The oriT region of the conjugative transfer system of plasmid pCU1 and specificity between it and the mob region of other N tra plasmids.

The oriT region of the conjugative IncN plasmid pCU1 has been localized to a 669-bp sequence extending from pCU1 coordinates 8.48 to 9.15 kb. The nucleotide sequence of this region was determined. The region is AT-rich (69% AT residues), with one 19-bp and one 81-bp sequence containing 79% or more AT residues. Prominent sequence features include one set of thirteen 11-bp direct repeats, a second set of two 14-bp direct repeats, six different inverted repeat sequences ranging from 6 to 10 bp in size, and two sequences showing 12 of 13 nucleotides identical to the consensus integration host factor binding sequence. Specificity between this oriT and mobilization (mob) functions encoded by the N tra system was demonstrated. This specificity is encoded by the region lying clockwise of the BglII site at coordinate 3.3 on the pCU1 map. Two N tra plasmids isolated in the preantibiotic era were unable to mobilize recombinant plasmids carrying the oriT region of pCU1 or to complement transposon Tn5 mutations in the mob region of the closely related plasmid pKM101.     

Herpes simplex virus: selection of origins of DNA replication.

A selection procedure was devised to study the role of cis -acting sequences at origins of DNA replication. Two regions in Herpes simplex virus oriS were examined: an AT-rich spacer sequence and a putative binding site, box III, for the origin binding protein. Plasmid libraries were generated using oligonucleotides with locally random sequences. The library, amplified in Escherichia coli , was used to transfect BHK cells followed by superinfection with HSV-1. Replicated plasmids resistant to Dpn I cleavage were amplified in E. coli. The selection scheme was repeated. Plasmids were isolated at different stages of the procedure and their replication efficiency was determined. Efficiently replicating plasmids had a high AT content in the spacer sequence as well as a low helical stability of this region. In contrast, this was not seen using the box III library. We also noted that the wild type sequence invariably dominated the library after five rounds of selection. These plasmids arose from recombination between plasmids and viral DNA. Our results imply that a large group of sequences can mechanistically serve as origins of DNA replication. In a viral system, however, where the initiation process might be rate-limiting, this potentially large group of sequences would always converge towards the most efficient replicator.     

Efficient production of a human tumour necrosis factor in Escherichia coli

To examine the effect of altering the nucleotide sequence of the Shine-Dalgarno (SD) region and the spacer sequence between the SD sequence and the AUG translation start signal, several plasmids were constructed which directed the synthesis of mature human tumour necrosis factor (TNF) under the control of the E. coli trp leader promoter. We found that the presence of the SD sequence, AAGGAGGT, which is complementary to the 3' end of 16S rRNA, gave the higher translational efficiency, and also, the presence of the spacer sequence which consists of only A and T residues raised the production of TNF 2-3 fold. The levels of expression of TNF were elevated over 20-fold by the alteration of the SD and spacer sequences and amounted to 20% of the total cellular proteins or approx. 5 X 10(6) molecules of TNF per cell.     

Enhancement of bovine growth hormone gene expression by increasing the plasmid copy number

Effect of copy number on the expression of bovine growth hormone gene (bGH) was investigated using the copy number mutants such as pKBJ10, pBJ( tet)10, pUBJ10-1, and pUBJ10 plasmids. The cells harboring plasmids below 84 copies/cell did not produced detectable levels of bGH. When the ColE1 replicon was replaced with the mutated ColE1 replicon originated from pUC19 plasmid, the copy number was increased to about 300 copies/cell and bGH production was enhanced by 11.5% (pUBJ10-1) and 12.3% of total cell protein (pUBJ10). A large amount of mRNA caused by increment of copy number would be needed to overcome some inhibitory threshold and might be an important factor for regulating bGH expression.     

Mutagenesis study to ameliorate the bacterial expression of phosphoprotein P of vesicular stomatitis virus

The phosphoprotein gene of vesicular stomatitis virus, a Rhabdovirus, has been inserted into bacterial expression plasmids containing the Escherichia coli tac promoter and ribosome binding site (RBS). A low level of expression of the protein was detected. Sequence analysis showed the presence of 15 nucleotides in the spacer region i.e., between the Shine-Dalgarno sequence and ATG. Alteration of the distance and the sequence in the spacer region by oligonucleotide-directed mutagenesis revealed a correlation among the expression levels, accessibility of the RBS and requirement for a minimum spacing of at least 7 nucleotides between the Shine-Dalgarno sequence and ATG for optimal gene expression.     

Post-transcriptional control of bacteriophage T4 gene 25 expression: mRNA secondary structure that enhances translational initiation.

Secondary structure of the mRNA in the translational initiation region is an important determinant of translation efficiency. However, the secondary structures that enhance or facilitate translation initiation are rare. We have previously proposed that such structure may exist in the case of bacteriophage T4 gene 25 translational initiation region, which contains three potential Shine-Dalgarno sequences (SD1, SD2, and SD3) with a spacing of 8, 17, and 27 nucleotides from the initiation codon of this gene, respectively. We now present results that clearly demonstrate the existence of a hairpin structure that includes SD1 and SD2 sequences and brings the SD3, the most typical of these Shine-Dalgarno sequences, to a favourable spacing with the initiation codon of gene 25.Using a phage T7 expression system, we show that mutations that prevent the formation of hairpin structure or eliminate the SD3 sequence result in a decreased level of gp25 synthesis. Double mutation in base-pair V restores the level of gene 25 expression that was decreased by either of the two mutations (C-to-G and G-to-C) alone, as predicted by an effect attributable to mRNA secondary structure. We introduced the mutations into the bacteriophage T4 by plasmid-phage recombination. Changes in the plaque and burst sizes of T4 mutants, carrying single and double mutations in the translational initiation region of gene 25, strongly suggest that the predicted mRNA secondary structure controls (enhances) the level of gene 25 expression in vivo. Hybridization of total cellular RNA with a gene 25 specific probe indicated that secondary structure or mutations in the translational initiation region do not notably affect the 25 mRNA stability. Immunoblot analysis of gp25 in Escherichia coli cells infected by T4 mutants showed that mRNA secondary structure increases the level of gp25 synthesis by three- to fourfold. Since the secondary structure increases the level of gp25 synthesis and does not affect mRNA stability, we conclude that this structure enhances translation initiation. We discuss some features of two secondary structures in the translational initiation regions of T4 genes 25 and 38.     

T7噬菌体转运真核表达载体入胞表达平台的构建

[目的]构建携带锚定序列的真核表达载体,研究T7噬菌体识别、包裹和转运真核表达载体进入细胞实现蛋白表达的可行性,为DNA疫苗研发建立新的技术平台.[方法]本研究通过重叠延伸PCR方法获得候选锚定序列并插入真核表达载体;建立荧光定量PCR方法比较T7噬菌体识别、包裹真核表达载体的效率;激光共聚焦显微镜观察T7噬菌体转运真...     

A novel autonomously replicating sequence (ARS) for multiple integration in the yeast Hansenula polymorpha DL-1.

Several autonomously replicating sequences of Hansenula polymorpha DL-1 (HARSs) with the characteristics of tandem integration were cloned by an enrichment procedure and analyzed for their functional elements to elucidate the mechanism of multiple integration in tandem repeats. All plasmids harboring newly cloned HARSs showed a high frequency of transformation and were maintained episomally before stabilization. After stabilization, the transforming DNA was stably integrated into the chromosome. HARS36 was selected for its high efficiency of transformation and tendency for integration. Several tandemly repeated copies of the transforming plasmid containing HARS36 (pCE36) integrated into the vicinity of the chromosomal end. Bal 31 digestion of the total DNA from the integrants followed by Southern blotting generated progressive shortening of the hybridization signal, indicating the telomeric localization of the transforming plasmids on the chromosome. The minimum region of HARS36 required for its HARS activity was analyzed by deletion analyses. Three important regions, A, B, and C, for episomal replication and integration were detected. Analysis of the DNA sequences of regions A and B required for the episomal replication revealed that region A contained several AT-rich sequences that showed sequence homology with the ARS core consensus sequence of Saccharomyces cerevisiae. Region B contained two directly repeated sequences which were predicted to form a bent DNA structure. Deletion of the AT-rich core in region A resulted in a complete loss of ARS activity, and deletion of the repeated sequences in region B greatly reduced the stability of the transforming plasmid and resulted in retarded cell growth. Region C was required for the facilitated chromosomal integration of transforming plasmids.     

A novel minisatellite repeat expansion identified at FRA16B in a Japanese carrier

Previously, the allelic expansion of a 33-bp AT-rich minisatellite repeat has been reported to cause FRA16B, a distamycin A-inducible fragile site. Here, we identified a novel 35-bp minisatellite repeat at FRA16B in a Japanese carrier. The nucleotide sequence of the 35-bp minisatellite was highly AT-rich and nearly identical to the 33-bp one but with insertion of two nucleotides, thymine and adenine. The copy number of the AT-rich minisatellite was 21 in total in the carrier, while only a few copies of the 33-bp minisatellite were present in a non-carrier Japanese subject. These results suggest that the molecular mechanism involved in the allelic expansion of the minisatellite repeat in FRA16B recognizes both minisatellites, the 33-bp one and the 35-bp one, as an amplicon. These observations were different from the ones at folate-sensitive fragile sites, where the CCG triplet repeat was commonly involved in the allelic expansion. Although a slight reduction in AT content (95% > 90%) in the region of minisatellite expansion in the carrier subject was observed, both AT-content and length of the highly AT-rich region seem to play important roles in the cytogenetic expression of the distamycin A-inducible fragile site. In another normal subject, without fragile site expression, allelic expansion involving the 33-bp minisatellite was observed, and the length of the AT-rich DNA region was increased up to approximately 1000 bp. Since the length of the AT-rich minisatellite region was increased up to approximately 1,100-bp in the carrier subject, the threshold length for the cytogenetic expression of the AT-rich DNA region may be between about 1,000-bp and 1,100-bp.     

Genes for tRNALys5 from Drosophila melanogaster.

The sequences of two cloned genes from Drosophila which hybridize with tRNALys5 are reported. One gene, in plasmid pDt39, has a sequence which corresponds to the sequence of tRNA. The other gene, in pDt59R, differs in three nucleotides pairs. Both plasmids are transcribed in vitro with extracts of Drosophila Kc cells to give full-sized tRNA precursors with four additional nucleotides at the 5'-end as well as truncated molecules containing 35 nucleotides. This premature termination occurs in a block of four T residues within the mature coding region. Sequences flanking the tRNA genes show little in common except for the blocks of five or more T-residues beyond the 3'-end of the gene. pDt39 hybridizes to 84AB on the polytene chromosomes of Drosophila and pDt59R hybridizes to 29A.     

A Petunia hybrida chloroplast DNA region, close to one of the inverted repeats, shows sequence homology with the Euglena gracilis chloroplast DNA region that carries the putative replication origin

Summary Three distinct chloroplast (cp) DNA fragments from Petunia hybrida, which promote autonomous replication in yeast, were mapped on the chloroplast genome. Sequence analysis revealed that these fragments (called ARS A, B and C) have a high AT content, numerous short direct and inverted repeats and at least one yeast ARS consensus sequence 5A/TTTTATPuTTTA/T, essential for yeast ARS activity. ARS A and B also showed the presence of (semi-)conserved sequences, present in all Chlamydomanas reinhardii cpDNA regions that promote autonomous replication in yeast (ARS sequences) or in C. reinhardii (ARC sequences). A 431 bp BamHI/EcoRI fragment, close to one of the inverted repeats and adjacent to the ARS B subfragment contains an AT-rich stretch of about 100 nucleotides that show extensive homology with an Euglena gracilis cpDNA fragment which is part of the replication origin region. This conserved region contains direct and inverted repeats, stem-and-loop structures can be folded and it contains an ARS consensus sequence. In the near vicinity a GC-rich block is present. All these features make this cpDNA region the best candidate for being the origin of replication of P. hybrida cpDNA.     

Construction of chimeric plasmids containing histone H5 cDNA from hen erythrocyte. DNA sequence of a fragment derived from the 5' region of H5 mRNA.

We report that construction and characterization of chicken erythrocyte histone H5 cDNA recombinant plasmids. cDNA was synthesized from poly(A)+ polysomal RNA enriched in H5 mRNA and inserted into the PstI site of pBR322. Several clones containing H5 cDNA sequences were obtained and one of them (p541), expressing H5 antigenic determinants, was sequenced. The DNA insert of p541 contains 118 nucleotides from the 5' non-translated region of H5 mRNA and sequences coding for up to residue 46 of the N-terminus of the arginine (position 15) H5 variant. There is a strikingly high number of repeated sequences both in the leader and coding region; among these, the octanucleotide 5' GCG GCG GC 3' is found five times along the sequence. Although the H5 mRNA 5' leader is GC-rich (66%), there is an AT-rich region, about 16 nucleotides long, which shares strong homology with the leaders of sea urchin histone H1 mRNAs.