClaviceps purpurea strain. Transformants could be obtained by using plasmid pAN 7-1 carrying a bacterial gene for hygromycin (hph) resistance fused to a fungal promoter or by plasmid p3SR2 which carries the acetamidase gene (amdS) fromAspergillus nidulans.     

Transformation of Aspergillus nidulans by the orotidine-5'-phosphate decarboxylase gene of Neurospora crassa

Relief of an auxotrophic requirement for uridine in Aspergillus nidulans strain G191 has been achieved by transformation with a segment of Neurospora crassa DNA containing the corresponding gene coding for orotidine-5'-phosphate decarboxylase. The mitotic stability of such transformants suggests that the DNA has integrated into the genome. Southern hybridisation analysis of DNA isolated from transformants revealed the presence of pBR322 sequences which have integrated into the host genome along with the N. crassa DNA.     

The function of the tet gene in the plasmid pBR322 is not inhibited by expression of an anti-tet gene

The possibility of gene suppression by the expression of anti-sense sequences has been tested for tet gene of pBR322 plasmid. Anti-tet gene has been inserted into lac-promoter regulated site of M13mp 10 single-stranded high copy phage vector. To achieve that, HihdIII-BamHI fragment of pBR322 carrying part of the tet gene was inserted into poly-linker of mp 10. The influence of the anti-tet gene expression on growth parameters of cells with or without tetracycline in the growth media was monitored for JM103 cells. The results indicate that in this system the detectable suppression of the tet gene by anti-tet expression was not manifested.     

噬菌体T7基因6.5和6.7的克隆及其缺失变种的构建

 用适当的限制性内切酶,将噬菌体T7基因6.5和6.7从整个噬菌体T7基因组中分离出来,插入到质粒pBR322中去,转化E.Coli HMS174,筛选出这两个基因的成功克隆。运用同样手段,从整个噬菌体T7基因组中分离出含有部分基因6编码序列,而不含基因6.5和6.7编码序列的T7DNA片段,插入到pBR322的衍生质粒中去,转化Ecoli C1757,再用含有基因6和基因7的双突变噬菌体T7去感染这一转化菌,通过同源交叉而得到缺失基因6.5和6.7的噬菌体T7缺失变种。这种噬菌体只能在载有噬菌体T7基因6.5和6.7,或者只载有基因6.7质粒的寄主中增殖。通过噬菌体结构蛋白电泳分析证明,这种噬菌体丢失了野生型菌体T7所具有的两条结构蛋白带。     

Isolation of a new plasmid pIRL19: its use in construction of small vectors and detection of specific sequence in a foreign DNA

A new plasmid, pIRL19, was constructed by ligating a 1875-bp HaeII fragment carrying the ampicillin-resistance (Apr) gene to a 370-bp HaeII fragment containing the replication origin of the plasmid pBR322. The plasmid essentially contains only the basic replicator and the Apr gene. This basic replicator provides a valuable initial building block for in vitro construction of other very small vectors with antibiotic-resistance determinants. To illustrate this potential, we have transferred the chloramphenicol-resistance (Cmr) gene and a part of the Apr gene from the plasmid pBR329 into pIRL19 such that the new plasmid pIRL20 acquired the Cmr gene and maintained the integrity of its Apr structural gene.     

Generation of deletions in the 3'-flanking sequences of the Escherichia coli crp gene that induce cyclic AMP suppressor functions

The crp structural gene and its 3'-flanking sequences were subcloned into M13mp8, and in vitro deletions were constructed in both the 5' and 3' ends of the gene by using Bal 31 nuclease. Deletions ranged in size from 24 to 250 base pairs at the 5' end of crp. Sixteen deletions generated at the 3' end of the gene ranged in size from 133 to 675 base pairs. The majority of deletions extended into the crp structural gene. Another class of deletions, i.e., delta crp-4, delta crp-17, and delta crp-2, had endpoints extending in the 3'-flanking sequences external to the crp structural gene. Deletions were subcloned into pBR322 and transformed into the Escherichia coli cya crp deletion strain NCR438. Transformants containing plasmid pBM4 with the delta crp4 mutation, a deletion of 133 base pairs, were cyclic AMP independent. Strain NCR440 harboring this plasmid expressed beta-galactosidase and threonine dehydratase activities and fermented lactose, ribose, arabinose, and xylose in the absence of exogenous cyclic AMP. The delta crp-4 mutation also caused strain NCR440 to be hypersensitive to exogenous cyclic AMP. The cylic AMP receptor protein expressed in maxicells from pBM4 carrying the delta crp-4 mutation comigrated with the wild-type protein on electrophoretic gels. The delta crp-4 mutation demonstrates that sequences distal to the crp structural gene can mediate cyclic AMP suppressor functions.     

Genetic analysis of Saccharomyces cerevisiae transformed by plasmid containing a supressor transfer ribonucleic acid gene.

The behavior in Saccharomyces cerevisiae of plasmid pYTE1, which contains yeast tyrosine-inserting ochre suppressor SUP4.o, a 4-kilobase EcoRI fragment of yeast 2muDNA, and the bacterial plasmid pBR322, has been studied. Selection of yeast transformants was by suppression of multiple ochre mutations. About 10(3) to 10(4) transformants per microgram of pYTE1 dfeoxyribonucleic acid were obtained. The majority of transformants contained both an integrated copy of the SUP4.o gene plus pBR322 deoxyribonucleic acid sequences and autonomously replicating forms of the plasmid. The integrated copy was extremely stable mitotically and meiotically, but the associated nonintegrated copies were lost at meiosis. The chromosomally integrated pBR322 sequences were linked to the SUP4.o gene. The integration site was at the SUP4+ locus. In transformants with only nonintegrated copies of pYTE1, the expression of suppression was reduced, and the plasmid was unstable in mitosis. Plasmid deoxyribonucleic acid preparations from both types of transformant could be used to retransform yeast cells. Plasmid pYTE1 has restriction enzyme sites useful for the high frequency and stable transformation of other genes into yeasts. The potential uses of this plasmid for transformation of other organisms is discussed.     

Transformation of Aspergillus niger by the amdS gene of Aspergillus nidulans.

Aspergillus niger grows poorly on acetamide as a nitrogen or carbon source and lacks sequences detectably homologous to the amdS gene encoding the acetamidase of Aspergillus nidulans. We have taken advantage of these observations to develop a transformation system for A. niger using the amdS gene as a dominant heterologous marker for selecting transformants on the basis of acetamide utilization. Transformants varied in their ability to grow on amide media and the number of integrated copies of the amdS plasmid ranged from 1 or 2 to greater than 100. Southern analysis of transformants revealed that the multiple copies were integrated into the chromosome in tandem arrays. This result indicates that transformation of A. niger is more similar to mammalian cells than to yeast. Analysis of enzyme activity levels and RNA levels showed that most of the copies of amdS were expressed. Mitotic stabilities of transformants were found to be high. A transformant containing greater than 100 copies of the amdS gene was impaired in omega-amino acid utilization, a result that has also been found in A. nidulans. Since, in A. nidulans, omega-amino acids induce acetamidase via a characterizied regulatory gene (amdR/intA) this observation implies that titration of an analogous A. niger regulatory gene product by multiple amdS copies has occurred. Additional evidence suggested that the amdS gene is regulated in A. niger. It has also been shown that an unselected plasmid can be co-transformed with the amdS plasmid into A. niger.     

The metC gene in Escherichia coli K-12: Isolation and studies of relatedness in Enterobacteriaceae

Abstract A specialized transducing phage λ carrying the structural gene for β-cystathionase ( metC ) of Escherichia coli was isolated. The phage carries a 21-kb fragment of the E. coli K-12 chromosome, and its structure was analyzed using restriction enzymes. The metC gene was recloned into resistance plasmid pBR322, using Eco RI or Hin dIII. The information for the metC gene is contained in a 1.3-kb fragment, which shows a high degree of homology with representative strains of all tribes of Enterobacteriaceae.